Insecticidal protein toxins from Photorhabdus

Purification of Toxin from Photorhabdus luminescens and Demonstration of Toxicity After Oral Delivery of Purified Toxin

The insecticidal protein toxin of the present invention was purified from Photorhabdus luminescens strain W-14, ATCC Accession Number 55397. Stock cultures of Photorhabdus luminescens were maintained on petri dishes containing 2% Proteose Peptone No. 3 (i.e., PP3, Difco Laboratories, Detroit Mich.) in 1.5% agar, incubated at 25° C. and transferred weekly. Colonies of the primary form of the bacteria were inoculated into 200 ml of PP3 broth supplemented with 0.5% polyoxyethylene sorbitan mono-stearate (TWEEN-60, Sigma Chemical Company, St. Louis, Mo.) in a one liter flask. The broth cultures were grown for 72 hours at 30° C. on a rotary shaker. The toxin proteins can be recovered from cultures grown in the presence or absence of TWEEN surfactant; however, the absence of Tween can affect the form of the bacteria grown and the profile of-proteins produced by the bacteria. In the absence of TWEEN surfactant, a variant shift occurs insofar as the molecular weight of at least one identified toxin subunit shifts from about 200 kDa to about 185 kDa.

The 72 hour cultures were centrifuged at 10,000×g for 30 minutes to remove cells and debris. The supernatant fraction that contained the insecticidal activity was decanted and brought to 50 mM K 2 HPO 4 by adding an appropriate volume of 1.0 M K 2 HPO 4 . The pH was adjusted to 8.6 by adding potassium hydroxide. This supernatant fraction was then mixed with DEAE-SEPHACEL resin (Pharmacia LKB Biotechnology) which had been equilibrated with 50 mM K 2 HPO 4 . The toxic activity was adsorbed to the DEAE resin. This mixture was then poured into a 2.6×40 cm column and washed with 50 mM K 2 HPO 4 at room temperature at a flow rate of 30 ml/hr until the effluent reached a steady baseline UV absorbance at 280 nm. The column was then washed with 150 mM KCl until the effluent again reached a steady 280 nm baseline. Finally the column was washed with 300 mM KCl and fractions were collected.

Fractions containing the toxin were pooled and filter sterilized using a 0.2 micron pore membrane filter. The toxin was then concentrated and equilibrated to 100 mM KPO 4 , pH 6.9, using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa at 4° C. (Centriprep 100, Amicon Division-W.R. Grace and Company). A 3 ml sample of the toxin concentrate was applied to the top of a 2.6×95 cm SEPHACRYL resin S-400 HR gel filtration column (Pharmacia LKB Biotechnology). The eluent buffer was 100 mM KPO 4 , pH 6.9, which was run at a flow rate of 17 ml/hr, at 4° C. The effluent was monitored at 280 nm.

Fractions were collected and tested for toxic activity. Toxicity of chromatographic fractions was examined in a biological assay using Manduca sexta larvae. Fractions were either applied directly onto the insect diet (Gypsy moth wheat germ diet, ICN Biochemicals Division—ICN Biomedicals, Inc.) or administered by intrahemocelic injection of a 5 μl sample through the first proleg of 4th or 5th instar larva using a 30 gauge needle. The weight of each larva within a treatment group was recorded at 24 hour intervals. Toxicity was presumed if the insect ceased feeding and died within several days of consuming treated insect diet or if death occurred within 24 hours after injection of a fraction.

The toxic fractions were pooled and concentrated using the Centriprep-100 and were then analyzed by HPLC using a 7.5 mm×60 cm TSK-GEL G-4000 SW gel permeation column with 100 mM potassium phosphate, pH 6.9 eluent buffer running at 0.4 ml/min. This analysis revealed the toxin protein to be contained within a single sharp peak that eluted from the column with a retention time of approximately 33.6 minutes. This retention time corresponded to an estimated molecular weight of 1,000 kDa. Peak fractions were collected for further purification while fractions not containing this protein were discarded. The peak eluted from the HPLC absorbs UV light at 218 and 280 nm but did not absorb at 405 nm. Absorbance at 405 nm was shown to be an attribute of xenorhabdin antibiotic compounds.

Electrophoresis of the pooled peak fractions in a non-denaturing agarose gel (Metaphor Agarose, FMC BioProducts) showed that two protein complexes are present in the peak. The peak material, buffered in 50 mM Tris-HCl, pH 7.0, was separated on a 1.5% agarose stacking gel buffered with 100 mM Tris-HCl at pH 7.0 and 1.9% agarose resolving gel buffered with 200 mM Tris-borate at pH 8.3 under standard buffer conditions (anode buffer 1M Tris-HCl, pH 8.3; cathode buffer 0.025 M Tris, 0.192 M glycine). The gels were run at 13 mA constant current at 15° C. until the phenol red tracking dye reached the end of the gel. Two protein bands were visualized in the agarose gels using Coomassie brilliant blue staining.

The slower migrating band was referred to as “protein band 1” and faster migrating band was referred to as “protein band 2.” The two protein bands were present in approximately equal amounts. The Coomassie stained agarose gels were used as a guide to precisely excise the two protein bands from unstained portions of the gels. The excised pieces containing the protein bands were macerated and a small amount of sterile water was added. As a control, a portion of the gel that contained no protein was also excised and treated in the same manner as the gel pieces containing the protein. Protein was recovered from the gel pieces by electroelution into 100 mM Tris-borate pH 8.3, at 100 volts (constant voltage) for two hours. Alternatively, protein was passively eluted from the gel pieces by adding an equal volume of 50 mM Tris-HCl, pH 7.0, to the gel pieces, then incubating at 30° C. for 16 hours. This allowed the protein to diffuse from the gel into the buffer, which was then collected.

Results of insect toxicity tests using HPLC-purified toxin (33.6 min. peak) and agarose gel purified toxin demonstrated toxicity of the extracts. Injection of 1.5 μg of the HPLC purified protein kills within 24 hours. Both protein bands 1 and 2, recovered from agarose gels by passive elution or electroelution, were lethal upon injection. The protein concentration estimated for these samples was less than 50 ng/larva. A comparison of the weight gain and the mortality between the groups of larvae injected with protein bands 1 or 2 indicate that protein band 1 was more toxic by injection delivery.

When HPLC-purified toxin was applied to larval diet at a concentration of 7.5 μg/larva, it caused a halt in larval weight gain (24 larvae tested). The larvae begin to feed, but after consuming only a very small portion of the toxin treated diet they began to show pathological symptoms induced by the toxin and the larvae cease feeding. The insect frass became discolored and most larva showed signs of diarrhea. Significant insect mortality resulted when several 5 μg toxin doses were applied to the diet over a 7-10 day period.

Agarose-separated protein band 1 significantly inhibited larval weight gain at a dose of 200 ng/larva. Larvae fed similar concentrations of protein band 2 were not inhibited and gained weight at the same rate as the control larvae. Twelve larvae_were fed eluted protein and 45 larvae were fed protein-containing agarose pieces. These two sets of data indicate that protein band 1 was orally toxic to Manduca sexta . In this experiment it appeared that protein band 2 was not toxic to Manduca sexta.

Further analysis of protein bands 1 and 2 by SDS-PAGE under denaturing conditions showed that each band was composed of several smaller protein subunits. Proteins were visualized by Coomassie brilliant blue staining followed by silver staining to achieve maximum sensitivity.

The protein subunits in the two bands were very similar. Protein band 1 contains 8 protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical profile except that the 25.1, 60.8, and 65.6 kDa proteins were not present. The 56.2, 60.8, 65.6, and 184 kDa proteins were present in the complex of protein band 1 at approximately equal concentrations and represent 80% or more of the total protein content of that complex.

The native HPLC-purified toxin was further characterized as follows. The toxin was heat labile in that after being heated to 60° C. for 15 minutes it lost its ability to kill or to inhibit weight gain when injected or fed to Manduca sexta larvae. Assays were designed to detect lipase, type C phospholipase, nuclease or red blood cell hemolysis activities and were performed with purified toxin. None of these activities were present. Antibiotic zone inhibition assays were also done and the purified toxin failed to inhibit growth of Gram-negative or -positive bacteria, yeast or filamentous fungi, indicating that the toxic is not a xenorhabdin antibiotic.

The native HPLC-purified toxin was tested for ability to kill insects other than Manduca sexta . Table 3 lasts insects killed by the HPLC-purified Photorhabdus luminescens toxin in this study.

Further Characterization of the High Molecular Weight Toxin Complex

In yet further analysis, the toxin protein complex was subjected to further characterization from W-14 growth medium. The culture conditions and initial purification steps through the S-400 HR column were identical to those described above. After isolation of the high molecular weight toxin complex from the S-400 HR column fractions, the toxic fractions were equilibrated with 10 mM Tris-HCl, pH 8.6, and concentrated in the centriplus 100 (Amicon) concentrators. The protein toxin complex was then applied to a weak anion exchange (WAX) column, Vydac 301VPH575 (Hesparia, Calif.), at a flow rate of 0.5 ml/min. The proteins were eluted with a linear potassium chloride gradient, 0-250 mM KCl, in 10 mM Tris-HCl pH 8.6 for 50 min. Eight protein peaks were detected by absorbance at 280 nm.

Bioassays using neonate southern corn rootworm ( Diabrotica undecimpunctata howardi , SCR) larvae and tobacco horn worm ( Manduca sexta , THW) were performed on all fractions eluted from the HPLC column. THW were grown on Gypsy Moth wheat germ diet (ICN) at 25° C. with a 16 hr light 8 hr dark cycle. SCR were grown on Southern Corn Rootworm Larval Insecta-Diet (BioServ) at 25° C. with a 16 hr light/8 hr dark cycle.

The highest mortality for SCR and THW larvae was observed for peak 6, which eluted with ca. 112 mM to 132mM KCl. SDS-PAGE analysis of peak 6 showed predominant peptides of 170 kDa, 66 kDa, 63 kDa, 59.5 kDa and 31 kDa. Western blot analysis was performed on peak 6 protein fraction with a mixture of polyclonal antibodies made against TcaA ii -syn, TcaA iii -syn, TcaB ii -syn, TcaC-syn, and TcbA ii -syn peptides (described in Example 21) and C5F2, a monoclonal antibody against the TcbA iii peptide. Peak 6 contained immuno-reactive bands of 170 kDa, 90 kDa, 66 kDa, 59.5 kDa and 31 kDa. These are very close to the predicted sizes for the TcaC (166 kDa), TcaA ii +TcaA iii (92 kDa), TcaA iii (66 kDa), TcaB ii (60 kDa) and TcaA ii (25 kDa), respectively. Peak 6 which was further analyzed by native agarose gel electrophoresis, as described herein, migrated as a single band with similar mobility to that of band 1.

The protein concentration of the purified peak 6 toxin protein was determined using the BCA reagents (Pierce). Dilutions of the protein were made in 10 mM Tris, pH 8.6 and applied to the diet bioassays. After 240 hours all neonate larvae on diet bioassays that received 450 ng or greater of the peak 6 protein fraction were dead. The group of larvae that received 90 ng of the same fraction had 40% mortality. After 240 hrs the survivors that received 90 ng and 20 ng of peak 6 protein fraction were ca. 10% and 70%, respectively, of the control weight.

Insecticide Utility

The Photorhabdus luminescens utility and toxicity were further characterized. Photorhabdus luminescens (strain W-14) culture broth was produced as follows. The production medium was 2% Bacto Proteose Peptone® Number 3 (PP3, Difco Laboratories, Detroit, Mich.) in Milli-Q® deionized water. Seed culture flasks consisted of 175 ml medium placed in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput and autoclaved for 20 minutes, T=250° F. Production flasks consisted of 500 mls in a 2.8 liter 500 ml tribaffled flask with a Delong neck, covered by a Shin-etsu silicon foam closure. These were autoclaved for 45 minutes, T=250° F. The seed culture was incubated at 28° C. at 150 rpm in a gyrotory shaking incubator with a 2 inch throw. After 16 hours of growth, 1% of the seed culture was placed in the production flask which was allowed to grow for 24 hours before harvest. Production of the toxin appears to be during log phase growth. The microbial broth was transferred to a 1 L centrifuge bottle and the cellular biomass was pelleted (30 minutes at 2500 RPM at 4° C., [R.C.F.=about 1600] HG-4L Rotor RC3 Sorval centrifuge, Dupont, Wilmington, Del.). The primary broth was chilled at 4° C. for 8-16 hours and recentrifuged at least 2 hours (conditions above) to further clarify the broth by removal of a putative mucopolysaccharide which precipitated upon standing. (An alternative processing method combined both steps and involved the use of a 16 hour clarification centrifugation, same conditions as above.) This broth was then stored at 4° C. prior to bioassay or filtration.

Photorhabdus culture broth and protein toxin(s) purified from this broth showed activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects. More specifically, the activity is seen against corn rootworm (larvae and adult), Colorado potato beetle, and turf grubs, which are members of the insect order Coleoptera. Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and weevils. Activity has also been observed against aster leafhopper, which is a member of the order, Homoptera. Other members of the Homoptera include planthoppers, pear pyslla, apple sucker, scale insects, whiteflies, and spittle bugs, as well as numerous host specific aphid species. The broth and purified fractions are also active against beet armyworm, cabbage looper, black cutworm, tobacco budworm, European corn borer, corn earworm, and codling moth, which are members of the order Lepidoptera. Other typical members of this order are clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm, and fall armyworm. Activity is also seen against fruitfly and mosquito larvae, which are members of the order Diptera. Other members of the order Diptera are pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly, house fly, and various mosquito species. Activity is seen against carpenter ant and Argentine ant, which are members of the order that also includes fire ants, oderous house ants, and little black ants.

The broth/fraction is useful for reducing populations of insects and were used in a method of inhibiting an insect population. The method may comprise applying to a locus of the insect an effective insect inactivating amount of the active described. Results are reported in Table 4.

Activity against corn rootworm larvae was tested as follows. Photorhabdus culture broth (filter sterilized, cell-free) or purified HPLC fractions were applied directly to the surface (about 1.5 cm 2 ) of 0.25 ml of artificial diet in 30 μl aliquots following dilution in control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from sterilized eggs, with second instar SCR grown on artificial diet or with second instar Diabrotica virgifera virgifera (Western corn rootworm, WCR) reared on corn seedlings grown in Metromix®. Second instar larvae were weighed prior to addition to the diet. The plates were sealed, placed in a humidified growth chamber and maintained at 27° C. for the appropriate period (4 days for neonate and adult SCR, 2-5 days for WCR larvae, 7-14 days for second instar SCR). Mortality and weight determinations were scored as indicated. Generally, 16 insects per treatment were used in all studies. Control mortalities were as follows: neonate larvae, <5%, adult beetles, 5%.

Activity against Colorado potato beetle was tested as follows. Photorhabdus culture broth or control medium was applied to the surface (about 2.0 cm 2 ) of 1.5 ml of standard artificial diet held in the wells of a 24-well tissue culture plate. Each well received 50 μl of treatment and was allowed to air dry. Individual second instar Colorado potato beetle ( Leptinotarsa decemlineata , CPB) larvae were then placed onto the diet and mortality was scored after 4 days. Ten larvae per treatment were used in all studies. Control mortality was 3.3%.

Activity against Japanese beetle grubs and beetles was tested as follows. Turf grubs ( Popillia japonica , 2-3rd instar) were collected from infested lawns and maintained in the laboratory in soil/peat mixture with carrot slices added as additional diet. Turf beetles were pheromone-trapped locally and maintained in the laboratory in plastic containers with maple leaves as food. Following application of undiluted Photorhabdus culture broth or control medium to corn rootworm artificial diet (30 μl/1.54 cm 2 , beetles) or carrot slices (larvae), both stages were placed singly in a diet well and observed for any mortality and feeding. In both cases there was a clear reduction in the amount of feeding (and feces production) observed.

Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well contained 200 μl of aqueous solution (Photorhabdus culture broth, control medium or H 2 O) and approximately 20, 1-day old larvae ( Aedes aegypti ). There were 6 wells per treatment. The results were read at 2 hours after infestation and did not change over the three day observation period. No control mortality was seen.

Activity against fruitflies was tested as follows. Purchased Drosophila melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium or Photorhabdus culture broth. This was accomplished by placing 8.0 ml of dry medium in each of 3 rearing vials per treatment and adding 8.0 ml of the appropriate liquid. Ten late instar Drosophila melanogaster maggots were then added to each vial. The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 3, 7 and 10 days of exposure. Incorporation of Photorhabdus culture broth into the diet media for fruitfly maggots caused a slight (17%) but significant reduction in day-10 adult emergence as compared to water and control medium (3% reduction).

Activity against aster leafhopper was tested as follows. The ingestion assay for aster leafhopper ( Macrosteles severini ) is designed to allow ingestion of the active without other external contact. The reservoir for the active/“food” solution is made by making 2 holes in the center of the bottom portion of a 35×10 mm Petri dish. A 2 inch Parafilm M® square is placed across the top of the dish and secured with an “O” ring. A 1 oz. plastic cup is then infested with approximately 7 leafhoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes. In tests using undiluted Photorhabdus culture broth, the broth and control medium were dialyzed against water to reduce control mortality. Mortality is reported at day 2 where 26.5% control mortality was seen. In the tests using purified fractions (200 mg protein/ml) a final concentration of 5% sucrose was used in all treatments to improve survivability of the aster leafhoppers. The assay was held in an incubator at 28° C., 70% RH with a 16/8 photoperiod. The assay was graded for mortality at 72 hours. Control mortality was 5.5%.

Activity against Argentine ants was tested as follows. A 1.5 ml aliquot of 100% Photorhabdus culture broth, control medium or water was pipetted into 2.0 ml clear glass vials. The vials were plugged with a piece of cotton dental wick that was moistened with the appropriate treatment. Each vial was placed into a separate 60×16mm Petri dish with 8 to 12 adult Argentine ants ( Linepithema humile ). There were three replicates per treatment. Bioassay plates were held on a laboratory bench, at room temperature under fluorescent ceiling lights. Mortality readings were made after 5 days of exposure. Control mortality was 24%.

Activity against carpenter ant was tested as follows. Black carpenter ant workers ( Camponotus pennsylvanicus ) were collected from trees on DowElanco property in Indianapolis, Ind. Tests with Photorhabdus culture broth were performed as follows. Each plastic bioassay container (7⅛″×3″) held fifteen workers, a paper harborage and 10 ml of broth or control media in a plastic shot glass. A cotton wick delivered the treatment to the ants through a hole in the shot glass lid. All treatments contained 5% sucrose. Bioassays were held in the dark at room temperature and graded at 19 days. Control mortality was 9%. Assays delivering purified fractions utilized artificial ant diet mixed with the treatment (purified fraction or control solution) at a rate of 0.2 ml treatment/2.0 g diet in a plastic test tube. The final protein concentration of the purified fraction was less than 10 μg/g diet. Ten ants per treatment, a water source, harborage and the treated diet were placed in sealed plastic containers and maintained in the dark at 27° C. in a humidified incubator. Mortality was scored at day 10. No control mortality was seen.

Activity against various lepidopteran larvae was tested as follows. Photorhabdus culture broth or purified fractions were applied directly to the surface (about 1.5 cm 2 ) of 0.25 ml of standard artificial diet in 30 μl aliquots following dilution in control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate larva. European corn borer ( Ostrinia nubilalis ) and corn earworm ( Helicoverpa zea ) eggs were supplied from commercial sources and hatched in-house, whereas beet armyworm ( Spodoptera exigua ), cabbage looper ( Trichoplusia ni ), tobacco budworm ( Heliothis virescens ), codling moth ( Laspeyresia pomonella ) and black cutworm ( Agrotis ipsilon ) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27° C. for the appropriate period. Mortality and weight determinations were scored at days 5-7 for Photorhabdus culture broth and days 4-7 for the purified fraction. Generally, 16 insects per treatment were used in all studies. Control mortality ranged from 4-12.5% for control medium and was less than 10% for phosphate buffer.

Insecticide Utility upon Soil Application

Photorhabdus luminescens (strain W-14) culture broth was shown to be active against corn rootworm when applied directly to soil or a soil-mix (Metromix®). Activity against neonate SCR and WCR in Metromix® was tested as follows (Table 5). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the light on moist filter paper for 6 days. After roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastic cup with 50 gm of dry Metromix®. Twenty neonate SCR or WCR were then placed directly on the roots of the seedling and covered with Metromix®. Upon infestation, the seedlings were then drenched with 50 ml total volume of a diluted broth solution. After drenching, the cups were sealed and left at room temperature in the light for 7 days. Afterwards, the seedlings were washed to remove all Metromix® and the roots were excised and weighed. Activity was rated as the percentage of corn root remaining relative to the control plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either −, +, ++, or +++, with − representing no damage and +++ representing severe damage.

Activity against neonate SCR in soil was tested as follows (Table 6). The test was run using corn seedlings (United Agriseeds brand CL614) that were germinated in the light on moist filter paper for 6 days. After the roots were approximately 3-6 cm long, a single kernel/seedling was planted in a 591 ml clear plastic cup with 150 gm of soil from a field in Lebanon, Ind. planted the previous year with corn. This soil had not been previously treated with insecticides. Twenty neonate SCR were then placed directly on the roots of the seedling and covered with soil. After infestation, the seedlings were drenched with 50 ml total volume of a diluted broth solution. After drenching, the unsealed cups were incubated in a high relative humidity chamber (80%) at 78° F. Afterwards, the seedlings were washed to remove all soil and the roots were excised and weighed. Activity was rated as the percentage of corn root remaining relative to the control plants and as leaf damage induced by feeding. Leaf damage was scored visually and rated as either −, +, ++, or +++, with − representing no damage and +++ representing severe damage.

Activity of Photorhabdus luminescens (strain W-14) culture broth against second instar turf grubs in Metromix® was observed in tests conducted as follows (Table 7). Approximately 50 gm of dry Metromix® was added to a 591 ml clear plastic cup. The Metromix® was then drenched with 50 ml total volume of a 50% (v/v) diluted Photorhabdus broth solution. The dilution of crude broth was made with water, with 50% broth being prepared by adding 25 ml of crude broth to 25 ml of water for 50 ml total volume. A 1% (w/v) solution of proteose peptone #3 (PP3), which is a 50% dilution of the normal media concentration, was used as a broth control. After drenching, five second instar turf grubs were placed on the top of the moistened Metromix®. Healthy turf grub larvae burrowed rapidly into the Metromix®. Those larvae that did not burrow within 1 h were removed and replaced with fresh larvae. The cups were sealed and placed in a 28° C. incubator, in the dark. After seven days, larvae were removed from the Metromix® and scored for mortality. Activity was rated the percentage of mortality relative to control.

Insecticide Utility upon Leaf Application

Activity of Photorhabdus broth against European corn borer was seen when the broth was applied directly to the surface of maize leaves (Table 8). In these assays Photorhabdus broth was diluted 100-fold with culture medium and applied manually to the surface of excised maize leaves at a rate of about 6.0 μl/cm 2 of leaf surface. The leaves were air dried and cut into equal sized strips approximately 2×2 inches. The leaves were rolled, secured with paper clips and placed in 1 oz plastic shot glasses with 0.25 inch of 2% agar on the bottom surface to provide moisture. Twelve neonate European corn borers were then placed onto the rolled leaf and the cup was sealed. After incubation for 5 days at 27° C. in the dark, the samples were scored for feeding damage and recovered larvae.

Activity of the culture broth against neonate tobacco budworm ( Heliothis virescens ) was demonstrated using a leaf dip methodology. Fresh cotton leaves were excised from the plant and leaf disks were cut with an 18.5 mm cork-borer. The disks were individually emersed in control medium (PP3) or Photorhabdus luminescens (strain W-14) culture broth which had been concentrated approximately 10-fold using an Amicon (Beverly, Mass.), Proflux M12 tangential filtration system with a 10 kDa filter. Excess liquid was removed and a straightened paper clip was placed through the center of the disk. The paper clip was then wedged into a plastic, 1.0 oz shot glass containing approximately 2.0 ml of 1% Agar. This served to suspend the leaf disk above the agar. Following drying of the leaf disk, a single neonate tobacco budworm larva was placed on the disk and the cup was capped. The cups were then sealed in a plastic bag and placed in a darkened, 27° C. incubator for 5 days. At this time the remaining larvae and leaf material were weighed to establish a measure of leaf damage (Table 9).

Characterization of Toxin Peptide Components

In yet another analysis, the toxin protein complex was re-isolated from the Photorhabdus luminescens growth medium (after culture without TWEEN surfactant) by performing a 10%-80% ammonium sulfate precipitation followed by an ion exchange chromatography step (Mono Q) and two molecular sizing chromatography steps. These conditions were like those used in Example 1. During the first molecular sizing step, a second biologically active peak was found at about 100±10 kDa. Based upon protein measurements, this fraction was 20-50 fold less active than the larger, or primary, active peak of about 860±100 kDa (native). During this isolation experiment, a smaller active peak of about 325±50 kDa that retained a considerable portion of the starting biological activity was also resolved. It is thought that the 325 kDa peak is related to or derived from the 860 kDa peak.

A 56 kDa protein was resolved in this analysis. The N-terminal sequence of this protein is presented in SEQ ID NO:6. It is noteworthy that this protein shares significant identity and conservation with SEQ ID NO:5 at the N-terminus, suggesting that the two may be encoded by separate members of a gene family and that the proteins produced by each gene are sufficiently similar to both be operable in the insecticidal toxin complex.

A second, prominent 185 kDa protein was consistently present in amounts comparable to that of protein 3 from Table 10, and may be the same protein or protein fragment. The N-terminal sequence of this 185 kDa protein is shown at SEQ ID NO:7.

Additional N-terminal amino acid sequence data were also obtained from isolated proteins. None of the determined N-terminal sequences appear identical to a protein identified in Table 10. Other proteins were present in isolated preparation. One such protein has an estimated molecular weight of 108 kDa and an N-terminal sequence as shown in SEQ ID NO:8. A second such protein has an estimated molecular weight of 80 kDa and an N-terminal sequence as shown in SEQ ID NO:9.

When the protein material in the approximately 325 kDa active peak was analyzed by size, bands of approximately 51, 31, 28, and 22 kDa were observed. As in all cases in which a molecular weight was determined by analysis of electrophoretic mobility, these molecular weights were subject to error effects introduced by buffer ionic strength differences, electrophoresis power differences, and the like. One of ordinary skill would understand that definitive molecular weight values cannot be determined using these standard methods and that each was subject to variation. It was hypothesized that proteins of these sizes are degradation products of the larger protein species (of approximately 200 kDa size) that were observed in the larger primary toxin complex.

Finally, several preparations included a protein having the N-terminal sequence shown in SEQ ID NO:10. This sequence was strongly homologous to known chaperonin proteins, accessory proteins known to function in the assembly of large protein complexes. Although the applicants could not ascribe such an assembly function to the protein identified in SEQ ID NO:10, it was consistent with the existence of the described toxin protein complex that such a chaperonin protein could be involved in its assembly. Moreover, although such proteins have not directly been suggested to have toxic activity, this protein may be important to determining the overall structural nature of the protein toxin, and thus, may contribute to the toxic activity or durability of the complex in vivo after oral delivery.

Subsequent analysis of the stability of the protein toxin complex to proteinase K was undertaken. It was determined that after 24 hour incubation of the complex in the presence of a 10-fold molar excess of proteinase K, activity was virtually eliminated (mortality on oral application dropped to about 5%). These data confirm the proteinaceous nature of the toxin.

The toxic activity was also retained by a dialysis membrane, again confirming the large size of the native toxin complex.

Isolation, Characterization and Partial Amino Acid Sequencing of Photorhabdus Toxins

Isolation and N-Terminal Amino Acid Sequencing

In a set of experiments conducted in parallel to Examples 5 and 6, ammonium sulfate precipitation of Photorhabdus proteins was performed by adjusting Photorhabdus broth, typically 2-3 liters, to a final concentration of either 10% or 20% by the slow addition of ammonium sulfate crystals. After stirring for 1 hour at 4° C., the material was centrifuged at 12,000×g for 30 minutes. The supernatant was adjusted to 80% ammonium sulfate, stirred at 4° C. for 1 hour, and centrifuged at 12,000×g for 60 minutes. The pellet was resuspended in one-tenth the volume of 10 mM Na 2 .PO 4 , pH 7.0 and dialyzed against the same phosphate buffer overnight at 4° C. The dialyzed material was centrifuged at 12,000×g for 1 hour prior to ion exchange chromatography.

A.HR 16/50 Q Sepharose (Pharmacia) anion exchange column was equilibrated with 10 mM Na 2 .PO 4 , pH 7.0. Centrifuged, dialyzed ammonium sulfate pellet was applied to the Q Sepharose column at a rate of 1.5 ml/min and washed extensively at 3.0 ml/min with equilibration buffer until the optical density (O.D. 280) reached less than 0.100. Next, either a 60 minute NaCl gradient ranging from 0 to 0.5 M at 3 ml/min, or a series of step elutions using 0.1 M, 0.4 M and finally 1.0 NaCl for 60 minutes each was applied to the column. Fractions were pooled and concentrated using a Centriprep 100. Alternatively, proteins could be eluted by a single 0.4 M NaCl wash without prior elution with 0.1 M NaCl.

Two milliliter aliquots of concentrated Q Sepharose samples were loaded at 0.5 ml/min onto a HR 16/50 Superose 12 (Pharmacia) gel filtration column equilibrated with 10 mM Na 2 .PO 4 , pH 7.0. The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected. The void volume material was collected and concentrated using a centriprep 100. Two milliliter aliquots of concentrated Superose 12 samples were loaded at 0.5 ml/min onto a HR 16/50 Sepharose 4B-CL (Pharmacia) gel filtration column equilibrated with 10 mM Na 2 .PO 4 , pH 7.0. The column was washed with the same buffer for 240 min at 0.5 ml/min and 2 min samples were collected.

The excluded protein peak was subjected to a second fractionation by application to a gel filtration column that used a Sepharose CL-4B resin, which separates proteins ranging from about 30 kDa to 1000 kDa. This fraction was resolved into two peaks; a minor peak at the void volume (>1000 kDa) and a major peak which eluted at an apparent molecular weight of about 860 kDa. Over a one week period subsequent samples subjected to gel filtration showed the gradual appearance of a third peak (approximately 325 kDa) that seemed to arise from the major peak, perhaps by limited proteolysis. Bioassays performed on the three peaks showed that the void peak had no activity, while the 860 kDa toxin complex fraction was highly active, and the 325 kDa peak was less active, although quite potent. SDS PAGE analysis of Sepharose CL-4B toxin complex peaks from different fermentation productions revealed two distinct peptide patterns, denoted “P” and “S”. The two patterns had marked differences in the molecular weights and concentrations of peptide components in their fractions. The “S” pattern, produced most frequently, had 4 high molecular weight peptides (>150 kDa) while the “P” pattern had 3 high molecular weight peptides. In addition, the “S” peptide fraction was found to have 2-3 fold more activity against European Corn Borer. This shift may be related to variations in protein expression due to age of inoculum and/or other factors based on growth parameters of aged cultures.

Milligram quantities of peak toxin complex fractions determined to be “P” or “S” peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (Seprabuff™ to PVDF membranes (ProBlott™, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides in the “S” pattern had unique N-terminal amino acid sequences compared to the sequences identified in the previous example. A 201 kDa (TcdA ii ) peptide set forth as SEQ ID NO:13 below shared between 33% amino acid identity and 50% similarity (similarity and identity were calculated by hand) with SEQ ID NO:1 (TcbA ii )(in Table 10 vertical lines denote amino acid identities and colons indicate conservative amino acid substitutions). A second peptide of 197 kDa, SEQ ID NO:14 (TcdB), had 42% identity and 58% similarity with SEQ ID NO:2 (TcaC) (similarity and identity were calculated by hand). Yet a third peptide of 205 kDa was denoted TcdA ii . In addition, a limited N-terminal amino acid sequence, SEQ ID NO:16 (TcbA), of a peptide of at least 235 kDa was identical with the amino acid sequence, SEQ ID NO:12, deduced from a cloned gene (tcbA), SEQ ID NO:11, containing a deduced amino acid sequence corresponding to SEQ ID NO:1 (TcbA ii ). This indicates that the larger 235+ kDa peptide was proteolytically processed to the 201 kDa peptide, (TcbA ii ), (SEQ ID NO:1) during fermentation, possibly resulting in activation of the molecule. In yet another sequence, the sequence originally reported as SEQ ID NO:5 (TcaB ii ) reported in Example 5 above, was found to contain an aspartic acid residue (Asp) at the third position rather than glycine (Gly) and two additional amino acids Gly and Asp at the eighth and ninth positions, respectively. In yet two other sequences, SEQ ID NO:2 (TcaC) and SEQ ID NO:3 (TcaB i ), additional amino acid sequence was obtained. Densitometric quantitation was performed using a sample that was identical to the “S” preparation sent for N-terminal analysis. This analysis showed that the 201 kDa and 197 kDa peptides represent 7.0% and 7.2%, respectively, of the total Coomassie brillant blue stained protein in the “S” pattern and are present in amounts similar to the other abundant peptides. It was speculated that these peptides may represent protein homologs, analogous to the situation found with other bacterial toxins, such as various CryI Bt toxins. These proteins vary from 40-90% similarity at their N-terminal amino acid sequence, which encompasses the toxic fragment.

Internal Amino Acid Sequencing

To facilitate cloning of toxin peptide genes, internal amino acid sequences of selected peptides were obtained as followed. Milligram quantities of peak 2A fractions determined to be “P” or “S” peptide patterns were subjected to preparative SDS PAGE, and transblotted with TRIS-glycine (Seprabuff™ to PVDF membranes (ProBlott™, Applied Biosystems) for 3-4 hours. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. Three peptides, referred to as TcbA ii (containing SEQ ID NO:1), TcdA ii , and TcaB i (containing SEQ ID NO:3) were subjected to trypsin digestion by Harvard MicroChem followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal peptides were sequenced for the peptide TcdA ii (205 kDa peptide) referred to as TcdA ii -PT111 (SEQ ID NO:17) and TcdA ii -PT79 (SEQ ID NO:18). Two internal peptides were sequenced for the peptide TcaB i (68 kDa peptide) referred to as TcaB i -PT158 (SEQ ID NO:19) and TcaB i -PT108 (SEQ ID NO:20). Four internal peptides were sequenced for the peptide TcbA ii (201 kDa peptide) referred to as TcbA ii -PT103 (SEQ ID NO:21), TcbA ii -PTS6 (SEQ ID NO:22), TcbA ii -PT81(a) (SEQ ID NO:23), and TcbA ii -PT81(b) (SEQ ID NO:24).

Construction of a Cosmid Library of Photorhabdus luminescens W-14 Genomic DNA and its Screening to Isolate Genes Encoding Peptides Comprising the Toxic Protein Preparation

As a prerequisite for the production of Photorhabdus insect toxic proteins in heterologous hosts, and for other uses, it is necessary to isolate and characterize the genes that encode those peptides. This objective was pursued in parallel. One approach, described later, was based on the use of monoclonal and polyclonal antibodies raised against the purified toxin which were then used to isolate clones from an expression library. The other approach, described in this example, is based on the use of the N-terminal and internal amino acid sequence data to design degenerate oligonucleotides for use in PCR amplication. Either method can be used to identify DNA clones that contain the peptide-encoding genes so as to permit the isolation of the respective genes, and the determination of their DNA base sequence.

Genomic DNA Isolation

Photorhabdus luminescens strain W-14 (ATCC accession number 55397) was grown on 2% proteose peptone #3 agar (Difco Laboratories, Detroit, Mich.) and insecticidal toxin competence was maintained by repeated bioassay after passage, using the method described in Example 1 above. A 50 ml shake culture was produced in a 175 ml baffled flask in 2% proteose peptone #3 medium, grown at 28° C. and 150 rpm for approximately 24 hours. 15 ml of this culture was pelleted and frozen in its medium at −20° C. until it was thawed for DNA isolation. The thawed culture was centrifuged, (700×g, 30 min) and the floating orange mucopolysaccharide material was removed. The remaining cell material was centrifuged (25,000×g, 15 min) to pellet the bacterial cells, and the medium was removed and discarded.

Genomic DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Current Protocols in Molecular Biology (Ausubel et al. eds, John Wiley & Sons, 1994) [modified to include a salt shock and with all volumes increased 10-fold]. The pelleted bacterial cells were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12 ml of 5 M NaCl was added; this mixture was centrifuged 20 min at 15,000×g. The pellet was resuspended in 5.7 ml TE and 300 ml of 10% SDS and 60 ml of 20 mg/ml proteinase K (Gibco BRL Products, Grand Island, N.Y.; in sterile distilled water) were added to the suspension. This mixture was incubated at 37° C. for 1 hr; then approximately 10 mg lysozyme (Worthington Biochemical Corp., Freehold, N.J.) was added. After an additional 45 min, 1 ml of 5 M NaCl and 800 ml of CTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) were added. This preparation was incubated 10 min at 65° C., then gently agitated and further incubated and agitated for approximately 20 min to assist clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently and centrifuged. After two extractions with an equal volume of PCI (phenol/chloroform/isoamyl alcohol; 50:49:1, v/v/v; equilibrated with 1 M Tris-HCl, pH 8.0; Intermountain Scientific Corporation, Kaysville, Utah), the DNA was precipitated with 0.6 volume of isopropanol. The DNA precipitate was gently removed with a glass rod, washed twice with 70% ethanol, dried, and dissolved in 2 ml STE (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA). This preparation contained 2.5 mg/ml DNA, as determined by optical density at 260 nm (i.e., OD 260 )

The molecular size range of the isolated genomic DNA was evaluated for suitability for library construction. CHEF gel analysis was performed in 1.5% agarose (Seakem® LE, FMC BioProducts, Rockland, Me.) gels with 0.5×TBE buffer (44.5 mM Tris-HCl pH 8.0, 44.5 mM H 3 BO 3 , 1 mM EDTA) on a BioRad CHEF-DR II apparatus with a Pulsewave 760 Switcher (Bio-Rad Laboratories, Inc., Richmond, Calif.). The running parameters were: initial A time, 3 sec; final A time, 12 sec; 200 volts; running temperature, 4-18° C.; run time, 16.5 hr. Ethidium bromide staining and examination of the gel under ultraviolet light indicated the DNA ranged from 30-250 kbp in size.

Construction of Library

A partial Sau3A 1 digest was made of this Photorhabdus genomic DNA preparation. The method was based on section 3.1.3 of Ausubel (supra.). Adaptions included running smaller scale reactions under various conditions until nearly optimal results were achieved. Several scaled-up large reactions with varied conditions were run, the results analyzed on CHEF gels, and only the best large scale preparation was carried forward. In the optimal case, 200 μg of Photorhabdus genomic DNA was incubated with 1.5 units of Sau3A 1 (New England Biolabs, “NEB”, Beverly, Mass.) for 15 min at 37° C. in 2 ml total volume of 1×NEB 4 buffer (supplied as 10× by the manufacturer). The reaction was stopped by adding 2 ml of PCI and centrifuging at 8000×g for 10 min. To the supernatant were added 200 μl of 5 M NaCl plus 6 ml of ice-cold ethanol. This preparation was chilled for 30 min at −20° C., then centrifuged at 12,000×g for 15 min. The supernatant was removed and the precipitate was dried in a vacuum oven at 40° C., then resuspended in 400 μl STE. Spectrophotometric assay indicated about 40% recovery of the input DNA. The digested DNA was size fractionated on a sucrose gradient according to section 5.3.2 of CPMB (op. cit.). A 10% to 40% (w/v) linear sucrose gradient was prepared with a gradient maker in Ultra-Clear™ tubes (Beckman Instruments, Inc., Palo Alto, Calif.) and the DNA sample was layered on top. After centrifugation, (26,000 rpm, 17 hr, Beckman SW41 rotor, 20° C.), fractions (about 750 μl) were drawn from the top of the gradient and analyzed by CHEF gel electrophoresis (as described earlier). Fractions containing Sau3A 1 fragments in the size range 20-40 kbp were selected and DNA was precipitated by a modification (amounts of all solutions increased approximately 6.3-fold) of the method in section 5.3.3 of Ausubel (supra.). After overnight precipitation, the DNA was collected by centrifugation (17,000×g, 15 min), dried, redissolved in TE, pooled into a final volume of 80 μl, and reprecipitated with the addition of 8 μl 3 M sodium acetate and 220 μl ethanol. The pellet collected by centrifugation as above was resuspended in 12 μl TE. Concentration of the DNA was determined by Hoechst 33258 dye (Polysciences, Inc., Warrington, Pa.) fluorometry in a Hoefer TKO100 fluorimeter (Hoefer Scientific Instruments, San Francisco, Calif.). Approximately 2.5 μg of the size-fractionated DNA was recovered.

Thirty μg of cosmid pWE15 DNA (Stratagene, La Jolla, Calif.) was digested to completion with 100 units of restriction enzyme BamH 1 (NEB) in the manufacturer's buffer (final volume of 200 μl, 37° C., 1 hr). The reaction was extracted with 100 μl of PCI and DNA was precipitated from the aqueous phase by addition of 20 μl 3M sodium acetate and 550 μl −20° C. absolute ethanol. After 20 min at −70° C., the DNA was collected by centrifugation (17,000×g, 15 min), dried under vacuum, and dissolved in 180 μl of 10 mM Tris-HCl, pH 8.0. To this were added 20 μl of 10×CIP buffer (100 mM Tris-HCl, pH 8.3; 10 mM ZnCl 2 ; 10 mM MgCl 2 ), and 1 μl (0.25 units) of 1:4 diluted calf intestinal alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, Ind.). After 30 min at 37° C., the following additions were made: 2 μl 0.5 M EDTA, pH 8.0; 10 μl 10% SDS; 0.5 μl of 20 mg/ml proteinase K (as above), followed by incubation at 55° C. for 30 min. Following sequential extractions with 100 μl of PCI and 100 μl phenol (Intermountain Scientific Corporation, equilibrated with 1 M Tris-HCl, pH 8.0), the dephosphorylated DNA was precipitated by addition of 72 μl of 7.5 M ammonium acetate and 550 μl −20° C. ethanol, incubation on ice for 30 min, and centrifugation as above. The pelleted DNA was washed once with 500 μl −20° C. 70% ethanol, dried under vacuum, and dissolved in 20 μl of TE buffer.

Ligation of the size-fractionated Sau3A 1 fragments to the BamH 1-digested and phosphatased pWE15 vector was accomplished using T4 ligase (NEB) by a modification (i.e., use of premixed 10×ligation buffer supplied by the manufacturer) of the protocol in section 3.33 of Ausubel. Ligation was carried out overnight in a total volume of 20 μl at 15° C., followed by storage at −20° C.

Four μl of the cosmid DNA ligation reaction, containing about 1 μl of DNA, was packaged into bacteriophage lambda using a commercial packaging extract (Gigapack® III Gold Packaging Extract, Stratagene), following the manufacturer's directions. The packaged preparation was stored at 4° C. until use. The packaged cosmid preparation was used to infect Escherichia coli XL1 Blue MR cells (Stratagene) according to the Gigapack® III Gold protocols (“Titering the Cosmid Library”), as follows. XL1 Blue MR cells were grown in LB medium (g/L: Bacto-tryptone, 10; Bacto-yeast extract, 5; Bacto-agar, 15; NaCl, 5; [Difco Laboratories, Detroit, Mich.] containing 0.2% (w/v) maltose plus 10 mM MgSO 4 , at 37° C. After 5 hr growth, cells were pelleted at 700×g (15 min) and resuspended in 6 ml of 10 mM MgSO 4 . The culture density was adjusted with 10 mM MgSO 4 to OD 600 =0.5. The packaged cosmid library was diluted 1:10 or 1:20 with sterile SM medium (0.1 M NaCl, 10 mM MgSO 4 , 50 mM Tris-HCl pH 7.5, 0.01% w/v gelatin), and 25 μl of the diluted preparation was mixed with 25 μl of the diluted XL1 Blue MR cells. The mixture was incubated at 25° C. for 30 min (without shaking), then 200 μl of LB broth was added, and incubation was continued for approximately 1 hr with occasional gentle shaking. Aliquots (20-40 μl) of this culture were spread on LB agar plates containing 100 mg/l ampicillin (i.e., LB-Amp 100 ) and incubated overnight at 37° C. To store the library without amplification, single colonies were picked and inoculated into individual wells of sterile 96-well microwell plates; each well containing 75 μl of Terrific Broth (TB media: 12 g/l Bacto-tryptone, 24 g/l Bacto-yeast extract, 0.4% v/v glycerol, 17 mM KH 2 PO 4 , 72 mM K 2 HPO 4 ) plus 100 mg/l ampicillin (i.e., TB-Amp 100 ) and incubated (without shaking) overnight at 37° C. After replicating the 96-well plate into a copy plate, 75 μl/well of filter-sterilized TB:glycerol (1:1, v/v; with, or without, 100 mg/l ampicillin) was added to the plate, it was shaken briefly at 100 rpm, 37° C., and then closed with Parafilm® (American National Can, Greenwich, Conn.) and placed in a −70° C. freezer for storage. Copy plates were grown and processed identically to the master plates. A total of 40 such master plates (and their copies) were prepared.

Screening of the Library with Radiolabeled DNA Probes

To prepare colony filters for probing with radioactively labeled probes, ten 96-well plates of the library were thawed at 25° C. (bench top at room temperature). A replica plating tool with 96 prongs was used to inoculate a fresh 96-well copy plate containing 75 μl/well of TB-Amp 100 . The copy plate was grown overnight (stationary) at 37° C., then shaken about 30 min at 100 rpm at 37° C. A total of 800 colonies was represented in these copy plates, due to nongrowth of some isolates. The replica tool was used to inoculate duplicate impressions of the 96-well arrays onto Magna NT (MSI, Westboro, Mass.) nylon membranes (0.45 micron, 220×250 mm) which had been placed on solid LB-Amp 100 (100 ml/dish) in Bio-assay plastic dishes (Nunc, 243×243×18 mm; Curtin Mathison Scientific, Inc., Wood Dale, Ill.). The colonies were grown on the membranes at 37° C. for about 3 hr.

A positive control colony (a bacterial clone containing a GZ4 sequence insert, see below) was grown on a separate Magna NT membrane (Nunc, 0.45 micron, 82 mm circle) on LB medium supplemented with 35 mg/l chloramphenicol (i.e., LB-Cam 35 ), and processed alongside the library colony membranes. Bacterial colonies on the membranes were lysed, and the DNA was denatured and neutralized according to a protocol taken from the Genius™ System User's Guide version 2.0 (Boehringer Mannheim, Indianapolis, Ind.). Membranes were placed colony side up on filter paper soaked with 0.5 N NaOH plus 1.5 M NaCl for 15 min to denature, and neutralized on filter paper soaked with 1 M Tris-HCl pH 8.0, 1.5 M NaCl for 15 min. After UV-crosslinking using a Stratagene UV Stratalinker set on auto crosslink, the membranes were stored dry at 25° C. until use. Membranes were trimmed into strips containing the duplicate impressions of a single 96-well plate, then washed extensively by the method of section 6.4.1 in CPMB (op. cit.): 3 hr at 25° C. in 3×SSC, 0.1% (w/v) SDS, followed by 1 hr at 65° C. in the same solution, then rinsed in 2×SSC in preparation for the hybridization step (20×SSC=3 M NaCl, 0.3 M sodium citrate, pH 7.0).

Amplification of a Specific Genomic Fragment of a TcaC Gene

Based on the N-terminal amino acid sequence determined for the purified TcaC peptide fraction [disclosed herein as SEQ ID NO:2], a pool of degenerate oligonucleotides (pool S4Psh) was synthesized by standard β-cyanoethyl chemistry on an Applied BioSystem ABI394 DNA/RNA Synthesizer (Perkin Elmer, Foster City, Calif.). The oligonucleotides were deprotected 8 hours at 55° C., dissolved in water, quantitated by spectrophotometric measurement, and diluted for use. This pool corresponds to the determined N-terminal amino acid sequence of the TcaC peptide. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents inosine:

Another set of degenerate oligonucleotides was synthesized (pool P2.3.5R), representing the complement of the coding strand for determined amino acid sequence of the SEQ ID NO:17:

These oligonucleotides were used as primers in Polymerase Chain Reactions (PCR®, Roche Molecular Systems, Branchburg, N.J.) to amplify a specific DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above). A typical reaction (50 μl) contained 125 pmol of each primer pool P2Psh and P2.3.5R, 253 ng of genomic template DNA, 10 nmol each of dATP, dCTP, dGTP, and dTTP, 1×GeneAmp® PCR buffer, and 2.5 units of AmpliTaq® DNA polymerase (both from Roche Molecular Systems; 10×GeneAmp buffer is 100 mM Tris-HCl pH 8.3, 500 mM KCl, 0.01% w/v gelatin). Amplifications were performed in a Perkin Elmer Cetus DNA Thermal Cycler (Perkin Elmer, Foster City, Calif., using 35 cycles of 94° C. (1.0 min), 55° C. (2.0 min), 72° C. (3.0 min), followed by an extension period of 7.0 min at 72° C. Amplification products were analyzed by electrophoresis through 2% w/v NuSieve® 3:1 agarose (FMC BioProducts) in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0). A specific product of estimated size 250 bp was observed amongst numerous other amplification products by ethidium bromide (0.5 μg/ml) staining of the gel and examination under ultraviolet light.

The region of the gel containing an approximately 250 bp product was excised, and a small plug (0.5 mm dia.) was removed and used to supply template for PCR amplification (40 cycles). The reaction (50 μl) contained the same components as above, minus genomic template DNA. Following amplification, the ends of the fragments were made blunt and were phosphorylated by incubation at 25° C. for 20 min with 1 unit of T4 DNA polymerase (NEB), 1 nmol ATP, and 2.15 units of T4 kinase (Pharmacia Biotech Inc., Piscataway, N.J.).

DNA fragments were separated from residual primers by electrophoresis through 1% w/v GTG® agarose (FMC) in TEA. A gel slice containing fragments of apparent size 250 bp was excised, and the DNA was extracted using a Qiaex kit (Qiagen Inc., Chatsworth, Calif.).

The extracted DNA fragments were ligated to plasmid vector pBC KS(+) (Stratagene) that had been digested to completion with restriction enzyme Sma 1 and extracted in a manner similar to that described for pWE15 DNA above. A typical ligation reaction (16.3 μl) contained 100 ng of digested pBC KS(+) DNA, 70 ng of 250 bp fragment DNA, 1 nmol [Co(NH 3 ) 6 ]Cl 3 , and 3.9 Weiss units of T4 DNA ligase (Collaborative Biomedical Products, Bedford, Mass.), in 1×ligation buffer (50 mM Tris-HCl, pH 7.4; 10 mM MgCl 2 ; 10 M dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin). Following overnight incubation at 14° C., the ligated products were transformed into frozen, competent Escherichia coli DHSα cells (Gibco BRL) according to the suppliers' recommendations, and plated on LB-Cam 35 plates, containing IPTG (119 μg/ml) and X-gal (50 μg/ml). Independent white colonies were picked, and plasmid DNA was prepared by a modified alkaline-lysis/PEG precipitation method (PRISM™ Ready Reaction DyeDeoxy™ Terminator Cycle Sequencing Kit Protocols; ABI/Perkin Elmer). The nucleotide sequence of both strands of the insert DNA was determined, using T7 primers [pBC KS(+) bases 601-623: TAAAACGACGGCCAGTGAGCGCG) and LacZ primers [pBC KS(+) bases 792-816: ATGACCATGATTACGCCAAGCGCGC) and protocols supplied with the PRISM™ sequencing kit (ABI/Perkin Elmer) Nonincorporated dye-terminator dideoxyribonucleotides were removed by passage through Centri-Sep 100 columns (Princeton Separations, Inc., Adelphia, N.J.) according to the manufacturer's instructions. The DNA sequence was obtained by analysis of the samples on an ABI Model 373A DNA Sequencer (ABI/Perkin Elmer). The DNA sequences of two isolates, GZ4 and HB14, were found to be as illustrated in FIG. 1 .

This sequence illustrates the following features: 1) bases 1-20 represent one of the 64 possible sequences of the S4Psh degenerate oligonucleotides, ii) the sequence of amino acids 1-3 and 6-12 correspond exactly to that determined for the N-terminus of TcaC (disclosed as SEQ ID NO:2), iii) the fourth amino acid encoded is a cysteine residue rather than serine. This difference is encoded within the degeneracy for the serine codons (see above), iv) the fifth amino acid encoded is proline, corresponding to the TcaC N-terminal sequence given as SEQ ID NO:2, v) bases 257-276 encode one of the 192 possible sequences designed into the degenerate pool, vi) the TGA termination codon introduced at bases 268-270 is the result of complementarity to the degeneracy built into the oligonucleotide pool at the corresponding position, and does not indicate a shortened reading frame for the corresponding gene.

Labeling of a TcaC Peptide Gene-specific Probe

DNA fragments corresponding to the above 276 bases were amplified (35 cycles) by PCR® in a 100 μl reaction volume, using 100 pmol each of P2Psh and P2.3.5R primers, 10 ng of plasmids GZ4 or HB14 as templates, 20 nmol each of DATP, dCTP, dGTP, and dTTP, 5 units of AmpliTAq® DNA polymerase, and 1× concentration of GeneAmp® buffer, under the same temperature regimes as described above. The amplification products were extracted from a 1% GTG® agarose gel by Qiaex kit and quantitated by fluorometry.

The extracted amplification products from plasmid HB14 template (approximately 400 ng) were split into five aliquots and labeled with 32 P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) according to the manufacturer's instructions. Nonincorporated radioisotope was removed by passage through NucTrap® Probe Purification Columns (Stratagene), according to the supplier's instructions. The specific activity of the labeled DNA product was determined by scintillation counting to be 3.11×10 8 dpm/μg. This labeled DNA was used to probe membranes prepared from 800 members of the genomic library.

Screening with a TcaC-peptide Gene Specific Probe

The radiolabeled HB14 probe was boiled approximately 10 min, then added to “minimal hyb” solution. [Note: The “minimal hyb” method is taken from a CERES protocol; “Restriction Fragment Length Polymorphism Laboratory Manual version 4.0”, sections 4-40 and 4-47; CERES/NPI, Salt Lake City, Utah. NPI is now defunct, with its successors operating as Linkage Genetics]. “Minimal hyb” solution contains 10% w/v PEG (polyethylene glycol, M.W. approx. 8000), 7% w/v SDS; 0.6×SSC, 10 mM sodium phosphate buffer (from a 1M stock containing 95 g/l NaH 2 PO 4 .1H 2 O and 84.5 g/l Na 2 HPO 4 .7H 2 O), 5 mM EDTA, and 100 mg/ml denatured salmon sperm DNA. Membranes were blotted dry briefly then, without prehybridization, 5 strips of membrane were placed in each of 2 plastic boxes containing 75 ml of “minimal hyb” and 2.6 ng/ml of radiolabeled HB14 probe. These were incubated overnight with slow shaking (50 rpm) at 60° C. The filters were washed three times for approximately 10 min each at 25° C. in “minimal hyb wash solution” (0.25×SSC, 0.2% SDS), followed by two 30-min washes with slow shaking at 60° C. in the same solution. The filters were placed on paper covered with Saran Wrap® (Dow Brands, Indianapolis, Ind.) in a light-tight autoradiographic cassette and exposed to X-Omat X-ray film (Kodak, Rochester, N.Y.) with two DuPont Cronex Lightning-Plus C1 enhancers (Sigma Chemical Co., St. Louis, Mo.), for 4 hr at −70° C. Upon development (standard photographic procedures), significant signals were evident in both replicates amongst a high background of weaker, more irregular signals. The filters were again washed for about 4 hr at 68° C. in “minimal hyb wash solution” and then placed again in the cassettes and film was exposed overnight at −70° C. Twelve possible positives were identified due to strong signals on both of the duplicate 96-well colony impressions. No signal was seen with negative control membranes (colonies of XL1 Blue MR cells containing pWE15), and a very strong signal was seen with positive control membranes (DH5α cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.

The twelve putative hybridization-positive colonies were retrieved from the frozen 96-well library plates and grown overnight at 37° C. on solid LB-Amp 100 medium. They were then patched (3/plate, plus three negative controls: XL1 Blue MR cells containing the pWE15 vector) onto solid LB-Amp 100 . Two sets of membranes (Magna NT nylon, 0.45 micron) were prepared for hybridization. The first set was prepared by placing a filter directly onto the colonies on a patch plate, then removing it with adherent bacterial cells, and processing as below. Filters of the second set were placed on plates containing LB-Amp 100 medium, then inoculated by transferring cells from the patch plates onto the filters. After overnight growth at 37° C., the filters were removed from the plates and processed.

Bacterial cells on the filters were lysed and DNA denatured by placing each filter colony-side-up on a pool (1.0 ml) of 0.5 N NaOH in a plastic plate for 3 min. The filters were blotted dry on a paper towel, then the process was repeated with fresh 0.5 N NaOH. After blotting dry, the filters were neutralized by placing each on a 1.0 ml pool of 1 M Tris-HCl, pH 7.5 for 3 min, blotted dry, and reneutralised with fresh buffer. This was followed by two similar soakings (5 min each) on pools of 0.5 M Tris-HCl pH 7.5 plus 1.5 M NaCl. After blotting dry, the DNA was UV crosslinked to the filter (as above), and the filters were washed (25° C., 100 rpm) in about 100 ml of 3×SSC plus 0.1%(w/v) SDS (4 times, 30 min each with fresh solution for each wash). They were then placed in a minimal volume of prehybridization solution [6×SSC plus 1% w/v each of Ficoll 400 (Pharmacia), polyvinylpyrrolidone (av. M.W. 360,000; Sigma) and bovine serum albumin Fraction V; (Sigma)] for 2 hr at 65° C., 50 rpm. The prehybridization solution was removed, and replaced with the HB14 32 P-labeled probe that had been saved from the previous hybridization of the library membranes and which had been denatured at 95° C. for 5 min. Hybridization was performed at 60° C. for 16 hr with shaking at 50 rpm.

Following removal of the labeled probe solution, the membranes were washed 3 times at 25° C. (50 rpm, 15 min) in 3×SSC (about 150 ml each wash). They were then washed for 3 hr at 68° C. (50 rpm) in 0.25×SSC plus 0.2% SDS (minimal hyb wash solution), and exposed to X-ray film as described above for 1.5 hr at 25° C. (no enhancer screens). This exposure revealed very strong hybridization signals to cosmid isolates 22G12, 25A10, 26A5, and 26B10, and a very weak signal with cosmid isolate 8B10. No signal was seen with the negative control (pWE15) colonies, and a very strong signal was seen with positive control membranes (DH5a cells containing the GZ4 isolate of the PCR product) that had been processed concurrently with the experimental samples.

Amplification of a Specific Genomic Fragment of a TcaB Gene

Based on the N-terminal amino acid sequence determined for the purified TcaB i peptide fraction (disclosed here as SEQ ID NO:3) a pool of degenerate oligonucleotides (pool P8F) was synthesized as described for peptide TcaC. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, G, and T are the standard DNA bases, and I represents inosine:

Another set of degenerate oligonucleotides was synthesized (pool P8.108.3R), representing the complement of the coding strand for the determined amino acid sequence of the TcaB i -PT108 internal peptide (disclosed herein as SEQ ID NO:20):

These oligonucleotides were used as primers for PCR® using HotStart 50 Tubes™ (Molecular Bio-Products, Inc., San Diego, Calif.) to amplify a specific DNA fragment from genomic DNA prepared from Photorhabdus strain W-14 (see above). A typical reaction (50 μl) contained (bottom layer) 25 pmol of each primer pool P8F and PB.108.3R, with 2 nmol each of DATP, dCTP, dGTP, and dTTP, in 1×GeneAmp® PCR buffer, and (top layer) 230 ng of genomic template DNA, 8 nmol each of DATP, dCTP, dGTP, and dTTP, and 2.5 units of AmpliTaq® DNA polymerase, in 1×GeneAmp® PCR buffer. Amplifications were performed by 35 cycles as described for the TcaC peptide. Amplification products were analyzed by electrophoresis through 0.7% w/v SeaKem® LE agarose (FMC) in TEA buffer. A specific product of estimated size 1600 bp was observed.

Four such reactions were pooled, and the amplified DNA was extracted from a 1.0% SeaKem® LE gel by Qiaex kit as described for the TcaC peptide. The extracted DNA was used directly as the template for sequence determination (PRISM™ Sequencing Kit) using the P8F and P8.108.3R primer pools. Each reaction contained about 100 ng template DNA and 25 pmol of one primer pool, and was processed according to standard protocols as described for the TcaC peptide. An analysis of the sequence derived from extension of the P8F primers revealed the short DNA sequence (and encoded amino acid sequence):

which corresponds to a portion of the N-terminal peptide sequence disclosed as SEQ ID NO:3 (TcaB i ).

Labeling of a TcaB i -peptide Gene-specific Probe

Approximately 50 ng of gel-purified TcaB i DNA fragment was labeled with 32 P-dCTP as described above, and nonincorporated radioisotopes were removed by passage through a NICK Column® (Pharmacia). The specific activity of the labelled DNA was determined to be 6×10 9 dpm/μg. This labeled DNA was used to probe colony membranes prepared from members of the genomic library that had hybridized to the TcaC-peptide specific probe.

The membranes containing the 12 colonies identified in the TcaC-probe library screen (see above) were stripped of radioactive TcaC-specific label by boiling twice for approximately 30 min each time in 1 liter of 0.1×SSC plus 0.1% SDS. Removal of radiolabel was checked with a 6 hr film exposure. The stripped membranes were then incubated with the TcaB i peptide-specific probe prepared above. The labeled DNA was denatured by boiling for 10 min, and then added to the filters that had been incubated for 1 hr in 100 ml of “minimal hyb” solution at 60° C. After overnight hybridization at this temperature, the probe solution was removed, and the filters were washed as follows (all in 0.3×SSC plus 0.1% SDS): once for 5 min at 25° C., once for 1 hr at 60° C. in fresh solution, and once for 1 hr at 63° C. in fresh solution. After 1.5 hr exposure to X-ray film by standard procedures, 4 strongly-hybridizing colonies were observed. These were, as with the TcaC-specific probe, isolates 22G12, 25A10, 26A5, and 26B10.

The same TcaB i probe solution was diluted with an equal volume (about 100 ml) of “minimal hyb” solution, and then used to screen the membranes containing the 800 members of the genomic library. After hybridization, washing, and exposure to X-ray film as described above, only the four cosmid clones 22G12, 25A10, 26A5, and 26B10, were found to hybridize strongly to this probe.

Isolation of Subclones Containing Genes Encoding TcaC and YcaB i Peptides, and Determination of DNA Base Sequence Thereof

Three hybridization-positive cosmids in strain XL1 Blue MR were grown with shaking overnight (200 rpm) at 30° C. in 100 ml TB-Amp 100 . After harvesting the cells by centrifugation, cosmid DNA was prepared using a commercially available kit (BIGprep™, 5 Prime 3 Prime, Inc., Boulder, Colo.), following the manufacturer's protocols. Only one cosmid, 26A5, was successfully isolated by this procedure. When digested with restriction enzyme EcoR 1 (NEB) and analyzed by gel electrophoresis, fragments of approximate sizes 14, 10, 8 (vector), 5, 3.3, 2.9, and 1.5 kbp were detected. A second attempt to isolate cosmid DNA from the same three strains (8 ml cultures; TB-Amp 100 , 30° C.) utilized a boiling miniprep method (Evans G. and G. Wahl., 1987, “Cosmid vectors for genomic walking and rapid restriction mapping.” in Guide to Molecular Cloning Techniques, Meth. Enzymology, Vol. 152, S. Berger and A. Kimmel, eds., pgs. 604-610). Only one cosmid, 25A10, was successfully isolated by this method. When digested with restriction enzyme EcoR I (NEB) and analyzed by gel electrophoresis, this cosmid showed a fragmentation pattern identical to that previously seen with cosmid 26A5.

A 0.15 μg sample of 26A5 cosmid DNA was used to transform 50 ml of E. coli DH5α cells (Gibco BRL), by the supplier's protocols. A single colony isolate of that strain was inoculated into 4 ml of TB-Amp 100 , and grown for 8 hr at 37° C. Chloramphenicol was added to a final concentration of 225 μg/ml, incubation was continued for another 24 hr, then cells were harvested by centrifugation and frozen at −20° C. Isolation of the 26A5 cosmid DNA was by a standard alkaline lysis miniprep (Maniatis et al., op. cit., p. 382), modified by increasing all volumes by 50% and with stirring or gentle mixing, rather than vortexing, at every step. After washing the DNA pellet in 70% ethanol, it was dissolved in TE containing 25 μg/ml ribonuclease A (Boehringer Mannheim).

Identification of EcoR I Fragments Hybridizing to GZ4-derived and TcaB i -Probes

Approximately 0.4 μg of cosmid 25A10 (from XL1 Blue MR cells) and about 0.5 μg of cosmid 26A5 (from chloramphenicol-amplified DH5α cells) were each digested with about 15 units of EcoR I(NEB) for 85 min, frozen overnight, then heated at 65° C. for five min, and electrophoresed in a 0.7% agarose gel (Seakem® LE, 1×TEA, 80 volts, 90 min). The DNA was stained with ethidium bromide as described above, and photographed under ultraviolet light. The EcoR I digest of cosmid 25A10 was a complete digestion, but the sample of cosmid 26A5 was only partially digested under these conditions. The agarose gel containing the DNA fragments was subjected to depurination, denaturation and neutralization, followed by Southern blotting onto a Magna NT nylon membrane, using a high salt (20×SSC) protocol, all as described in section 2.9 of Ausubel et al. (CPMB, op. cit.). The transferred DNA was then UV-crosslinked to the nylon membrane as before.

An TcaC-peptide specific DNA fragment corresponding to the insert of plasmid isolate GZ4 was amplified by PCR® in a 100 ml reaction volume as described previously above. The amplification products from three such reactions were pooled and were extracted from a 1% GTR® agarose gel by Qiaex kit, as described above, and quantitated by fluorometry. The gel-purified DNA (100 ng) was labeled with 32 P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) as described above, to a specific activity of 6.34×10 8 dpm/μg.

The 32 P-labeled GZ4 probe was boiled 10 min, then added to “minimal hyb” buffer (at 1 ng/ml), and the Southern blot membrane containing the digested cosmid DNA fragments was added, and incubated for 4 hr at 60° C. with gentle shaking at 50 rpm. The membrane was then washed 3 times at 25° C. for about 5 min each (minimal hyb wash solution), followed by two washes for 30 min each at 60° C. The blot was exposed to film (with enhancer screens) for about 30 min at −70° C. The GZ4 probe hybridized strongly to the 5.0 kbp (apparent size) EcoR I fragment of both these two cosmids, 26A5 and 25A10.

The membrane was stripped of radioactivity by boiling for about 30 min in 0.1×SSC plus 0.1% SDS, and absence of radiolabel was checked by exposure to film. It was then hybridized at 60° C. for 3.5 hours with the (denatured) TcaB i probe in “minimal hyb” buffer previously used for screening the colony membranes (above), washed as described previously, and exposed to film for 40 min at −70° C. with two enhancer screens. With both cosmids, the TcaB i probe hybridized lightly with the about 5.0 kbp EcoR 1 fragment, and strongly with a fragment of approximately 2.9 kbp.

The sample of cosmid 26A5 DNA previously described, (from DH5α cells) was used as the source of DNA from which to subclone the bands of interest. This DNA (2.5 μg) was digested with about 3 units of EcoR I (NEB) in a total volume of 30 μl for 1.5 hr, to give a partial digest, as confirmed by gel electrophoresis. Ten μg of pBC KS (+) DNA (Stratagene) were digested for 1.5 hr with 20 units of EcoR I in a total volume of 20 μl, leading to total digestion as confirmed by electrophoresis. Both EcoR I-cut DNA preparations were diluted to 50 μl with water, to each an equal volume of PCI was added, the suspension was gently mixed, spun in a microcentrifuge and the aqueous supernatant was collected. DNA was precipitated by 150 μl ethanol, and the mixture was placed at −20° C. overnight. Following centrifugation and drying, the EcoR I -digested pBC KS (+) was dissolved in 100 μl TE; the partially digested 26A5 was dissolved in 20 μl TE. DNA recovery was checked by fluorometry.

In separate reactions, approximately 60 ng of EcoR I-digested pBC KS(+) DNA was ligated with approximately 180 ng or 270 ng of partially digested cosmid 26A5 DNA. Ligations were carried out in a volume of 20 μl at 15° C. for 5 hr, using T4 ligase and buffer from New England BioLabs. The ligation mixture, diluted to 100 μl with sterile TE, was used to transform frozen, competent DH5α cells (Gibco BRL) according to the supplier's instructions. Varying amounts (25-200 μl) of the transformed cells were plated on freshly prepared solid LB-Cam 35 medium with 1 mM IPTG and 50 mg/l X-gal. Plates were incubated at 37° C. about 20 hr, then chilled in the dark for approximately 3 hr to intensify color for insert selection. White colonies were picked onto patch plates of the same composition and incubated overnight at 37° C.

Two colony lifts of each of the selected patch plates were prepared as follows. After picking white colonies to fresh plates, round Magna NT nylon membranes were pressed onto the patch plates, the membrane was lifted off, and subjected to denaturation, neutralization and UV crosslinking as described above for the library colony membranes. The crosslinked colony lifts were vigorously washed, including gently wiping off the excess cell debris with a tissue. One set was hybridized with the GZ4(TcaC) probe solution described earlier, and the other set was hybridized with the TcaB i probe solution described earlier, according to the ‘minimal hyb’ protocol, followed by washing and film exposure as described for the library colony membranes.

Colonies showing hybridization signals either only with the GZ4 probe, with both GZ4 and TcaB i probes, or only with the TcaB i probe, were selected for further work and cells were streaked for single colony isolation onto LB-Cam 35 media with IPTG and X-gal as before. Approximately 35 single colonies, from 16 different isolates, were picked into liquid LB-Cam 35 media and grown overnight at 37° C.; the cells were collected by centrifugation and plasmid DNA was isolated by a standard alkaline lysis miniprep according to Maniatis et al. (op. cit. p. 368). DNA pellets were dissolved in TE+25 μg/ml ribonuclease A and DNA concentration was determined by fluorometry. The EcoR I digestion pattern was analyzed by gel electrophoresis. The following isolates were picked as useful. Isolate A17.2 contains religated pBC KS(+) only and was used for a (negative) control. Isolates D38.3 and C44.1 each contain only the 2.9 kbp, TcaB i -hybridizing EcoR I fragment inserted into pBC KS(+). These plasmids, named pDAB2000 and pDAB2001, respectively, are illustrated in FIG. 2 .

Isolate A35.3 contains only the approximately 5 kbp, GZ4)-hybridizing EcoR 1 fragment, inserted into pBC KS(+). This plasmid was named pDAB2002 (also FIG. 2 ). These isolates provided templates for DNA sequencing.

Plasmids pDAB2000 and pDAB2001 were prepared using the BIGprep™ kit as before. Cultures (30 ml) were grown overnight in TB-Cam 35 to an OD 600 of 2, then plasmid was isolated according to the manufacturer's directions. DNA pellets were redissolved in 100 μl TE each, and sample integrity was checked by EcoR I digestion and gel electrophoretic analysis.

Sequencing reactions were run in duplicate, with one replicate using as template pDAB2000 DNA, and the other replicate using as template pDAB2001 DNA. The reactions were carried out using the dideoxy dye terminator cycle sequencing method, as described above for the sequencing of the GZ4/HB14 DNAs. Initial sequencing runs utilized as primers the LacZ and T7 primers described above, plus primers based on the determined sequence of the TcaB i PCR amplification product (TH1=ATTGCAGACTGCCAATCGCTTCGG, TH12=GAGAGTATCCAGACCGCGGATGATCTG).

After alignment and editing of each sequencing output, each was truncated to between 250 to 350 bases, depending on the integrity of the chromatographic data as interpreted by the Perkin Elmer Applied Biosystems Division SeqEd 675 software. Subsequent sequencing “steps” were made by selecting appropriate sequence for new primers. With a few exceptions, primers (synthesized as described above) were 24 bases in length with a 50% G+C composition. Sequencing by this method was carried out on both strands of the approximately 2.9 kbp EcoR I fragment.

To further serve as template for DNA sequencing, plasmid DNA from isolate pDAB2002 was prepared by BIGprep™ kit. Sequencing reactions were performed and analyzed as described above. Initially, a T3 primer (pBS SK (+) bases 774-796: CGCGCAATTAACCCTCACTAAAG) and a T7 primer (pBS KS (+) bases 621-643: GCGCGTAATACGACTCACTATAG) were used to prime the sequencing reactions from the flanking vector sequences, reading into the insert DNA. Another set of primers, (GZ4F: GTATCGATTACAACGCTGTCACTTCCC; TH13: GGGAAGTGACAGCGTTGTAATCGATAC; TH14: ATGTTGGGTGCGTCGGCTAATGGACATAAC; and LW1-204: GGGAAGTGACAGCGTTGTAATCGATAC) was made to prime from internal sequences, which were determined previously by degenerate oligonucleotide-mediated sequencing of subcloned TcaC-peptide PCR products. From the data generated during the initial rounds of sequencing, new sets of primers were designed and used to walk the entire length of the about 5 kbp fragment. A total of 55 oligo primers was used, enabling the identification of 4832 total bp of contiguous sequence.

When the DNA sequence of the EcoR I fragment insert of pDAB2002 is combined with part of the determined sequence of the pDAB2000/pDAB2001 isolates, a total contiguous sequence of 6005 bp was generated (disclosed herein as SEQ ID NO:25). When long open reading frames were translated into the corresponding amino acids, the sequence clearly shows the TcaB i N-terminal peptide (disclosed as SEQ ID NO:3), encoded by bases 68-124, immediately following a methionine residue (start of translation). Upstream lies a potential ribosome binding site (bases 51-58), and downstream, at bases 215-277 is encoded the TcaB i -PT158 internal peptide (disclosed herein as SEQ ID NO:19). Further downstream, in the same reading frame, at bases 1787-1822, exists a sequence encoding the TcaB i -PT108 internal peptide (disclosed herein as SEQ ID NO:20). Also in the same reading frame, at bases 1946-1972, is encoded the TcaB ii N-terminal peptide (disclosed herein as SEQ ID NO:5), and the reading frame continues uninterrupted to a translation termination codon at nucleotides 3632-3634.

The lack of an in-frame stop codon between the end of the sequence encoding TcaB i -PT108 and the start of the TcaB ii encoding region, and the lack of a discernible ribosome binding site immediately upstream of the TcaB ii coding region, indicate that peptides TcaB ii and TcaB i are encoded by a single open reading frame of 3567 bp beginning at base pair 65 in SEQ ID NO:25), and are most likely derived from a single primary gene product TcaB of 1189 amino acids (131,586 Daltons; disclosed herein as SEQ ID NO:26) by post-translational cleavage. If the amino acid immediately preceding the TcaB ii N-terminal peptide represents the C-terminal amino acid of peptide TcaB i , then the predicted mass of TcaB ii (627 amino acids) is 70,814 Daltons (disclosed herein as SEQ ID NO:28), somewhat higher than the size observed by SDS-PAGE (68 kDa). This peptide would be encoded by a contiguous stretch of 1881 base pairs (disclosed herein as SEQ ID NO:27). It is thought that the native C-terminus of TcaB i lies somewhat closer to the C-terminus of TcaB i -PT108. The molecular mass of PT108 [3.438 kDa; determined during N-terminal amino acid sequence analysis of this peptide] predicts a size of 30 amino acids. Using the size of this peptide to designate the C-terminus of the TcaB i coding region [Glu at position 604 of SEQ ID NO:28], the derived size of TcaB i is determined to be 604 amino acids or 68,463 Daltons, more in agreement with experimental observations.

Translation of the TcaB ii peptide coding region of 1686 base pairs (disclosed herein as SEQ ID NO:29) yields a protein of 562 amino acids (disclosed herein as SEQ ID NO:30) with predicted mass of 60,789 Daltons, which corresponds well with the observed 61 kDa.

A potential ribosome binding site (bases 3682-3687) is found 48 bp downstream of the stop codon for the tcaB open reading frame. At bases 3694-3726 is found a sequence encoding the N-terminus of peptide TcaC, (disclosed as SEQ ID NO.2). The open reading frame initiated by this N-terminal peptide continues uninterrupted to base 6005 (2361 base pairs, disclosed herein as the first 2361 base pairs of SEQ ID NO.31). A gene (tcaC) encoding the entire TcaC peptide, (apparent size about 165 kDa; about 1500 amino acids), would comprise about 4500 bp.

Another isolate containing cloned EcoR I fragments of cosmid 26A5, E20.6, was also identified by its homology to the previously mentioned GZ4 and TcaB i probes. Agarose gel analysis of EcoR I digests of the DNA of the plasmid harbored by this strain (pDAB2004, FIG. 2 ), revealed insert fragments of estimated sizes 2.9, 5, and 3.3 kbp. DNA sequence analysis initiated from primers designed from the sequence of plasmid pDAB2002 revealed that the 3.3 kbp EcoR I fragment of pDAB2004 lies adjacent to the 5 kbp EcoR I fragment represented in pDAB2002. The 2361 base pair open reading frame discovered in pDAB2002 continues uninterrupted for another 2094 bases in pDAB2004 [disclosed herein as base pairs 2362 to 4458 of SEQ ID NO:31]. DNA sequence analysis using the parent cosmid 26A5 DNA as template confirmed the continuity of the open reading frame. Altogether, the open reading frame (tcaC SEQ ID NO:31) comprises 4455 base pairs, and encodes a protein (TcaC) of 1485 amino acids [disclosed herein as SEQ ID NO:32]. The calculated molecular size of 166,214 Daltons is consistent with the estimated size of the TcaC peptide (165 kDa), and the derived amino acid sequence matches exactly that disclosed for the TcaC N-terminal sequence [SEQ ID NO:2].

The lack of an amino acid sequence corresponding to SEQ ID NO:17; used to design the degenerate oligonucleotide primer pool in the discovered sequence indicates that the generation of the PCR® products found in isolates GZ4 and HB14, which were used as probes in the initial library screen, were fortuitously generated by reverse-strand priming by one of the primers in the degenerate pool. Further, the derived protein sequence does not include the internal fragment disclosed herein as SEQ ID NO:18. These sequences reveal that plasmid pDAB2004 contains the complete coding region for the TcaC peptide.

Further analysis of SEQ ID NO:25 reveals the end of an open reading frame (bases 1-43), which encodes the final 13 amino acids of the TcaA iii peptide, disclosed herein as SEQ ID NO:35. Only 24 bases separate the end of the TcaA iii coding region and the start of the TcaB i coding region. Included within the 24 bases are sequences that may serve as a ribosome binding site. Although possible, it is not likely that a Photorhabdus gene promoter is encoded within this short region. We propose that genomic region tca, which includes three long open reading frames [tcaA (SEQ ID NO:33), tcaB (SEQ ID NO:25, bases 65-36334), and tcaC (SEQ ID NO:31),which is separated from the end of tcaB by only 59 bases] is regulated as an operon, with transcription initiating upstream of the start of the tcaA gene (SEQ ID NO:33), and resulting in a polycistronic messenger RNA.

Screening of the Photorhabdus Genomic Library for Genes Encoding the TcbA ii Peptide

This example describes a method used to identify DNA clones that contain the TcbA ii peptide-encoding genes, the isolation of the gene, and the determination of its partial DNA base sequence.

Primers and PCR Reactions

The TcbA ii polypeptide of the insect active preparation is about 206 kDa. The amino acid sequence of the N-terminus of this peptide is disclosed as SEQ ID NO:1. Four pools of degenerate oligonucleotide primers (“Forward primers”: TH-4, TH-5, TH-6, and TH-7) were synthesized to encode a portion of this amino acid sequence, as described in Example 8, and are shown below.

In addition, a primary (“a”) and a secondary (“b”) sequence of an internal peptide preparation (TcbA ii -PT81) have been determined and are disclosed herein as SEQ ID NO:23 and SEQ ID NO:24, respectively. Four pools of degenerate oligonucleotides (“Reverse Primers”: TH-8, TH-9, TH-10 and TH-11) were similarly designed and synthesized to encode the reverse complement of sequences that encode,a portion of the peptide of SEQ ID NO:23, as shown below.

Sets of these primers were used in PCR® reactions to amplify TcbA ii -encoding gene fragments from the genomic Photorhabdus luminescens W-14 DNA prepared in Example 6. All PCR® reactions were run with the “Hot Start” technique using AmpliWax™ gems and other Perkin Elmer reagents and protocols. Typically, a mixture (total volume 11 μl) of MgCl 2 , dNTP's, 10×GeneAmp® PCR Buffer II, and the primers were added to tubes containing a single wax bead. [10×GeneAmp® PCR Buffer II is composed of 100 mM Tris-HCl, pH 8.3; and 500 mM KCl.] The tubes were heated to 80° C. for 2 minutes and allowed to cool. To the top of the wax seals, a solution containing 10×GeneAmp PCR Buffer II, DNA template, and AmpliTaq® DNA polymerase were added. Following melting of the wax seal and mixing of components by thermal cycling, final reaction conditions (volume of 50 μl) were: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl 2 ; 200 μM each in dATP, dCTP, dGTP, dTTP; 1.25 mM in a single Forward primer pool; 1.25 μM in a single Reverse primer pool, 1.25 units of AmpliTaq® DNA polymerase, and 170 ng of template DNA.

The reactions were placed in a thermocycler (as in Example 8) and run with the following program:

A series of amplifications was run at three different annealing temperatures (55°, 60°, 65° C.) using the degenerate primer pools. Reactions with annealing at 65° C. had no amplification products visible following agarose gel electrophoresis. Reactions having a 60° C. annealing regime and containing primers TH-5+TH-10 produced an amplification product that had a mobility corresponding to 2.9 kbp. A lesser amount of the 2.9 kbp product was produced under these conditions with primers TH-7+TH-10. When reactions were annealed at 55° C.,these primer pairs produced more of the 2.9 kbp product, and this product was also produced by primer pairs TH-5+TH-8 and TH-5+TH-11. Additional very faint 2.9 kbp bands were seen in lanes containing amplification products from primer pairs TH-7 plus TH-8, TH-9, TH-10, or TH-11.

To obtain sufficient PCR amplification product for cloning and DNA sequence determination, 10 separate PCR reactions were set up using the primers TH-5+TH-10, and were run using the above conditions with a 55° C. annealing temperature. All reactions were pooled and the 2.9 kbp product was purified by Qiaex extraction from an agarose gel as described above.

Additional sequences determined for TcbA ii internal peptides are disclosed herein as SEQ ID NO:21 and SEQ ID NO:22. As before, degenerate oligonucleotides (Reverse primers TH-17 and TH-18) were made corresponding to the reverse complement of sequences that encode a portion of the amino acid sequence of these peptides.

Degenerate oligonucleotides TH-18 and TH-17 were used in an amplification experiment with Photorhabdus luminescens W-14 DNA as template and primers TH-4, TH-5, TH-6, or TH-7 as the 5′-(Forward) primers. These reactions amplified products of approximately 4 kbp and 4.5 kbp, respectively. These DNAs were transferred from agarose gels to nylon membranes and hybridized with a 32 P-labeled probe (as described above) prepared from the 2.9 kbp product amplified by the TH-5+TH10 primer pair. Both the 4 kbp and the 4.5 kbp amplification products hybridized strongly to the 2.9 kbp probe. These results were used to construct a map ordering the TcbA ii internal peptide sequences as shown in FIG. 3 . Approximate distances between the primers are shown in nucleotides in FIG. 3 .

DNA Sequence of the 2.9 kbp TcbA ii -encoding Fragment

Approximately 200 ng of the purified 2.9 kbp fragment (prepared above) was precipitated with ethanol and dissolved in 17 ml of water. One-half of this was used as sequencing template with 25 pmol of the TH-5 pool as primers, the other half was used as template for TH-10 priming. Sequencing reactions were as given in Example 8. No reliable sequence was produced using the TH-10 primer pool; however, reactions with TH-5 primer pool produced the sequence disclosed below:.

1 AATCGTGTTG ATCCCTATGC CGNGCCGGGT TCGGTGGAAT CGATGTCCTC ACCGGGGGTT

61 TATTNGAGGG ANTNGTCCCG TGAGGCCAAA AANTGGAATG AAAGAAGTTC AATTTNTTAC

121 CTAGATAAAC GTCGCCCGGN TTTAGAAAGN TTANTGNTCA GCCAGAAAAT TTTGGTTGAG

181 GAAATTCCAC CGNTGGTTCT CTCTATTGAT TNGGGCCTGG CCGGGTTCGA ANNAAAACNA

241 GGAAATNCAC AAGTTGAGGT GATGGNTTTG TNGCNANCTT NTCGTTTAGG TGGGGAGAAA

301 CCTTNTCANC ACGNTTNTGA AACTGTCCGG GAAATCGTCC ATGANCGTGA NCCAGGNTTN

361 CGCCATTGG

Based on this sequence, a sequencing primer (TH-21, 5′-CCGGGCGACGTTTATCTAGG-3′) was designed to reverse complement bases 120-139, and initiate polymerization towards the 5′ end (i.e., TH-5 end) of the gel-purified 2.9 kbp TcbA ii -encoding PCR fragment. The determined sequence is shown below, and is compared to the biochemically determined N-terminal peptide sequence of TcbA ii SEQ ID NO:1.

TcbA ii 2.9 kbp PCR Fragment Sequence Confirmation

[Underlined amino acids=encoded by degenerate oligonucleotides]

SEQ ID NO:1 F   I   O   G   Y   S   D   L   F    G   -   -   A

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2.9 kbp seq GC ATG CAG GGG TAT AGT GAC CTG TTT GGT AAT CGT GCT

Characterization of Zinc-metalloproteases in Photorhabdus Broth: Protease Inhibition, Classification, and Purification

Protease Inhibition and Classification Assays: Protease assays were performed using FITC-casein dissolved in water as substrate (0.08% final assay concentration). Proteolysis reactions were performed at 25° C. for 1 h in the appropriate buffer with 25 μl of Photorhabdus broth (150 μl total reaction volume). Samples were also assayed in the presence and absence of dithiothreitol. After incubation, an equal volume of 12% trichloroacetic acid was added to precipitate undigested protein. Following precipitation for 0.5 h and subsequent centrifugation, 100 μl of the supernatant was placed into a 96-well microtiter plate and the pH of the solution was adjusted by addition of an equal volume of 4N NaOH. Proteolysis was then quantitated using a Fluoroskan II fluorometric plate reader at excitation and emission wavelengths of 485 and 538 nm, respectively. Protease activity was tested over a range from pH 5.0-10.0 in 0.5 units increments. The following buffers were used at 50 mM final concentration: sodium acetate (pH 5.0-6.5); Tris-HCL (pH 7.0-8.0); and bis-Tris propane (pH 8.5-10.0). To identify the class of protease(s) observed, crude broth was treated with a variety of protease inhibitors (0.5 μg/μl final concentration) and then examined for protease activity at pH 8.0 using the substrate described above. The protease inhibitors used included E-64 (L-trans-expoxysaccinylleucylamido[4-,-guanidino]-butane), 3,4 dichloroisocoumarin, Leupeptin, pepstatin, amastatin, ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline.

Protease assays performed over a pH range revealed that indeed protease(s) were present which exhibited maximal activity at about pH 8.0 (Table 17). Addition of DTT did not have any effect on protease activity. Crude broth was then treated with a variety of protease inhibitors (Table 18). Treatment of crude broth with the inhibitors described above revealed that 1,10 phenanthroline caused complete inhibition of all protease activity when added at a final concentration of 50 μg, with the IC 50 =5 μg in 100 μl of a 2 mg/ml crude broth solution. These data indicate that the most abundant protease(s) found in the Photorhabdus broth are from the zinc-metalloprotease class of enzymes.

The isolation of a zinc-metalloprotease was performed by applying dialyzed 10-80% ammonium sulfate pellet to a Q Sepharose column equilibrated at 50 mM Na 2 PO 4 , pH 7.0 as described in Example 5 for Photorhabdus toxin. After extensive washing, a 0 to 0.5 M NaCl gradient was used to elute toxin protein. The majority of biological activity and protein was eluted from 0.15-0.45 M NaCl. However, it was observed that the majority of proteolytic activity was present in the 0.25-0.35 M NaCl fraction with some activity in the 0.15-0.25 M NaCl fraction. SDS PAGE analysis of the 0.25-0.35 M NaCl fraction showed a major peptide band of approximately 60 kDa. The 0.15-0.25 M NaCl fraction contained a similar 60 kDa band but at lower relative protein concentration. Subsequent gel filtration of this fraction using a Superose 12 HR 16/50 column resulted in a major peak migrating at 57.5 kDa that contained a predominant (>90% of total stained protein) 58.5 kDa band by SDS PAGE analysis. Additional analysis of this fraction using various protease inhibitors as described above determined that the protease was a zinc-metalloprotease. Nearly all of the protease activity present in Photorhabdus broth at day 1 of fermentation corresponded to the about 58 kDa zinc-metalloprotease.

In yet a second isolation of zinc-metalloprotease(s), W-14 Photorhabdus broth grown for three days was taken and protease activity was visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) laced with gelatin as described in Schmidt, T. M., Bleakley, B. and Nealson, K. M. 1988. SDS running gels (5.5×8 cm) were made with 12.5% polyacrylamide (40% stock solution of acrylamide/bis-acrylamide; Sigma Chemical Co., St. Louis, Mo.) into which 0.1% gelatin final concentration (Biorad EIA grade reagent; Richmond Calif.) was incorporated upon dissolving in water. SDS-stacking gels (1.0×8 cm) were made with 5% polyacrylamide, also laced with 0.1% gelatin. Typically, 2.5 μg of protein to be tested was diluted in 0.03 ml of SDS-PAGE loading buffer without dithiothreitol (DTT) and loaded onto the gel. Proteins were electrophoresed in SDS running buffer (Laemmli, U.K. 1970. Nature 227, 680) at 0° C. and at 8 mA. After electrophoresis was complete, the gel was washed for 2 h in 2.5% (v/v) Triton X-100. Gels were then incubated for 1 h at 37° C. in 0.1 M glycine (pH 8.0). After incubation, gels were fixed and stained overnight with 0.1% amido black in methanol-acetic acid- water (30:10:60, vol./vol./vol.; Sigma Chemical Co.). Protease activity was visualized as light areas against a dark, amido black stained background due to proteolysis and subsequent diffusion of incorporated gelatin. At least three distinct bands produced by proteolytic activity at 58-, 41-, and 38 kDa were observed.

Activity assays of the different proteases in W-14 day three culture broth were performed using FITC-casein dissolved in water as substrate (0.02% final assay concentration). Proteolysis experiments were performed at 37° C. for 0-0.5 h in 0.1M Tris-HCl (pH 8.0) with different protein fractions in a total volume of 0.15 ml. Reactions were terminated by addition of an equal volume of 12% trichloroacetic acid (TCA) dissolved in water. After incubation at room temperature for 0.25 h, samples were centrifuged at 10,000×9 for 0.25 h and 0.10 ml aliquots were removed and placed into 96-well microtiter plates. The solution was then neutralized by the addition of an equal volume of 2 N sodium hydroxide, followed by quantitation using a Fluoroskan II fluorometric plate reader with excitation and emission wavelengths of 485 and 538 nm, respectively. Activity measurements were performed using FITC-Casein with different protease concentrations at 37° C. for 0-10 min. A unit of activity was arbitrarily defined as the amount of enzyme needed to produce 1000 fluorescent units/min and specific activity was defined as units/mg of protease.

Inhibition studies were performed using two zinc-metalloprotease inhibitors; 1,10 phenanthroline and N-(a-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp(phosphoramidon) with stock solutions of the inhibitors dissolved in 100% ethanol and water, respectively. Stock concentrations were typically 10 mg/ml and 5 mg/ml for 1,10 phenanthroline and phosphoramidon, respectively, with final concentrations of inhibitor at 0.5-1.0 mg/ml per reaction. Treatment of three day W-14 crude broth with 1,10 phenanthroline, an inhibitor of all zinc metalloproteases, resulted in complete elimination of all protease activity while treatment with phosphoramidon, an inhibitor of thermolysin-like proteases (Weaver, L. H., Kester, W. R., and Matthews, B. W. 1977. J. Mol. Biol. 114, 119-132), resulted in about 56% reduction of protease activity. The residual proteolytic activity could not be further reduced with additional phosphoramidon.

The proteases of three day W-14 Photorhabdus broth were purified as follows: 4.0 liters of broth were concentrated using an Amicon spiral ultra filtration cartridge Type S1Y100 attached to an Amicon M-12 filtration device. The flow-through material having native proteins less than 100 kDa in size (3.8 L) was concentrated to 0.375 L using an Amicon spiral ultra filtration cartridge Type S1Y10 attached to an Amicon M-12 filtration device. The retentate material contained proteins ranging in size from 10-100 kDa. This material was loaded onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem (Framington, Mass.) Poros® 50 HQ strong anion exchange packing that had been equilibrated in 10 mM sodium phosphate buffer (pH 7.0). Proteins were loaded on the column at a flow rate of 5 ml/min, followed by washing unbound protein with buffer until A280=0.00. Afterwards, proteins were eluted using a NaCl gradient of 0-1.0 M NaCl in 40 min at a flow rate of 7.5 ml/min. Fractions were assayed for protease activity, supra., and active fractions were pooled. Proteolytically active fractions were diluted with 50% (v/v) 10 mM sodium phosphate buffer (pH 7.0) and loaded onto a Pharmacia HR 10/10 Mono Q column equilibrated in 10 mM sodium phosphate. After washing the column with buffer until A 280 =0.00, proteins were eluted using a NaCl gradient of 0-0.5 M NaCl for 1 h at a flow rate of 2.0 ml/min. Fractions were assayed for protease activity. Those fractions having the greatest amount of phosphoramidon-sensitive protease activity, the phosphoramidon sensitive activity being due to the 41/38 kDa protease, infra., were pooled. These fractions were found to elute at a range of 0.15-0.25 M NaCl. Fractions containing a predominance of phosphoramidon-insensitive protease activity, the 58 kDa protease, were also pooled. These fractions were found to elute at a range of 0.25-0.35 M NaCl. The phosphoramidon-sensitive protease fractions were then concentrated to a final volume of 0.75 ml using a Millipore Ultrafree®-15 centrifugal filter device Biomax-5K NMWL membrane. This material was applied at a flow rate of 0.5 ml/min to a Pharmacia HR 10/30 column that had been packed with Pharmacia Sephadex G-50 equilibrated in 10 mM sodium phosphate buffer (pH 7.0)/0.1 M NaCl. Fractions having the maximal phosphoramidon-sensitive protease activity were then pooled and centrifuged over a Millipore Ultrafree®-15 centrifugal filter device Biomax-50K NMWL membrane. Proteolytic activity analysis, supra., indicated this material to have only phosphoramidon-sensitive protease activity. Pooling of the phosphoramidon-insensitive protease, the 58 kDa protein, was followed by concentrating in a Millipore Ultrafree®-15 centrifugal filter device Biomax-50K NMWL membrane and further separation on a Pharmacia Superdex-75 column. Fractions containing the protease were pooled.

Analysis of purified 58—and 41/38 kDa purified proteases revealed that, while both types of protease were completely inhibited with 1,10 phenanthroline, only the 41/38 kDa protease was inhibited with phosphoramidon. Further analysis of crude broth indicated that protease activity of day 1 W-14 broth has 23% of the total protease activity due to the 41/38 kDa protease, increasing to 44% in day three W-14 broth.

Standard SDS-PAGE analysis for examining protein purity and obtaining amino terminal sequence was performed using 4-20% gradient MiniPlus SepraGels purchased from Integrated Separation Systems (Natick, Mass.). Proteins to be amino-terminal sequenced were blotted onto PVDF membrane following purification, infra., (ProBlott™ Membranes; Applied Biosystems, Foster City, Calif.), visualized with 0.1% amido black, excised, and sent to Cambridge Prochem; Cambridge, Mass., for sequencing.

Deduced amino terminal sequence of the 58-(SEQ ID NO:45) and 41/38 kDa (SEQ ID NO:44) proteases from three day old W-14 broth were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ ID NO:44), respectively.

Sequencing of the 41/38 kDa protease revealed several amino termini, each one having an additional amino acid removed by proteolysis. Examination of the primary, secondary, tertiary and quartenary sequences for the 38 and 41 kDa polypeptides allowed for deduction of the sequence shown above and revealed that these two proteases are homologous.

Characterization of Photorhabdus Strains

In order to establish that the collection described herein was comprised of Photorhabdus strains, the strains herein were assessed in terms of recognized microbiological traits that are characteristic of Photorhabdus and which differentiate it from other Enterobacteriaceae and Xenorhabdus spp. (Farmer, J. J. 1984. Bergey's Manual of Systemic Bacteriology, Vol 1. pp. 510-511. (ed. Kreig N. R. and Holt, J. G.). Williams & Wilkins, Baltimore; Akhurst and Boemare, 1988, Boemare et al., 1993). These characteristic traits are as follows: Gram's stain negative rods, organism size of 0.5-2 μm in width and 2-10 μm in length, red/yellow colony pigmentation, presence of crystalline inclusion bodies, presence of catalase, inability to reduce nitrate, presence of bioluminescence, ability to take up dye from growth media, positive for protease production, growth-temperature range below 37° C., survival under anaerobic conditions and positively motile. (Table 20). Reference Escherichia coli , Xenorhabdus and Photorhabdus strains were included in all tests for comparison. The overall results are consistent with all strains being part of the family Enterobacteriaceae and the genus Photorhabdus.

A luminometer was used to establish the bioluminescence of each strain and provide a quantitative and relative measurement of light production. For measurement of relative light emitting units, the broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (6, 12, and 24 hr) and compared to background luminosity (uninoculated media and water). Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlin, Ohio) spectrophotometer using a sipper cell. Appropriate dilutions were then made (to normalize optical density to 1.0 unit) before measuring luminosity. Aliquots of the diluted broths were then placed into cuvettes (300 μl each) and read in a Bio-Orbit 1251 Luminometer (Bio-Orbit Oy, Twiku, Finland). The integration period for each sample was 45 seconds. The samples were continuously mixed (spun in baffled cuvettes) while being read to provide oxygen availability. A positive test was determined as being ≧5-fold background luminescence (about 5-10 units). In addition, colony luminosity was detected with photographic film overlays and visually, after adaptation in a darkroom. The Gram's staining characteristics of each strain were established with a commercial Gram's stain kit (BBL, Cockeysville, Md.) used in conjunction with Gram's stain control slides (Fisher Scientific, Pittsburgh, Pa.). Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) 100×oil immersion objective lens (with 10×ocular and 2×body magnification). Microscopic examination of individual strains for organism size, cellular description and inclusion bodies (the latter after logarithmic growth) was performed using wet mount slides (10×ocular, 2×body and 40×objective magnification) with oil immersion and phase contrast microscopy with a micrometer (Akhurst, R. J. and Boemare, N. E. 1990. Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R. and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA.; Baghdiguian S., Boyer-Giglio M. H., Thaler, J. O., Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185.). Colony pigmentation was observed after inoculation on Bacto nutrient agar, (Difco Laboratories, Detroit, Mich.) prepared as per label instructions. Incubation occurred at 28° C. and descriptions were produced after 5-7 days. To test for the presence of the enzyme catalase, a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube. One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or within 5 seconds. Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined. To test for nitrate reduction, each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, Mich.). After 24 hours incubation at 28° C., nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, Mich., 1984). The generation of a distinct pink or red color indicates the formation of nitrite from nitrate. The ability of each strain to uptake dye from growth media was tested with Bacto MacConkey agar containing the dye neutral red; Bacto Tergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agar containing the dye eosin-Y (agars from Difco Laboratories, Detroit, Mich., all prepared according to label instructions). After inoculation on these media, dye uptake was recorded after incubation at 28° C. for 5 days. Growth on these latter media is characteristic for members of the family Enterobacteriaceae. Motility of each strain was tested using a solution of Bacto Motility Test Medium (Difco Laboratories, Detroit, Mich.) prepared as per label instructions. A butt-stab inoculation was performed with each strain and motility was judged macroscopically by a diffuse zone of growth spreading from the line of inoculum. In many cases, motility was also observed microscopically from liquid culture under wet mount slides. Biochemical nutrient evaluation for each strain was performed using BBL Enterotube II (Benton, Dickinson, Germany). Product instructions were followed with the exception that incubation was carried out at 28° C. for 5 days. Results were consistent with previously cited reports for Photorhabdus. The production of protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, Mich.) plates made as per label instructions. Cultures were inoculated and the plates were incubated at 28° C. for 5 days. To assess growth at different temperatures, agar plates [2% proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, Mich.) in deionized water] were streaked from a common source of inoculum. Plates were sealed with Nesco® film and incubated at 20, 28 and 37° C. for up to three weeks. Plates showing no growth at 37° C. showed no cell viability after transfer to a 28° C. incubator for one week. Oxygen requirements for Photorhabdus strains were tested in the following manner. A butt-stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, Mich.) was made. The tubes were incubated at room temperature for one week and cultures were then examined for type and extent of growth. The indicator resazurin demonstrates the level of medium oxidation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, Mich.). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism.

Cellular fatty acid analysis is a recognized tool for bacterial characterization at the genus and species level (Tornabene, T. G. 1985. Lipid Analysis and the Relationship to Chemotaxonomy in Methods in Microbiology , Vol. 18, 209-234.; Goodfellow, M. and O'Donnell, A. G. 1993. Roots of Bacterial Systematics in Handbook of New Bacterial Systematics (ed. Goodfellow, M. & O'Donnell, A. G.) pp. 3-54. London: Academic Press Ltd.), these references are incorporated herein by reference, and were used to confirm that our collection was related at the genus level. Cultures were shipped to an external, contract laboratory for fatty acid methyl ester analysis (FAME) using a Microbial ID (MIDI, Newark, Del., USA) Microbial Identification System (MIS). The MIS system consists of a Hewlett Packard HP5890A gas chromatograph with a 25 mm×0.2 mm 5% methylphenyl silicone fused silica capillary column. Hydrogen is used as the carrier gas and a flame-ionization detector functions in conjunction with an automatic sampler, integrator and computer. The computer compares the sample fatty acid methyl esters to a microbial fatty acid library and against a calibration mix of known fatty acids. As selected by the contract laboratory, strains were grown for 24 hours at 28° C. on trypticase soy agar prior to analysis. Extraction of samples was performed by the contract lab as per standard FAME methodology. There was no direct identification of the strains to any luminescent bacterial group other than Photorhabdus. When the cluster analysis was performed, which compares the fatty acid profiles of a group of isolates, the strain fatty acid profiles were related at the genus level.

The evolutionary diversity of the Photorhabdus strains in our collection was measured by analysis of PCR (Polymerase Chain Reaction) mediated genomic fingerprinting using genomic DNA from each strain. This technique is based on families of repetitive DNA sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic, J., Schneider, M., Del. Bruijn, F. J. and Lupski, J. R. 1994. Methods Mol. Cell. Biol., 5, 25-40.). Three of these, repetitive extragenic palindromic sequence (REP), enterobacterial repetitive intergenic consensus (ERIC) and the BOX element are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F. J., Fulbright, D. W., Stephens, C. T. and D E Bruijn, F. J. 1994. Appl. Environ. Micro. 60, 2286-2295). Rep-PCR utilizes oligonucleotide primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA “fingerprint” for each strain.

To isolate genomic DNA from our strains, cell pellets were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and 12 ml of 5 M NaCl was then added. This mixture was centrifuged 20 min. at 15,000×g. The resulting pellet was resuspended in 5.7 ml of TE and 300 μl of 10% SDS and 60 μl 20 mg/ml proteinase K (Gibco BRL Products, Grand Island, N.Y.) were added. This mixture was incubated at 37° C. for 1 hr, approximately 10 mg of lysozyme was then added and the mixture was incubated for an additional 45 min. One milliliter of 5M NaCl and 800 μl of CTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) were then added and the mixture was incubated 10 min. at 65° C., gently agitated, then incubated and agitated for an additional 20 min. to aid in clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently then centrifuged. Two extractions were then performed with an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1). Genomic DNA was precipitated with 0.6 volume of isopropanol. Precipitated DNA was removed with a glass rod, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by optical density at 260 nm. To perform rep-PCR analysis of Photorhabdus genomic DNA the following primers were used, REP1R-I; 5′-IIIICGICGICATCIGGC-3′ and REP2-I; 5′-ICGICTTATCIGGCCTAC-3′. PCR was performed using the following 25 μl reaction: 7.75 μl H 2 O, 2.5 μl 10×LA buffer (PanVera Corp., Madison, Wis.), 16 μl dNTP mix (2.5 mM each), 1 μl of each primer at 50 pM/μl, 1 μl DMSO, 1.5 μl genomic DNA (concentrations ranged from 0.075-0.480 μg/μl) and 0.25 μl TaKaRa EX Taq (PanVera Corp., Madison, Wis.). The PCR amplification was performed in a Perkin Elmer DNA Thermal Cycler (Norwalk, Conn.) using the following conditions: 95° C./7 min. then 35 cycles of; 94° C./1 min.,44° C./1 min., 65° C./8 min., followed by 15 min. at 65° C. After cycling, the 25 μl reaction was added to 5 μl of 6×gel loading buffer (0.25% bromophenol blue, 40% w/v sucrose in H 2 O). A 15×20 cm 1%-agarose gel was then run in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 μl of each reaction. The gel was run for approximately 16 hours at 45 v. Gels were then stained in 20 μg/ml ethidium bromide for 1 hour and destained in TBE buffer for approximately 3 hours. Polaroid® photographs of the gels were then taken under UV illumination.

The presence or absence of bands at specific sizes for each strain was scored from the photographs and entered as a similarity matrix in the numerical taxonomy software program, NTSYS-pc (Exeter Software, Setauket, N.Y.). Controls of E. coli strain HB101 and Xanthomonas oryzae pv. oryzae assayed at the same time produced PCR “fingerprints” corresponding to published reports (Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C. M., Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson, R. J., Del. Bruijn, F. J., Rice, C. and Leach, J. E. 1995. Int. Rice Res. Notes, 20, 23-24.; Vera Cruz, C. M., Ardales, E. Y., Skinner, D. Z., Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T. W. and Leach, J. E. 1996. Phytopathology (in press, respectively). The data from Photorhabdus strains were then analyzed with a series of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data) to generate a matrix of similarity coefficients (using the Jaccard coefficient) and SAHN (Sequential, Agglomerative, Heirarchical and Nested) clustering [using the UPGMA (Unweighted Pair-Group Method with Arithmetic Averages) method] which groups related strains and can be expressed as a phenogram (FIG. 5 ). The COPH (cophenetic values) and MXCOMP (matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based. A resulting normalized Mantel statistic (r) was generated which is a measure of the goodness of fit for a cluster analysis (r=0.8-0.9 represents a very good fit). In our case r=0.919. Therefore, our collection is comprised of a diverse group of easily distinguishable strains representative of the Photorhabdus genus.

Insecticidal Utility of Toxin(s) Produced by Various Photorhabdus Strains

Initial “seed” cultures of the various Photorhabdus strains were produced by inoculating 175 ml of 2% Proteose Peptone #3 (PP3) (Difco Laboratories, Detroit, Mich.) liquid media with a primary variant subclone in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput. Inoculum for each seed culture was derived from oil-overlay agar slant cultures or plate cultures. After inoculation, these flasks were incubated for 16 hrs at 28° C. on a rotary shaker at 150 rpm. These seed cultures were then used as uniform inoculum sources for a given fermentation of each strain. Additionally, overlaying the post-log seed culture with sterile mineral oil, adding a sterile magnetic stir bar for future resuspension and storing the culture in the dark, at room temperature provided long-term preservation of inoculum in a toxin-competent state. The production broths were inoculated by adding 1% of the actively growing seed culture to fresh 2% PP3 media (e.g., 1.75 ml per 175 ml fresh media). Production of broths occurred in either 500 ml tribaffled flasks (see above), or 2800 ml baffled, convex bottom flasks (500 ml volume) covered by a silicon foam closure. Production flasks were incubated for 24-48 hrs under the above mentioned conditions. Following incubation, the broths were dispensed into sterile 1 L polyethylene bottles, spun at 2600×g for 1 hr at 10° C. and decanted from the cell and debris pellet. The liquid broth was then vacuum filtered through Whatman GF/D (2.7 μM retention) and GF/B (1.0 μM retention) glass filters to remove debris. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron, Northborough, Mass.) using a 0.5 M open-channel filter. When necessary, additional clarification could be obtained by chilling the broth (to 4° C.) and centrifuging for several hours at 2600×g. Following these procedures, the broth was filter sterilized using a 0.2 μM nitrocellulose membrane filter. Sterile broths were then used directly for biological assay, biochemical analysis or concentrated (up to 15-fold) using a 10,000 MW cut-off, M12 ultra-filtration device (Amicon, Beverly Mass.) or centrifugal concentrators (Millipore, Bedford, Mass. and Pall Filtron, Northborough, Mass.) with a 10,000 MW pore size. In the case of centrifugal concentrators, the broth was spun at 2000×g for approximately 2 hr. The 10,000 MW permeate was added to the corresponding retentate to achieve the desired concentration of components greater than 10,000 MW. Heat inactivation of processed broth samples was acheived by heating the samples at 100° C. in a sand-filled heat block for 10 minutes.

The broth(s) and toxin complex(es) from different Photorhabdus strains are useful for reducing populations of insects and were used in a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described. A demonstration of the breadth of insecticidal activity observed from broths of a selected group of Photorhabdus strains fermented as described above is shown in Table 20. It is possible that additional insecticidal activities could be detected with these strains through increased concentration of the broth or by employing different fermentation methods. Consistent with the activity being associated with a protein, the insecticidal activity of all strains tested was heat labile (see above).

Culture broth(s) from diverse Photorhabdus strains show differential insecticidal activity (mortality and/or growth inhibition, reduced adult emergence) against a number of insects. More specifically, the activity is seen against corn rootworm larvae and boll weevil larvae which are members of the insect order Coleoptera. Other members of the Coleoptera include wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle. Activity is also observed against aster leafhopper and corn plant hopper, which are members of the order Homoptera. Other members of the Homoptera include planthoppers, pear psylla, apple sucker, scale insects, whiteflies, spittle bugs as well as numerous host specific aphid species. The broths and purified toxin complex(es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera. Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm and fall armyworm. Activity is also seen against fruitfly and mosquito larvae which are members of the order Diptera. Other members of the order Diptera are, pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly and house fly and various mosquito species. Activity with broth(s) and toxin complex(es) is also seen against two-spotted spider mite which is a member of the order Acarina which includes strawberry spider mites, broad mites, citrus red mite, European red mite, pear rust mite and tomato russet mite.

Activity against corn rootworm larvae was tested as follows. Photorhabdus culture broth(s) (0-15 fold concentrated, filter sterilized), 2% Proteose Peptone #3, purified toxin complex(es), 10 mM sodium phosphate buffer , pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) of artificial diet (Rose, R. I. and McCabe, J. M. (1973). J. Econ. Entomol. 66, (398-400) in 40 μl aliquots. Toxin complex was diluted in 10 mM sodium phosphate buffer, pH 7.0. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber and maintained at 27° C. for the appropriate period (3-5 days). Mortality and larval weight determinations were then scored. Generally, 16 insects per treatment were used in all studies. Control mortality was generally less than 5%.

Activity against boll weevil ( Anthomonas grandis ) was tested as follows. Concentrated (1-10 fold) Photorhabdus broths, control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 were applied in 60 μl aliquots to the surface of 0.35 g of artificial diet (Stoneville Yellow lepidopteran diet) and allowed to dry. A single, 12-24 hr boll weevil larva was placed on the diet, and the wells were sealed and held at 25° C., 50% RH for 5 days. Mortality and larval weights were then assessed. Control mortality ranged between 0-13%.

Activity against mosquito larvae was tested as follows. The assay was conducted in a 96-well microtiter plate. Each well contained 200 μl of aqueous solution (10-fold concentrated Photorhabdus culture broth(s), control medium (2% Proteose Peptone #3), 10 mM sodium phosphate buffer, toxin complex(es) @ 0.23 mg/ml or H 2 O) and approximately 20, 1-day old larvae ( Aedes aegypti ). There were 6 wells per treatment. The results were read at 3-4 days after infestation. Control mortality was between 0-20%.

Activity against fruitflies was tested as follows. Purchased Drosophila melanogaster medium was prepared using 50% dry medium and a 50% liquid of either water, control medium (2% Proteose Peptone #3), 10-fold concentrated Photorhabdus culture broth(s), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer , pH 7.0. This was accomplished by placing 4.0 ml of dry medium in each of 3 rearing vials per treatment and adding 4.0 ml of the appropriate liquid. Ten late instar Drosophila melanogaster maggots were then added to each 25 ml vial. The vials were held on a laboratory bench, at room temperature, under fluorescent ceiling lights. Pupal or adult counts were made after 15 days of exposure. Adult emergence as compared to water and control medium (0-16% reduction).

Activity against aster leafhopper adults ( Macrosteles severini ) and corn planthopper nymphs ( Peregrinus maidis ) was tested with an ingestion assay designed to allow ingestion of the active without other external contact. The reservoir for the active/“food” solution is made by making 2 holes in the center of the bottom portion of a 35×10 mm Petri dish. A 2 inch Parafilm M® square is placed across the top of the dish and secured with an “O” ring. A 1 oz. plastic cup is then infested with approximately 7 hoppers and the reservoir is placed on top of the cup, Parafilm down. The test solution is then added to the reservoir through the holes. In tests using 10-fold concentrated Photorhabdus culture broth(s), the broth and control medium (2% Proteose Peptone #3) were dialyzed against 10 mM sodium phosphate buffer, pH 7.0 and sucrose (to 5%) was added to the resulting solution to reduce control mortality. Purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 was also tested. Mortality is reported at day 3. The assay was held in an incubator at 28° C., 70% RH with a 16/8 photoperiod. The assays were graded for mortality at 72 hours. Control mortality was less than 6%.

Activity against lepidopteran larvae was tested as follows. Concentrated (10-fold) Photorhabdus culture broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 μl aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva. European corn borer ( Ostrinia nubilalis ) and tobacco hornworm ( Manduca sexta ) eggs were obtained from commercial sources and hatched in-house, whereas tobacco budworm ( Heliothis virescens ) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27° C. for the appropriate period. Mortality and weight determinations were scored at day 5. Generally, 16 insects per treatment were used in all studies. Control mortality generally ranged from about 4 to about 12.5% for control medium and was less than 10% for phosphate buffer.

Activity against two-spotted spider mite ( Tetranychus urticae ) was determined as follows. Young squash plants were trimmed to a single cotyledon and sprayed to run-off with 10-fold concentrated broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es), 10 mM sodium phosphate buffer, pH 7.0. After drying, the plants were infested with a mixed population of spider mites and held at lab temperature and humidity for 72 hr. Live mites were then counted to determine levels of control.

Non W-14 Photorhabdus Strains: Purification, Characterization and Activity Spectrum

Purification

The protocol, as follows, is similar to that developed for the purification of W-14 and was established based on purifying those fractions having the most activity against Southern corn root worm (SCR), as determined in bioassays (see Example 13). Typically, 4-20 L of broth that had been filtered, as described in Example 13, were received and concentrated using an Amicon spiral ultra filtration cartridge Type S1Y100 attached to an Amicon M-12 filtration device. The retentate contained native proteins consisting of molecular sizes greater than 100 kDa, whereas the flow through material contained native proteins less than 100 kDa in size. The majority of the activity against-SCR was contained in the 100 kDa retentate. The retentate was then continually diafiltered with 10 mM sodium phosphate (pH=7.0) until the filtrate reached an A 280 <0.100. Unless otherwise stated, all procedures from this point were performed in buffer as defined by 10 mM sodium phosphate (pH 7.0). The retentate was then concentrated to a final volume of approximately 0.20 L and filtered using a 0.45 mm Nalgene™ Filterware sterile filtration unit. The filtered material was loaded at 7.5 ml/min onto a Pharmacia HR16/10 column which had been packed with PerSeptive Biosystem Poros® 50 HQ strong anion exchange matrix equilibrated in buffer using a PerSeptive Biosystem Sprint® HPLC system. After loading, the column was washed with buffer until an A 280 <0.100 was achieved. Proteins were then eluted from the column at 2.5 ml/min using buffer with 0.4 M NaCl for 20 min for a total volume of 50 ml. The column was then washed using buffer with 1.0 M NaCl at the same flow rate for an additional 20 min (final volume=50 ml). Proteins eluted with 0.4 M and 1.0 M NaCl were placed in separate dialysis bags (Spectra/Por® Membrane MWCO: 2,000) and allowed to dialyze overnight at 4° C. in 12 L buffer. The majority of the activity against SCR was contained in the 0.4 M fraction. The 0.4 M fraction was further purified by application of 20 ml to a Pharmacia XK 26/100 column that had been prepacked with Sepharose CL4B (Pharmacia) using a flow rate of 0.75 ml/min. Fractions were pooled based on A 280 peak profile and concentrated to a final volume of 0.75 ml using a Millipore Ultrafree®-15 centrifugal filter device Biomax-50K NMWL membrane. Protein concentrations were determined using a Biorad Protein Assay Kit with bovine gamma globulin as a standard.

Characterization

The native molecular weight of the SCR toxin complex was determined using a Pharmacia HR 16/50 that had been prepacked with Sepharose CL4B in buffer. The column was then calibrated using proteins of known molecular size thereby allowing for calculation of the toxin approximate native molecular size. As shown in Table 21, the molecular size of the toxin complex ranged from 777 kDa with strain Hb to 1,900 kDa with strain WX-14. The yield of toxin complex also varied, from strain WX-12 producing 0.8 mg/L to strain Hb, which produced 7.0 mg/L.

Proteins found in the toxin complex were examined for individual polypeptide size using SDS-PAGE analysis. Typically, 20 mg protein of the toxin complex from each strain was loaded onto a 2-15% polyacrylamide gel (Integrated Separation Systems) and electrophoresed at 20 mA in Biorad SDS-PAGE buffer. After completion of electrophoresis, the gels were stained overnight in Biorad Coomassie blue R-250 (0.2% in methanol: acetic acid: water; 40:10:40 v/v/v). Subsequently, gels were destained in methanol:acetic acid: water; 40:10:40 (v/v/v). The gels were then rinsed with water for 15 min and scanned using a Molecular Dynamics Personal Laser Densitometer®. Lanes were quantitated and molecular sizes were calculated as compared to Biorad high molecular weight standards, which ranged from 200-45 kDa.

Sizes of the individual polypeptides comprising the SCR toxin complex from each strain are listed in Table 22. The sizes of the individual polypeptides ranged from 230 kDa with strain WX-1 to a size of 16 kDa, as seen with strain WX-7. Every strain, with the exception of strain Hb, had polypeptides comprising the toxin complex that were in the 160-230 kDa range, the 100-160 kDa range, and the 50-80 kDa range. These data indicate that the toxin complex may vary in peptide composition and components from strain to strain, however, in all cases the toxin attributes appears to consist of a large, oligomeric protein complex.

Activity Spectrum

As shown in Table 23, the toxin complexes purified from strains Hm and H9 were tested for activity against a variety of insects, with the toxin complex from strain W-14 for comparison. The assays were performed as described in Example 13. The toxin complex from all three strains exhibited activity against tobacco bud worm, European corn borer, Southern corn root worm, and aster leafhopper. Furthermore, the toxin complex from strains Hm and W-14 also exhibited activity against two-spotted spider mite. In addition, the toxin complex from W-14 exhibited activity against mosquito larvae. These data indicate that the toxin complex, while having similarities in activities between certain orders of insects, can also exhibit differential activities against other orders of insects.

Sub-Fractionation of Photorhabdus Protein Toxin Complex

The Photorhabdus protein toxin complex was isolated as described in Example 14. Next, about 10 mg toxin was applied to a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0 at a flow rate of 1 ml/min. The column was washed with 20 mM Tris-HCl, pH 7.0 until the optical density at 280 nm returned to baseline absorbance. The proteins bound to the column were eluted with a linear gradient of 0 to 1.0 M NaCl in 20 mM Tris-HCl, pH 7.0 at 1 ml/min for 30 min. One ml fractions were collected and subjected to Southern corn rootworm (SCR) bioassay (see Example 13). Peaks of activity were determined by a series of dilutions of each fraction in SCR bioassays. Two activity peaks against SCR were observed and were named A (eluted at about 0.2-0.3 M NaCl) and B (eluted at 0.3-0.4 M NaCl). Activity peaks A and B were pooled separately and both peaks were further purified using a 3-step procedure described below.

Solid (NH 4 )2SO 4 was added to the above protein fraction to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 5/5 column equilibrated with 1.7 M (NH 4 ) 2 SO 4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH 4 )2SO 4 , 0% ethylene glycol, 50 mM potassium phosphate, pH 7.0 to 25% ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no (NH 4 ) 2 SO 4 ) at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. Activities in each fraction against SCR were determined by bioassay.

The fractions with the highest activity were pooled and applied to a MonoQ 5/5 column which was equilibrated with 20 mM Tris-HCl, pH 7.0 at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to 1M NaCl in 20 mM Tris-HCl, pH 7.0.

For the final step of purification, the most active fractions above (determined by SCR bioassay) were pooled and subjected to a second phenyl-Superose 5/5/column. Solid (NH 4 ) 2 SO 4 was added to a final concentration of 1.7 M. The solution was then loaded onto the column equilibrated with 1.7 M (NH 4 ) 2 SO 4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH 4 ) 2 SO 4 , 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. Activities in each fraction against SCR were determined by bioassay.

The final purified protein by the above 3-step procedure from peak A was named toxin A and the final purified protein from peak B was named toxin B.

Characterization and Amino Acid Sequencing of Toxin A and Toxin B

In SDS-PAGE, both toxin A and toxin B contained two major (>90% of total Commassie stained protein) peptides: 192 kDa (named A1 and B1, respectively) and 58 kDa (named A2 and B2, respectively). Both toxin A and toxin B revealed only one major band in native PAGE, indicating A1 and A2 were subunits of one protein complex, and B1 and B2 were subunits of one protein complex. Further, the native molecular weight of both toxin A and toxin B were determined to be 860 kDa by gel filtration chromatography. The relative molar concentrations of A1 to A2 was judged to be a 1 to 1 equivalence as determined by densiometric analysis of SDS-PAGE gels. Similarly, B1 and B2 peptides were present at the same molar concentration.

Toxin A and toxin B were electrophoresed in 10% SDS-PAGE and transblotted to PVDF membranes. Blots were sent for amino acid analysis and N-terminal amino acid sequencing at Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino sequence of B1 was determined to be identical to SEQ ID NO:1, the TcbA ii region of the tcbA gene (SEQ ID NO:12, position 87 to 99). A unique N-terminal sequence was obtained for peptide B2 (SEQ ID NO:40). The N-terminal amino acid sequence of peptide B2 was identical to the TcbA iii region of the derived amino acid sequence for the tcbA gene (SEQ ID NO:12, position 1935 to 1945). Therefore, the B toxin contained predominantly two peptides, TcbA ii and TcbA iii , that were observed to be derived from the same gene product, TcbA.

The N-terminal sequence of A2 (SEQ ID NO:41) was unique in comparison to the TcbA iii peptide and other peptides. The A2 peptide was denoted TcdA iii (see Example 17). SEQ ID NO:6 was determined to be a mixture of amino acid sequences SEQ ID NO:40 and 41.

Peptides A1 and A2 were further subjected to internal amino acid sequencing. For internal amino acid sequencing, 10 μg of toxin A was electrophoresized in 10% SDS-PAGE and transblotted to PVDF membrane. After the blot was stained with amido black, peptides A1 and A2, denoted TcdA ii and TcdA iii , respectively, were excised from the blot and sent to Harvard MicroChem and Cambridge ProChem. Peptides were subjected to trypsin digestion followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected tryptic peptide fragments. Two internal amino acid sequences of peptide A1 (TcdA ii -PK71, SEQ ID NO:38 and TcdA ii -PK44, SEQ ID NO:39) were found to have significant homologies with deduced amino acid sequences of the TcbA ii region of the tcbA gene (SEQ ID NO:12). Similarly, the N-terminal sequence (SEQ ID NO:41) and two internal sequences of peptides A2 (TcdA iii -PK57, SEQ ID NO:42 and TcdA iii -PK20, SEQ ID NO.43) also showed significant homology with deduced amino acid sequences of TcbA iii region of the tcbA gene (SEQ ID NO:12).

In summary of above results, the toxin complex has at least two active protein toxin complexes against SCR; toxin A and toxin B. Toxin A and toxin B are similar in their native and subunits molecular weight, however, their peptide compositions are different. Toxin A contained peptides TcdA ii and TcdA iii as the major peptides and the toxin B contains TcbA ii and TcbA iii as the major peptides.

Purification and Characterization of Toxin C, Tca Peptides

The Photorhabdus protein toxin complex was isolated as described above. Next, about 50 mg toxin was applied to a MonoQ 10/10 column equilibrated with 20 mM Tris-HCl, pH 7.0 at a flow rate of 2 ml/min. The column was washed with 20 mM Tris-HCl, pH7.0 until the optical density at 280 nm returned to baseline level. The proteins bound to the column were eluted with a linear gradient of 0 to 1M NaCl in 20 mM Tris-HCl, pH 7.0 at 2 ml/min for 60 min. 2 ml fractions were collected and subjected to Western analysis using pAb TcaB ii -syn antibody (see Example 21) as the primary antibody. Fractions reacted with pAb TcaB ii -syn antibody were combined and solid (NH 4 ) 2 SO 4 was added to a final concentration of 1.7 M. Proteins were then applied to a phenyl-Superose 10/10 column equilibrated with 1.7 M (NH 4 ) 2 SO 4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (NH 4 ) 2 SO 4 , 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 1 ml/min for 120 min. 2 ml Fractions were collected, dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0, and analyzed by Western blots using pAb TcaB ii -syn antibody as the primary antibody.

Fractions cross-reacted with the antibody were pooled and applied to a MonoQ 5/5 column which was equilibrated with 20 mM Tris-HCl, pH 7.0 at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to 1M NaCl in 20 mM Tris-HCl, pH 7.0 for 30 min.

Fractions above reacted with pAb TcaB ii -syn antibody were pooled and subjected to a phenyl-Superose 5/5/column. Solid (NH 4 ) 2 SO 4 added to a final concentration of 1.7 M. The solution was then applied onto the column equilibrated with 1.7 M (NH 4 ) 2 SO 4 in 50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins bound to the column were then eluted with a linear gradient of 1.7 M (NH 4 ) 2 SO 4 , 50 mM potassium phosphate, pH 7.0 to 10 mM potassium phosphate, pH 7.0 at 0.5 ml/min for 60 min. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0.

For the final purification step, fractions reacted with pAb TcaB ii -syn antibody above determined by Western analysis were combined and applied to a Mono Q 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0 at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to 1M NaCl in 20 mM Tris-HCl, pH 7.0 for 30 min.

The final purified protein fraction contained 6 major peptides examined by SDS-PAGE: 165 kDa, 90 kDa, 64 kDa, 62 kDa, 58 kDa, and 22 kDa. The LD50 of the insecticidal activities of this purified fraction were determined to be 100 ng and 500 ng against SCR and ECB, respectively.

The above peptides were blotted to PVDF membranes and blots were sent for amino acids analysis and 5 amino acid long N-terminal sequencing at Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino acid sequence of the 165 kDa peptide was determined to be identical to peptide TcaC (SEQ ID 2, position 1 to 5). The N-terminal amino acid sequence of the 90 kDa peptide was determined to be TcaA ii region of the derived amino acid sequence for the tcaA gene (SEQ ID NO 33, position 254 to 258). The N-terminal amino acid sequence of 64 kDa peptide was determined to be identical to peptide TcaB i (SEQ ID 3, position 1 to 5). The N-terminal amino acid sequence of the 62 kDa peptide was determined to be TcaA ii region of the derived amino acid sequence for the tcaA gene (SEQ ID NO 33, position 489 to 493). The N-terminal amino acid sequence of 58 kDa peptide was determined to be identical to peptide TcaB ii (SEQ ID 5, position 1 to 5). The N-terminal amino acid sequence of the 22 kDa peptide (SEQ ID NO 62) was determined to be TcaA i region, denoted TcaA iv , of the derived amino acid sequence for the tcaA gene (SEQ ID NO 34, position 98 to 102). It is noted that all tcaA, tcaB, and tcaC genes reside in the same tca operon (FIG. 6 A).

Five μg of purified Tca fraction, purified toxin A, and purified toxin B were analyzed by western blot using the following antibodies individually as primary antibody: pAb TcaBii-syn antibody, mAb CF52 antibody, pAb TcdAii-syn antibody, and pAb Tcd iii -syn antibody (Example 21). With pAb TcaB ii -syn antibody only the purified Tca peptides fraction reacted, but not toxin A or toxin B. With mAb CF52 antibody, only toxin B reacted but not Tca peptides fraction or toxin A. With either pAb TcdAii-syn antibody or pAb Tcdiii-syn antibody only toxin A reacted, but not Tca peptides fraction or toxin B. This indicated that the insecticidal activity observed in the purified Tca peptides fraction is independent of toxin A and toxin B. The purified Tca peptide fraction is a third unique protein toxin, denoted toxin C.

Cleavage and Activation of TcbA Peptide

In the toxin B complex, peptide TcbA ii and TcbA iii originate from the single gene product TcbA (Example 15). The processing of TcbA peptide to TcbA ii and TcbA iii is presumably by the action of Photorhabdus protease(s), and most likely, the metalloproteases described in Example 10. In some cases, it was noted that when Photorhabdus W-14 broth was processed, TcbA peptide was present in toxin B complex as a major component, in addition to peptides TcbA ii and TcbA iii . Identical procedures, described for the purification of toxin B complex (Example 15), were used to enrich peptide TcbA from toxin complex fraction of W-14 broth. The final purified material was analyzed in a 4-20% gradient SDS-PAGE and major peptides were quantified by densitometry. It was determined that TcbA, TcbA ii and TcbA iii comprised 58%, 36%, and 6%, respectively, of total protein. The identities of these peptides were confirmed by their respective molecular sizes in SDS-PAGE and Western blot analysis using monospecific antibodies. The native molecular weight of this fraction was determined to be 860 kDa.

The cleavage of TcbA was evaluated by treating the above purified material with purified 38 kDa and 58 kDa W-14 Photorhabdus metalloproteases (Example 10), and trypsin as a control enzyme (Sigma, MO.). The standard reaction consisted 17.5 μg the above purified fraction, 1.5 unit protease, and 0.1 M Tris buffer, pH 8.0 in a total volume of 100 μl. For the control reaction, protease was omitted. The reaction mixtures were incubated at 37° C. for 90 min. At the end of the reaction, 20 μl was taken and boiled with SDS-PAGE sample buffer immediately for electrophoresis analysis in a 4-20% gradient SDS-PAGE. It was determined from SDS-PAGE that in both 38 kDa and 58 kDa protease treatments, the amount of peptides TcbA ii and TcbA iii increased about 3-fold while the amount of TcbA peptide decreased proportionally (Table 24). The relative reduction and augmentation of selected peptides was confirmed by Western blot analyses. Furthermore, gel filtration of the cleaved material revealed that the native molecular size of the complex remained the same. Upon trypsin treatment, peptides TcbA and TcbA ii were nonspecifically digested into small peptides. This indicated that 38 kDa and 58 kDa Photorhabdus proteases can specifically process peptide TcbA into peptides TcbA ii and TcbA iii . Protease treated and untreated control of the remaining 80 μl reaction mixture were serial diluted with 10 mM sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay. By comparing activity in several dilution, it was determined that the 38 kDa protease treatment increased SCR insecticidal activity approximately 3 to 4 fold. The growth inhibition of remaining insects in the protease treatment was also more severe than control (Table 24).

Activation and Procession of Toxin B by SCR Gut Proteases

In yet a second demonstration of proteolytic activation, it was examined whether W-14 toxins are processed by insects. Toxin B purified from Photorhabdus W-14 broth (see Example 15) was comprised of predominantly intact TcbA peptides as judged by SDS-PAGE and Western blot analysis using monoclonal antibody. The LD50 of this fraction against SCR was determined to be around 700 ng.

SCR larva were grown on coleopteran diet until they reached the fourth instar stage (about 100-125 mg total weight each insect). SCR gut content was collected as follows: the guts were removed using dissecting scissors and forceps. After removing the excess fatty material that coats the gut lining, about 40 guts were homogenized in a microcentrifuge tube containing 100 μl sterile water. The tube was then centrifuged at 14,000 rpm for 10 minutes and the pellet discarded. The supernatant was stored at a −70° C. freezer until use.

The processing of toxin B by insect gut was evaluated by treating the above purified toxin B with the SCR gut content collected. The reaction consisted 40 μg toxin B (1 mg/ml), 50 μl SCR gut content, and 0.1M Tris buffer, pH 8.0 in a total volume of 100 μl. For the control reaction, SCR gut content was omitted. The reaction mixtures were incubated at 37° C. for overnight. At the end of reaction, 10 μl was withdraw and boiled with equal volume 2×SDS-PAGE sample buffer for SDS-PAGE analysis. The remaining 90 μl reaction mixture was serial diluted with 10 mM sodium phosphate buffer, pH 7.0 and analyzed by SCR bioassay. SDS-PAGE analysis indicated in SCR gut content treatment, peptide TcbA was digested completely into smaller peptides. Analysis of the undenatured toxin fraction showed that the native size, about 860 kDa, remained the same even though larger peptides were fragmented. In SCR bioassays, it was found that the LDSO of SCR gut treated toxin B to be about 70 ng; representing a 10-fold increase. In a separate experiment, protease K treatment completely eliminated toxin activity.

Screening of the Library for a Gene Encoding the TcdA ii Peptide

The cloning and characterization of a gene encoding the TcdA ii peptide, described as SEQ ID NO:17 (internal peptide TcdA ii -PT111 N-terminal sequence) and SEQ ID NO:18 (internal peptide TcdA ii -PT79 N-terminal sequence) was completed. Two pools of degenerate oligonucleotides, designed to encode the amino acid sequences of SEQ ID NO:17 (Table 25) and SEQ ID NO:18 (Table 26), and the reverse complements of those sequences, were synthesized as described in Example 8. The DNA sequence of the oligonucleotides is given below:

Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using as forward primers P2.3.6.CB or P2.3.5, and as reverse primers P2.79.R.1 or P2.79R.CB, in all forward/reverse combinations, using Photorhabdus W-14 genomic DNA as template. In another set of reactions, primers P2.79.2 or P2.79.3 were used as forward primers, and P2.3.5R, P2.3.5RI, and P2.3R.CB were used as reverse primers in all forward/reverse combinations. Only in the reactions containing P2.3.6.CB as the forward primers combined with P2.79.R.1 or P2.79R.CB as the reverse primers was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 2500 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdA ii -PT111 lies amino-proximal to the peptide fragment TcdA ii -PT79.

The 2500 bp PCR products were ligated to the plasmid vector pCR™II (Invitrogen, San Diego, Calif.) according to the supplier's instructions, and the DNA sequences across the ends of the insert fragments of two isolates (HS24 and HS27) were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of both isolates was the same. New primers were synthesized based on the determined sequence, and used to prime additional sequencing reactions to obtain a total of 2557 bases of the insert [SEQ ID NO:36]. Translation of the partial peptide encoded by SEQ ID No: 36 yields the 845 amino acid sequence disclosed as SEQ ID NO:37. Protein homology analysis of this portion of the TcdA ii peptide fragment reveals substantial amino acid homology ((68% similarity,and 53% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, Wis.) to residues 542 to 1390 of protein TcbA [SEQ ID NO:12] or (60% similarity, and 54% identity using the Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis. to residues 567 to 1389)). It is therefore apparent that the gene represented in part by SEQ ID NO:36 produces a protein of similar, but not identical, amino acid sequence as the TcbA protein, and which likely has similar, but not identical biological activity as the TcbA protein.

In yet another instance, a gene encoding the peptides TcdA ii -PK44 and the TcdA iii 58 kDa N-terminal peptide, described as SEQ ID NO:39 (internal peptide TcdA ii -PK44 sequence), and SEQ ID NO:41 (TcdA iii 58 kDa N-terminal peptide sequence) was isolated. Two pools of degenerate oligonucleotides, designed to encode the amino acid sequences described as SEQ ID NO:39 (Table 28) and SEQ ID NO:41 (Table 27), and the reverse complements of those sequences, were synthesized as described in Example 8, and their DNA sequences.

Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using as forward primers A1.44.1 or A1.44.2, and reverse primers A2.3R or A2.4R, in all forward/reverse combinations, using Photorhabdus W-14 genomic DNA as template. In another set of reactions, primers A2.1 or A2.2 were used as forward primers, and A1.44.1R, and A1.44.2R were used as reverse primers in all forward/reverse combinations. Only in the reactions containing A1.44.1 or A1.44.2 as the forward primers combined with A2.3R as the reverse primer was a non-artifactual amplified product seen, of estimated size (mobility on agarose gels) of 1400 base pairs. The order of the primers used to obtain this amplification product indicates that the peptide fragment TcdA ii -PK44 lies amino-proximal to the 58 kDa peptide fragment of TcdA iii .

The 1400 bp PCR products were ligated to the plasmid vector pCR™II according to the supplier's instructions. The DNA sequences across the ends of the insert fragments of four isolates were determined using primers similar in sequence to the supplier's recommended primers and using sequencing methods described previously. The nucleic acid sequence of all isolates differed as expected in the regions corresponding to the degenerate primer sequences, but the amino acid sequences deduced from these data were the same as the actual amino acid sequences for the peptides determined previously, (SEQ ID NOS:41 and 39).

Screening of the W-14 genomic cosmid library as described in Example 8 with a radiolabeled probe comprised of the DNA prepared above (SEQ ID NO:36) identified five hybridizing cosmid isolates, namely 17D9, 20B10, 21D2, 27B10, and 26D1. These cosmids were distinct from those previously identified with probes corresponding to the genes described as SEQ ID NO:11 or SEQ ID NO:25. Restriction enzyme analysis and DNA blot hybridizations identified three EcoR I fragments, of approximate sizes 3.7, 3.7, and 1.1 kbp, that span the region comprising the DNA of SEQ ID NO:36. Screening of the W-14 genomic cosmid library using as probe the radiolabeled 1.4 kbp DNA fragment prepared in this example identified the same five cosmids (17D9, 20B10, 21D2, 27B10, and 26D1). DNA blot hybridization to EcoR I-digested cosmid DNAs also showed hybridization to the same subset of EcoR I fragments as seen with the 2.5 kbp TcdA ii gene probe, indicating that both fragments are encoded on the genomic DNA.

DNA sequence determination of the cloned EcoR I fragments revealed an uninterrupted reading frame of 7551 base pairs (SEQ ID NO:46), encoding a 282.9 kDa protein of 2516 amino acids (SEQ ID NO:47). Analysis of the amino acid sequence of this protein revealed all expected internal fragments of peptides TcdA ii (SEQ ID NOS:17, 18, 37, 38 and 39) and the TcdA iii peptide N-terminus (SEQ ID NO:41) and all TcdA iii internal peptides (SEQ ID NOS:42 and 43). The peptides isolated and identified as TcdA ii and TcdA iii are each products of the open reading frame, denoted tcda, disclosed as SEQ ID NO:46. Further, SEQ ID NO:47 shows, starting at position 89, the sequence disclosed as SEQ ID NO:13, which is the N-terminal sequence of a peptide of size approximately 201 kDa, indicating that the initial protein produced from SEQ ID NO: 46 is processed in a manner similar to that previously disclosed for SEQ ID NO:12. In addition, the protein is further cleaved to generate a product of size 209.2 kDa, encoded by SEQ ID NO:48 and disclosed as SEQ ID NO:49 (TcdA ii peptide), and a product of size 63.6 kDa, encoded by SEQ ID NO:50 and disclosed as SEQ ID NO:51 (TcdA iii peptide). Thus, it is thought that the insecticidal activity identified as toxin A (Example 15) derived from the products of SEQ ID NO:46, as exemplified by the full-length protein of 282.9 kDa disclosed as SEQ ID NO:47, is processed to produce the peptides disclosed as SEQ ID NOS:49 and 51. It is thought that the insecticidal activity identified as toxin B (Example 15) derives from the products of SEQ ID NO:11, as exemplified by the 280.6 kDa protein disclosed as SEQ ID NO:12. This protein is proteolytically processed to yield the 207.6 kDa peptide disclosed as SEQ ID NO:53, which is encoded by SEQ ID NO:52, and the 62.9 kDa peptide having N-terminal sequence disclosed as SEQ ID NO:40, and further disclosed as SEQ ID NO:55, which is encoded by SEQ ID NO:54.

Amino acid sequence comparisons between the proteins disclosed as SEQ ID NO:12 and SEQ ID NO:47 reveal that they have 69% similarity and 54% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, Wis. or 60% similarity and 54% identity using version 9.0 of the program. This high degree of evolutionary relationship is not uniform throughout the entire amino acid sequence of these peptides, but is higher towards the carboxy-terminal end of the proteins, since the peptides disclosed as SEQ ID NO:51 (derived from SEQ ID NO:47) and SEQ ID NO:55 (derived from SEQ ID NO:12) have 76% similarity and 64% identity using the Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, Wis. or 71% similarity and 64% identity using version 9.0 of the program.

Control of European Cornborer-Induced Leaf Damage on Maize Plants by Spray Application of Photorhabdus (Strain W-14) Broth

The ability of Photorhabdus toxin(s) to reduce plant damage caused by insect larvae was demonstrated by measuring leaf damage caused by European corn borer ( Ostrinia nubilalis ) infested onto maize plants treated with Photorhabdus broth. Fermentation broth from Photorhabdus strain W-14 was produced and concentrated approximately 10-fold using ultrafiltration (10,000 MW pore-size) as described in Example 13. The resulting concentrated broth was then filter sterilized using 0.2 micron nitrocellulose membrane filters. A similarly prepared sample of uninoculated 2% proteose peptone #3 was used for control purposes. Maize plants (an inbred line) were grown from seed to vegetative stage 7 or 8 in pots containing a soilless mixture in a greenhouse (27° C. day; 22° C. night, about 50%RH, 14 hr day-length, watered/fertilized as needed). The test plants were arranged in a randomized complete block design (3 reps/treatment, 6 plants/treatment) in a greenhouse with temperature about 22° C. day; 18° C. night, no artificial light and with partial shading, about 50%RH and watered/fertilized as needed. Treatments (uninoculated media and concentrated Photorhabdus broth) were applied with a syringe sprayer, 2.0 mls applied from directly (about 6 inches) over the whorl and 2.0 additional mls applied in a circular motion from approximately one foot above the whorl. In addition, one group of plants received no treatment. After the treatments had dried (approximately 30 minutes), twelve neonate European corn borer larvae (eggs obtained from commercial sources and hatched in-house) were applied directly to the whorl. After one week, the plants were scored for damage to the leaves using a modified Guthrie Scale (Koziel, M. G., Beland, G. L., Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L., Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, M. Z., Merlin, E., Rhodes, R., Warren, G. W., Wright, M. and Evola, S. V. 1993).

Bio/Technology, 11, 194-195.) and the scores were compared statistically [T-test (LSD) p<0.05 and Tukey's Studentized Range (HSD) Test p<0.1]. The results are shown in Table 29. For reference, a score of 1 represents no damage, a score of 2 represents fine “window pane” damage on the unfurled leaf with no pinhole penetration and a score of 5 represents leaf penetration with elongated lesions and/or mid rib feeding evident on more than three leaves (lesions <1 inch). These data indicate that broth or other protein containing fractions may confer protection against specific insect pests when delivered in a sprayable formulation or when the gene or derivative thereof, encoding the protein or part thereof, is delivered via a transgenic plant or microbe.

Genetic Engineering of Genes for Expression in E. coli

Summary of Constructions

A series of plasmids were constructed to express the tcbA gene of Photorhabdus W-14 in Escherichia coli . A list of the plasmids is shown in Table 30. A brief description of each construction follows as well as a summary of the E. coli expression data obtained.

Construction of pDAB2025

In Example 9, a large EcoR I fragment which hybridizes to the TcbA ii probe is described. This fragment was subcloned into pBC (Stratagene, La Jolla Calif.) to create pDAB2025. Sequence analysis indicates that the fragment is 8816 base pairs. The fragment encodes the tcbA gene with the initiating ATG at position 571 and the terminating TAA at position 8086. The fragment therefore carries 570 base pairs of Photorhabdus DNA upstream of the ATG and 730 base pairs downstream of the TAA.

Construction of Plasmid pDAB2026

The tcbA gene was PCR amplified from plasmid pDAB202S using the following primers; 5′ primer (SlAc51) 5′ TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3′ and 3′ primer (SlAc31) 5′ TTT AAA GCG GCC GCT TAA CGG ATG GTA TAA CGA ATA TG 3′. PCR was performed using a TaKaRa LA PCR kit from PanVera (Madison, Wis.) in the following reaction: 57.5 microliters water, 10 microliters 10×LA buffer, 16 microliters dNTPs (2.5 mM each stock solution), 20 microliters each primer at 10 pmoles/microliters, 300 ng of the plasmid pDAB2025 containing the W-14 tcbA gene and one microliter of TaKaRa LA Taq polymerase. The cycling conditions were 98° C./20 sec, 68° C./5 min, 72° C./10 min for 30 cycles. A PCR product of the expected about 7526 bp was isolated in a 0.8% agarose gel in TBE (100 mM Tris, 90 mM boric acid, 1 mM EDTA) buffer and purified using a Qiaex II kit from Qiagen (Chatsworth, Calif.). The purified tcbA gene was digested with Nco I and Not I and ligated into the baculovirus transfer vector pAcGP67B (PharMingen (San Diego, Calif.)) and transformed into DH5 α E. coli. The resulting recombinant is called pDAB2026. The tcbA gene was then cut from pDAB2026 and transferred to pET27b to create plasmid pDAB2027. A missense mutation in the tcbA gene was repaired in pDAB2027.

The repaired tcbA gene contains two changes from the sequence shown in Sequence ID NO:11; an A>G at 212 changing an asparagine 71 to serine 71 and a G>A at 229 changing an alanine 77 to threonine 77. These changes are both upstream of the proposed TcbA ii N-terminus.

Construction of pDAB2028

The tcbA coding region of pDAB2027 was transferred to vector pET15b. This was accomplished using shotgun ligations, the DNAs were cut with restriction enzymes Nco I and Xho I. The resulting recombinant is called pDAB2028.

Expression of TcbA in E. coli from Plasmid pDAB2028

Expression of tcbA in E. coli was obtained by modification of the methods previously described by Studier et al. (Studier, F. W., Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol., 185: 60-89.). Competent E. coli cells strain BL21(DE3) were transformed with plasmid pDAB2028 and plated on LB agar containing 100 μg/mL ampicillin and 40 mM glucose. The transformed cells were plated to a density of several hundred isolated colonies/plate. Following overnight incubation at 37° C. the cells were scraped from the plates and suspended in LB broth containing 100 μg/mL ampicillin. Typical culture volumes were from 200-500 mL. At time zero, culture densities (OD600) were from 0.05-0.15 depending on the experiment. Cultures were shaken at one of three temperatures (22° C., 30° C. or 37° C.) until a density of 0.15-0.5 was obtained at which time they were induced with 1 mM isopropylthio-β-galactoside (IPTG). Cultures were incubated at the designated temperature for 4-5 hours and then were transferred to 4° C. until processing (12-72 hours).

Purification and Characterization of TcbA Expressed in E.coli from Plasmid pDAB2028

E. coli cultures expressing TcbA peptides were processed as follows. Cells were harvested by centrifugation at 17,000×G and the media was decanted and saved in a separate container.

The media was concentrated about 8× using the M12 (Amicon, Beverly Mass.) filtration system and a 100 kD molecular mass cut-off filter. The concentrated media was loaded onto an anion exchange column and the bound proteins were eluted with 1.0 M NaCl. The 1.0 M NaCl elution peak was found to cause mortality against Southern corn rootworm (SCR) larvae Table 30). The 1.0 M NaCl fraction was dialyzed against 10 mM sodium phosphate buffer pH 7.0, concentrated, and subjected to gel filtration on Sepharose CL-4B (Pharmacia, Piscataway, N.J.). The region of the CL-4B elution profile corresponding to calculated molecular weight (about 900 kDa) as the native W-14 toxin complex was collected, concentrated and bioassayed against larvae. The collected 900 kDa fraction was found to have insecticidal activity (see Table 31 below), with symptomology similar to that caused by native W-14 toxin complex. This fraction was subjected to Proteinase K and heat treatment, the activity in both cases was either eliminated or reduced, providing evidence that the activity is proteinaceous in nature. In addition, the active fraction tested immunologically positive for the TcbA and TcbA iii peptides in immunoblot analysis when tested with an anti-TcbA iii monoclonal antibody (Table 31).

The cell pellet was resuspended in 10 mM sodium phosphate buffer, pH=7.0, and lysed by passage through a Bio-Neb™ cell nebulizer (Glas-Col Inc., Terra Haute, Ind.). The pellets were treated with DNase to remove DNA and centrifuged at 17,000×g to separate the cell pellet from the cell supernatant. The supernatant fraction was decanted and filtered through a 0.2 micron filter to remove large particles and subjected to anion exchange chromatography. Bound proteins were eluted with 1.0 M NaCl, dialyzed and concentrated using Biomax™ (Millipore Corp, Bedford, Mass.) concentrators with a molecular mass cut-off of 50,000 Daltons. The concentrated fraction was subjected to gel filtration chromatography using Sepharose CL-4B beaded matrix. Bioassay data for material prepared in this way is shown in Table 30 and is denoted as “TcbA Cell Sup”.

In yet another method to handle large amounts of material, the cell pellets were re-suspended in 10 mM sodium phosphate buffer, pH=7.0 and thoroughly homogenized by using a Kontes Glass Company (Vineland, N.J.) 40 ml tissue grinder. The cellular debris was pelleted by centrifugation at 25,000×g and the cell supernatant was decanted, passed through a 0.2 micron filter and subjected to anion exchange chromatography using a Pharmacia 10/10 column packed with Poros HQ 50 beads. The bound proteins were eluted by performing a NaCl gradient of 0.0 to 1.0 M. Fractions containing the TcbA protein were combined and concentrated using a 50 kDa concentrator and subjected to gel filtration chromatography using Pharmacia CL-4B beaded matrix. The fractions containing TcbA oligomer, molecular mass of approximately 900 kDa, were collected and subjected to anion exchange chromatography using a Pharmacia Mono Q 10/10 column equilibrated with 20 mM Tris buffer pH=7.3. A gradient of 0.0 to 1.0 M NaCl was used to elute recombinant TcbA protein. Recombinant TcbA eluted from the column at a salt concentration of approximately 0.3-0.4 M NaCl, the same molarity at which native TcbA oligomer is eluted from the Mono Q 10/10 column. The recombinant TcbA fraction was found to cause SCR mortality in bioassay experiments similar to those in Table 31.

A second set of expression constructions were prepared and tested for expression of the TcbA protein toxin.

Construction of pDAB2030: An Expression Plasmid for the tcbA Coding Region

The plasmid pDAB2028 (see herein) contains the tcbA coding region in the commercial vector pET15 (Novagen, Madison, Wis.), encodes an ampicillin selection marker. The plasmid pDAB2030 was created to express the tcbA coding region from a plasmid which encodes a kanamycin selection marker. This was done by cutting pET27 (Novagen, Madison, Wis.) a kanamycin selection plasmid, and pDAB2028 with Xba I and Xho I. This releases the entire multiple cloning site, including the tcbA coding region from plasmid pDAB2028. The two cut plasmids, were mixed and ligated. Recombinant plasmids were selected on kanamycin and those containing the pDAB2028 fragment were identified by restriction analysis. The new recombinant plasmid is called pDAB2030.

Construction of Plasmid pDAB2031: Correction of Mutations in tcbA i

The two mutations in the N-terminus of the tcbA coding region as described in Example 19 (Sequence ID NO:11; A>G at 212 changing an asparagine 71 to serine 71; G>A at 229 changing an alanine 77 to threonine 77) were corrected as follows: A PCR product was generated using the primers TH50 (5′ ACC GTC TTC TTT ACG ATC AGT G 3′) and S1Ac51 (5′ TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3′) and pDAB2025 as template to generate a 1778 bp product. This PCR product was cloned into plasmid pCR2.1 (Invitrogen, San Diego, Calif.) and a clone was isolated and sequenced. The clone was digested with Nco I and Pin AI and a 1670 bp fragment was purified from a 1% agarose gel. A plasmid containing the mutated tcbA coding region (pDAB2030) was digested with Nco I and Not I and purified away from the 1670 bp fragment in a 0.8% agarose with Qiaex II (Qiagen, Chatsworth, Calif.). The corrected Nco I/Pin AI fragment was then ligated into pDAB2030. The ligated DNA was transformed into DH5 α E. coli. A clone was isolated, sequenced and found to be correct. This plasmid, containing the corrected tcbA coding region, is called pDAB2031.

Construction of pDAB2033 and pDAB2034: Expression Plasmids for tcbA

The expression plasmids pDAB2025 and pDAB2027-2031 all rely on the Bacteriophage T7 expression system. An additional vector system was used for bacterial expression of the tcbA gene and its derivatives. The expression vector Trc99a (Pharmacia Biotech, Piscataway, N.J.) contains a strong trc promoter upstream of a multiple cloning site with a 5′ Nco I site which is compatible with the tcbA coding region from pDAB2030 and 2031. However, the plasmid does not have a compatible 3′ site. Therefore, the Hind III site of Trc99a was cut and made blunt by treatment with T4 DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.). The vector plasmid was then cut by Nco I followed by treatment with alkaline phosphatase. The plasmids pDAB2030 and pDAB2031 were each cut with Xho I (cuts at the 3′ end of the tcbA coding region) followed by treatment with T4 DNA polymerase to blunt the ends. The plasmids were then cut with Nco I, the DNAs were extracted with phenol, ethanol precipitated and resuspended in buffer. The Trc99a and pDAB2030 and pDAB2031 plasmids were mixed separately, ligated and transformed into DH5α cells and plated on LB media containing ampicillin and 50 mM glucose. Recombinant plasmids were identified by restriction digestion. The new plasmids are called pDAB2033 (contains the tcbA coding sequence with the two mutations in tcbA i ) and pDAB2034 (contains the corrected version of tcbA from pDAB2031).

Construction of Plasmid pDAB2032: An Expression Plasmid for tcbA ii A iii

A plasmid encoding the TcbA ii A iii portion of TcbA was created in a similar way as plasmid pDAB2031. A PCR product was generated using TH42 (5′ TAG GTC TCC ATG GCT TTT ATA CAA GGT TAT AGT GAT CTG 3′) and TH50 (5′ ACC GTC TTC TTT ACG ATC AGT G 3′) primers and plasmid pDAB2025 as template. This yielded a product of 1521 bp having an initiation codon at the beginning of the coding sequence of tcbA ii . This PCR product was isolated in a 1% agarose gel and purified. The purified product was cloned into pCR2.1 as above and a correct clone was identified by DNA sequence analysis. This clone was digested with Nco I and Pin AI, a 1414 bp fragment was isolated in a 1% agarose gel and ligated into the Nco I and Pin AI sites of plasmid pDAB2030 and transformed into DHα E. coli . This new plasmid, designed to express TcbA ii A iii in E. coli , is called pDAB2032.

Expression of tcbA and tcbA ii A iii from Plasmids pDAB2030, pDAB2031 and pDAB2032

Expression of tcbA in E. coli from plasmids pDAB2030, pDAB2031 and pDAB2032 was as described herein, except expression of tcbA ii A iii was done in E. coli strain HMS174(DE3)(Novagen, Madison, Wis.).

Expression of tcbA from Plasmid pDAB2033

The plasmid pDAB2033 was transformed into BL21 cells (Novagen, Madison, Wis.) and plated on LB containing 100 micrograms/mL ampicillin and 50 mM glucose. The plates were spread such that several hundred well separated colonies were present on each plate following incubation at either 30° C. or 37° C. overnight. The colonies were scraped from the plates and suspended in LB containing 100 micrograms/mL ampicillin, but no glucose. Typical culture volume was 250 mL in a single 1 L baffle bottom flask. The cultures were induced when the culture reached a density of 0.3-0.6 OD600 nm. Most often this density was achieved immediately after suspension of the cells from the plates and did not require a growth period in liquid media. Two induction methods were used. Method 1: cells were induced with 1 mM IPTG at 37° C. The cultures were shaken at 200 rpm on a platform shaker for 5 hours and harvested. Method 2: The cultures were induced with 25 micromolar IPTG at 30° C. and shaken at 200 rpm for 15 hours at either 20° C. or 30° C. The cultures were stored at 4° C. until used for purification.

Purification of TcbA from E. coli

Purification, bioassay and immunoblot analysis of TcbA and TcbA ii A iii was as described herein. Results of several representative E. coli expression experiments are shown in Table 32. All materials shown in Table 32 were purified from the media fraction of the cultures. The predicted native molecular weight is approximately 900 kD as described herein. The purity of the samples, the amount of TcbA relative to contaminating proteins, varied with each preparation.

Characterization of Toxin Peptides with Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectroscopy

Toxins isolated from W-14 broth were purified as described in Example 15. In some cases, the TcaB protein toxin was pretreated with proteases (Example 16) that had been isolated from W-14 broth as previously described (Example 15). Protein molecular mass was determined using matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, hereinafter MALDI-TOF, on a VOYAGER BIOSPECTROMETRY workstation with DELAYED EXTRACTION technology (PerSeptive Biosystems, Framingham, Mass.). Typically, the protein of interest (100-500 pmoles in 5 μl) was mixed with 1 μl of acetonitrile and dialyzed for 0.5 to 1 h on a Millipore VS filter having a pore size of 0.025 μM (Millipore Corp. Bedford, Mass.). Dialysis was performed by floating the filter on water(shinny side up) followed by adding protein-acetonitrile mixture as a droplet to the surface of the filter. After dialysis, the dialyzed protein removed using a pipette and was then mixed with a matrix consisting of sinapinic acid and trifluoroacetic acid according to manufacturers instructions. The protein and matrix were allowed to co-crystallize on a about 3 cm 2 gold-plated sample plate (PerSeptive Corp.). Excitation of the crystals and subsequent mass analysis was performed using the following conditions: laser setting of 3050; pressure of 4.55e-07; low mass gate of 1500.0; negative ions off; accelerating voltage of 25,000; grid voltage of 90.0%; guide wire voltage of 0.010%; linear mode; and a pulse delay time of 350 ns.

Protein mass analysis data are shown in Table 33. The data obtained from MALDI-TOF was compared to that hypothesized from gene sequence information and as previously determined by SDS-PAGE.

Production of Peptide Specific Polyclonal Antibodies

Nine peptide components of the W-14 toxin complex, namely, TcaA, TcaA iii , TcaB i , TcaB ii , TcaC, TcbA ii , TcbA iii , TcdA ii , and TcdA iii were selected as targets against which antibodies were produced. Comprehensive DNA and deduced amino acid sequence data for these peptides indicated that the sequence homology between some of these peptides was substantial. If a whole peptide was used as the immunogen to induce antibody production, the resulting antibodies might bind to multiple peptides in the toxin preparation. To avoid this problem antibodies were generated that would bind specifically to a unique region of each peptide of interest. The unique region (subpeptide) of each target peptide was selected based on the analyses described below.

Each entire peptide sequence was analyzed using MacVector™ Protein Analysis Tool (IBI Sequence Analysis Software, International Biotechnologies, Inc:, P.O. Box 9558, New Haven, Conn. 06535) to determine its antigenicity index. This program was designed to locate possible externally-located amino acid sequences, i.e., regions that might be antigenic sites. This method combined information from hydrophilicity, surface probability, and backbone flexibility predictions along with the secondary structure predictions in order to produce a composite prediction of the surface contour of a protein. The scores for each of the analyses were normalized to a value between −1.0 and +1.0 (MacVector™ Manual). The antigenicity index value was obtained for the entire sequence of the target peptide. From each peptide, an area covering 19 or more amino acids that showed a high antigenicity index from the original sequence was re-analyzed to determine the antigenicity index of the subpeptide without the flanking residues. This re-analysis was necessary because the antigenicity index of a peptide could be influenced by the flanking amino acid residues. If the isolated subpeptide sequence did not maintain a high antigenicity index, a new region was chosen and the analysis was repeated.

Each selected subpeptide sequence was aligned and compared to all seven target peptide sequences using MacVector™ alignment program. If a selected subpeptide sequence showed identity (greater than 20%) to another target peptide, a new 19 or more amino acid region was isolated and re-analyzed. Unique subpeptide sequences covering 19 or more amino acid showing high antigenicity index were selected from all target peptides.

The sequences of seven subpeptides were sent to Genemed Biotechnology Inc. The last amino acid residue on each subpeptide was deleted because it showed no apparent effect on the antigenicity index. A cysteine residue was added to the N-terminal of each subpeptide sequence, except TcaB i -syn which contains an internal cysteine residue. The present of a cysteine residue facilitates conjugation of a carrier protein (KLH). The final peptide products corresponding to the appropriate toxin peptides and SEQ ID NO.s are shown in Table 34.

Each conjugated synthetic peptide was injected into two rabbits according to Genemed accelerated program. The pre- and post-immune sera were available for testing after one month.

The preliminary test of both pre- and post-immune sera from each rabbit was performed by Genemed Biotechnologies Inc. Genemed reported that by using both ELISA and Western blot techniques, they detected the reaction of post-immune sera to the respective synthetic peptides. Subsequently, the sera were tested with the whole target peptides, by Western blot analysis. Two batches of partially purified Photorhabdus strain W-14 toxin complex was used as the antigen. The two samples had shown activity against the Southern corn rootworm. Their peptide patterns on an SDS-PAGE gel were slightly different.

Pre-cast SDS-polyacrylamide gels with 4-20% gradient (Integrated Separation Systems, Natick, Mass. 01760) were used. Between 1 to 8 μg of protein was applied to each gel well. Electrophoresis was performed and the protein was electroblotted onto Hybond-ECL™ nitrocellulose membrane (Amersham International). The membrane was blocked with 10% milk, in TBST (25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% TWEEN 20 surfactant) for one hour at room temperature. Each rabbit serum was diluted in 10% milk/TBST to 1:500. Other dilutions between 1:50 to 1:1000 were also used. The serum was added to the membrane and placed on a platform rocker for at least one hour. The membrane was washed thoroughly with the blocking solution or TBST. A 1:2000 dilution of secondary antibodies (goat anti-mouse IgG conjugated to horse radish peroxidase; BioRad Laboratories) in 10% milk/TBST was applied to the membrane placed on a platform rocker for one hour. The membrane was subsequently washed with excess amount of TBST. The detection of the protein was performed by using an ECL (Enhanced Chemiluminescence) detection kit (Amersham International).

Western blot analyses were performed to identify binding specificity of each anti-synthetic peptide antibodies. All synthetic polyclonal antibodies showed specificity toward to processed and, when applicable, unprocessed target peptides from protein fractions derived from Photorhabdus culture broth. Various antibodies were shown to recognize either unprocessed or processed recombinant proteins derived from heterologous expression systems such as bacteria or insect cells, using baculovirus expression constructs. In one case, the anti-TcbA iii -syn antibody showed some cross-reactivity to anti-TcdA iii peptide. In a second case, the anti-TcaC-syn antibody, recognized an unidentified 190 kDa peptide in W-14 toxin complex fractions.

Characterization of Photorhabdus Strains

In order to establish that the collection described herein was comprised of Photorhabdus strains, the strains herein were assessed in terms of recognized microbiological traits that are characteristic of the bacterial genus Photorhabdus and which differentiate it from other Enterobacteriaceae and Xenorhabdus spp. (Farmer, J. J. 1984. Bergey's Manual of Systemic Bacteriology, Vol 1. pp. 510-511. (ed. Kreig N. R. and Holt, J. G.). Williams & Wilkins, Baltimore.; Akhurst and Boemare, 1988, J. Gen. Microbiol. 134, 1835-1845; Forst and Nealson, 1996. Microbiol. Rev. 60, 21-43). These characteristic traits are as follows: Gram stain negative rods, organism size of 0.3-2 μm in width and 2-10 μm in length [with occasional filaments (15-50 μm) and spheroplasts], yellow to orange/red colony pigmentation on nutrient agar, presence of crystalline inclusion bodies, presence of catalase, inability to reduce nitrate, presence of bioluminescence, ability to take up dye from growth media, positive for protease production, growth at temperatures below 37° C., survival under anaerobic conditions and positively motile. (Table 33). Test methods were checked using reference Escherichia coli , Xenorhabdus and Photorhabdus strains. The overall results are consistent with all strains being part of the family Enterobacteriaceae and the genus Photorhabdus. Note that DEP1, DEP2, and DEP3 refer to Photorhabdus strains obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA (#29304, 29999 and 51583, respectively).

A luminometer was used to establish the bioluminescence associated with these Photorhabdus strains. To measure the presence or absence of relative light emitting units, the broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (24, 48, 72 hr) and compared to background luminosity (uninoculated media). Several Xenorhabdus strains were tested as negative controls for luminosity. Prior to measuring light emission from the various broths, cell density was established by measuring light absorbance (560 nM) in a Gilford Systems (Oberlin, Ohio) spectrophotometer using a sipper cell. The resulting light emitting units could then be normalized to density of cells. Aliquots of the broths were placed into 96-well microtiter plates (100 μl each) and read in a Packard Lumicount™ luminometer (Packard Instrument Co., Meriden, Conn.). The measurement period for each sample was 0.1 to 1.0 second. The samples were agitated in the luminometer for 10 sec prior to taking readings. A positive test was determined as being about 5-fold background luminescence (about 1-15 relative light units). In addition, degree of colony luminosity was confirmed with photographic film overlays and by eye, after visual adaptation in a darkroom. The Gram's staining characteristics of each strain were established with a commercial Gram's stain kit (BBL, Cockeysville, Md.) used in conjunction with Gram's stain control slides (Fisher Scientific, Pittsburgh, Pa.). Microscopic evaluation was then performed using a Zeiss microscope (Carl Zeiss, Germany) 100×oil immersion objective lens (with 10×ocular and 2×body magnification). Microscopic examination of individual strains for organism size, cellular description and inclusion bodies (the latter two observations after logarithmic growth) was performed using wet mount slides (10×ocular, 2×body and 40×objective magnification) and phase contrast microscopy with a micrometer (Akhurst, R. J. and Boemare, N. E. 1990. Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R. and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA.; Baghdiguian S., Boyer-Giglio M. H., Thaler, J. O., Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185.). Colony pigmentation was observed after inoculation on Bacto nutrient agar, (Difco Laboratories, Detroit, Mich.) prepared as per label instructions. Incubation occurred at 28° C. and descriptions were produced after 5 days. To test for the presence of the enzyme catalase, a colony of the test organism was removed on a small plug from a nutrient agar plate and placed into the bottom of a glass test tube. One ml of a household hydrogen peroxide solution was gently added down the side of the tube. A positive reaction was recorded when bubbles of gas (presumptive oxygen) appeared immediately or within 5 seconds. Controls of uninoculated nutrient agar and hydrogen peroxide solution were also examined. To test for nitrate reduction, each culture was inoculated into 10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, Mich.). After 24 hours incubation with gentle agitation at 28° C., nitrite production was tested by the addition of two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, Mich., 1984). The generation of a distinct pink or red color indicates the formation of nitrite from nitrate whereas the lack of color formation indicates that the strain is nitrate reduction negative. In the latter case, finely powdered zinc was added to further confirm the presence of unreduced nitrate; established by the formation of nitrite and the resultant red color. The ability of each strain to uptake dye from growth media was tested with Bacto MacConkey agar containing the dye neutral red; Bacto Tergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agar containing the dye eosin-Y (formulated agars from Difco Laboratories, Detroit, Mich., all prepared according to label instructions). After inoculation on these media, dye uptake was recorded after incubation at 28° C. for 5 days. Growth on these latter media is characteristic for members of the family Enterobacteriaceae. Motility of each strain was tested using a solution of Bacto Motility Test Medium (Difco Laboratories, Detroit, Mich.) prepared as per label instructions. A butt-stab inoculation was performed with each strain and motility was judged macroscopically by a diffuse zone of growth spreading from the line of inoculum. The production of protease was tested by observing hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, Mich.) made as per label instructions. Cultures were inoculated and the tubes or plates were incubated at 28° C. for 5 days. Gelatin hydrolysis was then checked at room temperature, i.e. less than 22° C. To assess growth at different temperatures, agar plates [2% proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, Mich.) in deionized water] were streaked from a common source of inoculum. Plates were incubated at 20, 28 and 37° C. for up to three weeks. The incubator temperature levels were checked with an electronic thermocouple and meter to insure valid temperature settings. Oxygen requirements for Photorhabdus strains were tested in the following manner. A butt-stab inoculation into fluid thioglycolate broth medium (Difco, Detroit, Mich.) was made. The tubes were incubated at room temperature for one week and cultures were then examined for type and extent of growth. The indicator resazurin demonstrates the presence of medium oxygenation or the aerobiosis zone (Difco Manual, 10th edition, Difco Laboratories, Detroit, Mich.). Growth zone results obtained for the Photorhabdus strains tested were consistent with those of a facultative anaerobic microorganism. In the case of unclear. results, the final agar concentration of fluid thioglycolate broth medium was raised to 0.75% and the growth characteristics rechecked.

The evolutionary diversity of the Photorhabdus strains in our collection was measured by analysis of PCR (Polymerase Chain Reaction) mediated genomic fingerprinting using genomic DNA from each strain. This technique is based on families of repetitive DNA sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic, J., Schneider, M., D E. Bruijn, F. J. and Lupski, J. R. 1994. Methods Mol. Cell. Biol., 5, 25-40). Three of these, repetitive extragenic palindromic sequence (REP), enterobacterial repetitive intergenic consensus (ERIC) and the BOX element are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate these strains (e.g., Louws, F. J., Fulbright, D. W., Stephens, C. T. and D E Bruijn, F. J. 1994. Appl. Environ. Micro. 60, 2286-2295). Rep-PCR utilizes oligonucleotide primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA “fingerprint” for each strain.

To isolate genomic DNA from our strains, cell pellets were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and 12 ml of 5 M NaCl was then added. This mixture was centrifuged 20 min. at 15,000×g. The resulting pellet was resuspended in 5.7 ml of TE and 300 μl of 10% SDS and 60 μl 20 mg/ml proteinase K (Gibco BRL Products, Grand Island, N.Y.) were added. This mixture was incubated at 37° C. for 1 hr, approximately 10 mg of lysozyme was then added and the mixture was incubated for an additional 45 min. One milliliter of 5M NaCl and 800 μl of CTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) were then added and the mixture was incubated 10 min. at 65° C., gently agitated, then incubated and agitated for an additional 20 min. to aid in clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed gently then centrifuged. Two extractions were then performed with an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1). Genomic DNA was precipitated with 0.6 volume of isopropanol. Precipitated DNA was removed with a glass rod, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by optical density at 260 nm. To perform rep-PCR analysis of Photorhabdus genomic DNA the following primers were used, REP1R-I; 5′-IIIICGICGICATCIGGC-3′ and REP2-I; 5′-ICGICTTATCIGGCCTAC-3′. PCR was performed using the following 25 μl reaction: 7.75 μl H 2 O, 2.5 μl 10×LA buffer (PanVera Corp., Madison, Wis.), 16 μl dNTP mix (2.5 mM each), 1 μl of each primer at 50 pM/μl, 1 μl DMSO, 1.5 μl genomic DNA (concentrations ranged from 0.075-0.480 μg/μl) and 0.25 μl TaKaRa EX Taq (PanVera Corp., Madison, Wis.). The PCR amplification was performed in a Perkin Elmer DNA Thermal Cycler (Norwalk, Conn.) using the following conditions: 95° C./7 min. then 35 cycles of; 94° C./1 min.,44° C./1 min., 65° C./8 min., followed by 15 min. at 65° C. After cycling, the 25 μl reaction was added to 5 μl of 6×gel loading buffer (0.25% bromophenol blue, 40% w/v sucrose in H 2 O). A 15×20 cm 1%-agarose gel was then run in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 μl of each reaction. The gel was run for approximately 16 hours at 45 v. Gels were then stained in 20 μg/ml ethidium bromide for 1 hour and destained in TBE buffer for approximately 3 hours. Polaroid® photographs of the gels were then taken under UV illumination.

The presence or absence of bands at specific sizes for each strain was scored from the photographs and entered as a similarity matrix in the numerical taxonomy software program, NTSYS-pc (Exeter Software, Setauket, N.Y.). Controls of E. coli strain HB101 and Xanthomonas oryzae pv. oryzae assayed under the same conditions produced PCR fingerprints corresponding to published reports (Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C. M., Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson, R. J., D E. Bruijn, F. J., Rice, C. and Leach, J. E. 1995. Int. Rice Res. Notes, 20, 23-24.; Vera Cruz, C. M., Ardales, E. Y., Skinner, D. Z., Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T. W. and Leach, J. E. 1996. Phytopathology 86, 1352-1359). The data from Photorhabdus strains were then analyzed with a series of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative data) to generate a matrix of similarity coefficients (using the Jaccard coefficient) and SAHN (Sequential, Agglomerative, Heirarchical and Nested) clustering [using the UPGMA (Unweighted Pair-Group Method with Arithmetic Averages) method] which groups related strains and can be expressed as a phenogram (FIG. 7 ). The COPH (cophenetic values) and MXCOMP (matrix comparison) programs were used to generate a cophenetic value matrix and compare the correlation between this and the original matrix upon which the clustering was based. A resulting normalized Mantel statistic (r) was generated which is a measure of the goodness of fit for a cluster analysis (r=0.8-0.9 represents a very good fit). In our case r=0.924. Therefore, the collection is comprised of a diverse group of easily distinguishable strains representative of the Photorhabdus genus.

Insecticidal Utility of Toxin(s) Produced by Various Photorhabdus Strains

Initial “storage” cultures of the various Photorhabdus strains were produced by inoculating 175 ml of 2% Proteose Peptone #3 (PP3) (Difco Laboratories, Detroit, Mich.) liquid medium with a primary variant colony in a 500 ml tribaffled flask with a Delong neck, covered with a Kaput closure. After inoculation, the flask was incubated for between 24-72 hrs at 28° C. on a rotary shaker at 150 rpm, until stationary phase was reached. The culture was transferred to a sterile bottle containing a sterile magnetic stir bar and the culture was overlayered with sterile mineral oil, to limit exposure to air. The storage culture was kept in the dark, at room temperature. These cultures were then used as inoculum sources for the fermentation of each strain.

“Seed” flasks or cultures were produced by either inoculating 2 mls of an oil overlayered storage culture or by transferring a primary variant colony into 175 ml sterile medium in a 500 ml tribaffled flask covered with a Kaput closure. (The use of other inoculum sources is also possible.) Typically, following 16 hours incubation at 28° C. on a rotary shaker at 150 rpm, the seed culture was transferred into production flasks. Production flasks were usually inoculated by adding about 1% of the actively growing seed culture to sterile 2% PP3 medium (e.g. 2.0 ml per 175 ml sterile medium). Production of broths occurred in 500 ml tribaffled flasks covered with a Kaput. Production flasks were agitated at 28° C. on a rotary shaker at 150 rpm. Production fermentations were terminated after 24-72 hrs although successful fermentation is not confined to this time duration. Following appropriate incubation, the broths were dispensed into sterile 1.0 L polyethylene bottles, spun at 2600×g for 1 hr at 10° C. and decanted from the cell and debris pellet. Further broth clarification was achieved with a tangential flow microfiltration device (Pall Filtron, Northborough, Mass.) using a 0.5 μM open-channel poly-ether sulfone (PES) membrane filter. The resulting broths were then concentrated (up to 10-fold) using a 10,000 or 100,000 MW cut-off membrane, M12 ultra-filtration device (Amicon, Beverly Mass.) or centrifugal concentrators (Millipore, Bedford, Mass. and Pall Filtron, Northborough, Mass.) with a 10,000 or 100,000 MW pore size. In the case of centrifugal concentrators, the broth was spun at 2000×g for approximately 2 hr. The membrane permeate was added to the corresponding retentate to achieve the desired concentration of components greater than the pore size used. Following these procedures, the broth was used for biochemical analysis or filter sterilized using a 0.2 μM cellulose nitrate membrane filter for biological assessment. Heat inactivation of processed broth samples was achieved by heating the samples at 100° C. in a sand-filled heat block for 10 minutes.

The broth(s) and toxin complex(es) from different Photorhabdus strains are useful for reducing populations of insects and were used in a method of inhibiting an insect population which comprises applying to a locus of the insect an effective insect inactivating amount of the active described. A demonstration of the breadth of insecticidal activity observed from broths of a selected group of Photorhabdus strains fermented as described above is shown in Table 36. It is possible that improved or additional insecticidal activities could be detected with these strains through increased concentration of the broth or by employing different fermentation methods. Consistent with the activity being associated with a protein, the insecticidal activity of all strains tested was heat labile.

Culture broth(s) from diverse Photorhabdus strains show differential insecticidal activity (mortality and/or growth inhibition) against a number of insects. More specifically, the activity is seen against corn rootworm which is a member of the insect order Coleoptera. Other members of the Coleoptera include boll weevils, wireworms, pollen beetles, flea beetles, seed beetles and Colorado potato beetle. The broths and purified toxin complex(es) are also active against tobacco budworm, tobacco hornworm and European corn borer which are members of the order Lepidoptera. Other typical members of this order are beet armyworm, cabbage looper, black cutworm, corn earworm, codling moth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent caterpillar, sod webworm and fall armyworm. Activity is also observed against German cockroach which is a member of the order Dictyoptera (or Blattodea). Other members of this order are oriental cockroach and American cockroach.

Activity against corn rootworm larvae was tested as follows. Photorhabdus culture broth(s) (10 fold concentrated, filter sterilized), 2% Proteose Peptone #3 (10 fold concentrated), purified toxin complex(es), 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) of artificial diet (Rose, R. I. and McCabe, J. M. 1973. J. Econ. Entomol. 66, 398-400) in 40 μl aliquots. Toxin complex was diluted in 10 mM sodium phosphate buffer, pH 7.0. The diet plates were allowed to air-dry in a sterile flow-hood and the wells were infested with single, neonate Diabrotica undecimpunctata howardi (Southern corn rootworm, SCR) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber and maintained at 27° C. for the appropriate period (3-5 days). Mortality and larval weight determinations were then scored. Generally, 16 insects per treatment were used in all studies. Control mortality was generally less than 5%.

Activity against lepidopteran larvae was tested as follows. Concentrated (10-fold) Photorhabdus culture broth(s), control medium (2% Proteose Peptone #3), purified toxin complex(es), 10 mM sodium phosphate buffer, pH 7.0 were applied directly to the surface (about 1.5 cm 2 ) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 μl aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, neonate larva. European corn borer ( Ostrinia nubilalis ) and tobacco hornworm ( Manduca sexta ) eggs were obtained from commercial sources and hatched in-house, whereas tobacco budworm ( Heliothis virescens ) larvae were supplied internally. Following infestation with larvae, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27° C. for the appropriate period. Mortality and weight determinations were scored at day 5. Generally, 16 insects per treatment were used in all studies. Control mortality generally ranged from about 0 to about 12.5% for control medium and was less than 10% for phosphate buffer.

Activity against cockroach was tested as follows. Concentrated (10-fold) Photorhabdus culture broth(s) and control medium (2% Proteose Peptone #3) were applied directly to the surface (about 1.5 cm 2 ) of standard artificial lepidopteran diet (Stoneville Yellow diet) in 40 μl aliquots. The diet plates were allowed to air-dry in a sterile flow-hood and each well was infested with a single, CO 2 anesthetized first instar German cockroach ( Blatella germanica ). Following infestation, the diet plates were sealed, placed in a humidified growth chamber and maintained in the dark at 27° C. for the appropriate period. Mortality and weight determinations were scored at day 5. Control mortality less than 10%.

Southern Analysis of Non-W-14 Photorhabdus Strains Using W-14 Gene Probes

Photorhabdus strains were grown on 2% proteose peptone #3 agar (Difco Laboratories, Detroit, Mich.) and insecticidal toxin competence was maintained by repeated bioassay after passage. A 50 ml shake culture was produced in 175 ml baffled flasks in 2% proteose peptone #3 medium, grown at 28° and 150 rpm for approximately 24 hours. Fifteen ml of this culture were centrifuged (700×g, 30 min) and frozen in its medium at −20° until it was thawed (slowly in ice water) for DNA isolation. The thawed W-14 culture was centrifuged (900×g, 15 min 4°), and the floating orange mucopolysaccharide material was removed. The remaining cell material was centrifuged (25,000×g, 4°) to pellet the bacterial cells, and the medium was removed and discarded.

Total DNA was isolated by an adaptation of the CTAB method described in section 2.4.1 of Ausubel et al. (1994). The modifications included a high salt shock, and all volumes were increased ten-fold over the “miniprep” recommended volumes. All centrifugations were at 4° C. unless otherwise specified. The pelleted bacterial cells were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) to a final volume of 10 ml, then 12 ml 5 M NaCl were added; this mixture was centrifuged 20 min at 15,000×g. The pellet was resuspended in 5.7 ml TE, and 300 μl of 10% SDS and 60 μl of 20 mg/ml proteinase K (in sterile distilled water, Gibco BRL Products, Grand Island, N.Y.) were added to the suspension. The mixture was incubated at 37° C. for 1 hr; then approximately 10 mg lysozyme (Worthington Biochemical Corp., Freehold, N.J.) were added. After an additional 45 min incubation, 1 ml of 5 M NaCl and 800 Al of CTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) were added. This preparation was incubated 10 min at 65° C., then gently agitated and further incubated and agitated for approximately 20 min to assist clearing of the cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:1, v:v) was added, mixed very gently, and the phases separated by centrifugation at 12,000×g for 15 min. The upper (aqueous) phase was gently removed with a wide-bore pipette and extracted twice as above with an equal volume of PCI (phenol/choloroform/isoamyl alcohol; 50:49:1, v:v:v; equilibrated with 1M Tris-HCl, pH 8.0; Intermountain Scientific Corporation, Kaysville, Utah). The DNA precipitated with 0.6 volume of isopropanol was gently removed on a glass rod, washed twice with 70% ethanol, dried, and dissolved in 2 ml STE (10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 8). This preparation contained 2.5 mg/ml DNA, as determined by optical density at 260 nm.

Identification of Bgl II/Hind III Fragments Hybridizing to tc-gene Specific Probes

Approximately 10 μg of genomic DNA was digested to completion with about 30 units each of Bgl II and Hind III (NEB) for 180 min, frozen overnight, then heated at 65° C. for five min, and electrophoresed in a 0.8% agarose gel (Seakem® LE, 1×TEA, 80 volts, 90 min). The DNA was stained with ethidium bromide (50 μg/ml) as described earlier, and photographed under ultraviolet light. The DNA fragments in the agarose gel were subjected to depurination (5 min in 0.2 M HCl), denaturation (15 min in 0.5 M NaOH, 1.5 M NaCl), and neutralization (15 min in 0.5 M Tris HCl pH 8.0, 1.5 M NaCl), with 3 rinses of distilled water between each step. The DNA was transferred by Southern blotting from the gel onto a NYTRAN nylon membrane (Amersham, Arlington Heights, Ill.) using a high salt (20×SSC) protocol, as described in section 2.9 of Ausubel et al. (CPMB, op. cit.). The transferred DNA was then UV-crosslinked to the nylon membrane using a Stratagene UV Stratalinker set on auto crosslink. The membranes were stored dry at 25° C. until use.

Hybidization was performed using the ECL™direct (Amersham, Arlington Heights, Ill.) labeling and detection system following protocols provided by the manufacturer. In brief, probes were prepared by covalently linking the denatured DNA to the enzyme horseradish peroxidase. Once labeled the probe was used under hybridization conditions which maintain the enzymatic activity. Unhybridized probe was removed by two gentle washes 20 minutes each at 42° C. in 0.5×SSC, 0.4% SDS, and 6M Urea. This was followed by two washes 5 minutes each at room temperature in 2×SSC. As directed by the manufacturer, ECL™ reagents were used to detect the hybridizing DNA bands. There are several factors which influence the ability to detect gene relatedness between various Photorhabdus strains and strain W-14. First, high stringency conditions have not been employed in these hybridizations. It is known in the art that varying the stringency of hybridization and wash conditions will influence the pattern and intensity of hybridizing bands. Second, Southern blots' blot to blot variation will influence the mobility of hybridizing bands and molecular weight estamates. Therefore, W-14 was included as a standard on all Southern blots.

Gene specific probes derived from the W-14 toxin genes were used in these hybridizations. The following lists the specific coordinates within each gene sequence to which the probe corresponds. A probe specific for tcaB i /B ii : 1174 to 3642 of Sequence ID #25, a probe specific for tcaC: 3637 to 6005 of Sequence ID #25, a probe specific for tcbA: 2097 to 4964 of Sequence ID #11, and a probe specific for tcda: 1660 to 4191 of sequence ID #46. The following tables summarize Southern Blot analyses of Photorhabdus strains. In the event that hybridization of probes occurred, the hybridized fragment(s) were noted as either identical or different from the pattern observed for the W-14 strain.

From these analyses it is apparent that homologs of W-14 genes are dispersed throughout these diverse Photorhabdus strains, as evidenced by differences in gene fragment sizes between W-14 and the other strains.

N-Terminal Amino Acid Sequences of Toxin Complex Peptides from Different Photorhabdus Strains

The relationship of peptides isolated from different Photorhabdus strains, as described in Example 14, were subjected to N-terminal amino acid sequencing. The N-terminal amino acid sequences of toxin peptides in several strains were compared to W-14 toxin peptides. In Table 40, a comparison of toxin peptides compared to date showed that identical or homologous (at least 40% similarity to W14 gene/peptides) toxin peptides were present in all of the strains. For example, the N-terminal amino acid sequence of TcaC, SEQ ID NO: 2, was found to be identical to that for 160 kDa peptide in HP88 but also homologs were present in strains WIR, H9, Hb, WX-1, and Hm. Some W-14 peptides or homologs have not been observed in other strains; however, not all peptides have been sequenced for toxin complexes from other strains due to N-terminal blockage or low abundance. In addition, many other N-terminal amino acid sequences (SEQ ID NOS: 82 to 88) have been obtained for toxin complex peptides from other strains that have no similarity to peptides from W-14 and in some case were identical to each other. For example, an identical amino acid sequence, SEQ ID NO: 82, was obtained for 64 kDa peptide present in both HP88 and Hb strains and a homologous sequence for a 70 kDa peptide in NC-1 strain (SEQ ID NO: 83).

Immunological Analysis of Photorhabdus Strains

Culture broths of Photorhabdus strains were concentrated 10 to 15 times using Centriprep-10 ultrafiltration device (amicon, Inc. Beverly, Mass. 01915). The concentration of the protein ranges from 0.3 to 3.0 mg per ml. Ten to 20 μg of total protein was loaded in each well of a precast 4-20% polyacrylamide gel (Integrated Separation Systems, Natick, Mass. 01760). Gel electrophoresis was performed for 1.25 hours using a constant current set at 25 ma per gel. The gel was electro-blotted on to Hybond-ECL™ nitrocellulose membrane (Amersham Corporation, Arlington Hts, Ill. 60005) using a semi-dry electro-blotter (Pharmacia Biotech Inc., Piscataway , N.J. 08854). A constant current was applied at 0.75 ma per cm for 2.5 hours. The membrane was blocked with 10% milk in TBST (25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for one hour at room temperature. Each primary antibody was diluted in 10% milk/TBST to 1:500. Other dilution between 1:50 to 1:1000 was also used. The membrane was incubated in primary antibody for at least one hour. Then it was washed thoroughly with the blocking solution or TBST. A 1:2000 dilution of secondary antibodies (goat anti-mouse IgG or goat anti rabbit TgG conjugated to horseradish peroxidase; BioRad Laboratories, Hercules, Calif. 94547) in 10% milk/TBST was applied to the membrane which was placed on a platform rocker for one hour. The membrane was subsequently washed with excess amount of TBST. The detection of the protein was performed by using an ECL (Enhanced Chemiluminescence) detection kit (Amersham International).

A panel of peptide specific-antibodies generated against W-14 peptides were used to characterize the protein composition of broths from nine non-W-14 Photorhabdus strains using Western blot analysis. In addition, one monoclonal antibody (MAb-C5F2) which recognizes TcbA iii protein in W-14-derived toxin complex was used. The results (Table 39) showed cross recognition of the antibodies to some of the proteins in these broths. In some cases, the proteins that were recognized by the antibodies were the same size as the W-14 target peptides. In other cases, the proteins that were recognized by the antibodies were smaller than the W-14 target peptides. This data indicate that some of the non-W-14 Photorhabdus strains may produce similar proteins to the W-14 strain. The difference could be due to deletion or protein processing or degradation process. Some of the strains did not contain protein(s) that could be recognized by some antibodies, however, it is possible that the concentration is significantly lower than those observed for W-14 peptides. When compared for various toxin peptide homologs these results showed peptide diversity among the Photorhabdus strains.

Additional non-W-14 Photorhabdus strains were characterized by Western blot analysis using the culture broth and/or partial purified protein fractions as antigen. The panel of antibodies include MAb-C5F2, MAb-DE1 (recognizing TcdA ii ), Pab-DE2 (recognizing TcaB), Pab-TcbA ii -syn, Pab-TcaC-syn, Pab TcaB ii -syn, Pab-TcbA iii -syn, Pab-TcaB i -syn. These antibodies showed cross-reactivity with proteins in the broth and in the partial purified fractions of non-W-14 strains.

The data indicate that antibodies could be used to identify proteins in the broth as well as in the partially purified protein fractions.

Bacterial Expression of the tcdA Coding Region

Engineering of the tcdA Gene for Bacterial Expression

The 5′ and 3′ ends of the tcdA coding region (SEQ ID NO:46) were modified to add useful cloning sites for inserting the segment into heterologous expression vectors. The ends were modified using unique primers in Polymerase Chain Reactions (PCR), performed essentially as described in Example 8. Primer sets, as described below, were used in conjunction with cosmid 21D2.4 as template, to created products with the appropriately modified ends.

The first primer set was used to modify the 5′ end of the gene, to insert a unique Nco I site at the initiator codon using the forward primer A0F1 (5′ GAT CGA TCG ATC CAT GGC CAA CGA GTC TGT AAA AGA GAT ACC TGA TG TAT TAA AAA GCC AGT GTG 3′) and to add unique Bgl II, Sal I and Not I sites to facilitate insertion of the remainder of the gene using the reverse primer A0R1 (5′ GAT CGA TCG TAC GCG GCC GCT CGA TCG ATC GTC GAC CCA TTG ATT TGA GAT CTG GGC GGC GGG TAT CCA GAT AAT AAA CGG AGT CAC 3′).

Another PCR reaction was designed to modify the 3′ end of the gene by adding an additional stop codon and convenient restriction sites for cloning. The forward primer A0F2 (5′ ACT GGC TGC GTG GTC GAC TGG CGG CGA TTT ACT 3′) was used to amplify across a unique Sal I site in the gene, later used to clone the modified 3′ end. The reverse primer A0R2 (5′ CGA TGC ATG CTG CGG CCG CAG GCC TTC CTC GAG TCA TTA TTT AAT GGT GTA GCG AAT ATG CAA AAT 3′) was used to insert a second stop codon (TGA) and cloning sites Xho I, Stu I and Not I. Bacterial expression vector pET27b (Novagen, Madison, Wis.), was modified to delete the Bgl II site at position 446, according to standard molecular biology techniques.

The 497 bp PCR product from the first amplification reaction (AOF1+AOR1), to modify the 5′ end of the gene, was ligated to the modified pET27b vector according to the supplier's instructions. The DNA sequences of the amplified portion of three isolates were determined using the supplier's recommended primers and the sequencing methods described previously. The sequence of all isolates was the same.

One isolate was then used as a cloning vector to insert the middle portion of the tcdA gene on a 6341 bp Bgl II to Sal I fragment. The resulting clone was called MC4 and contained all but the 3′ most portion of the tcdA coding sequence. Finally, to complete the full-length coding region, the 832 bp PCR product from the second PCR amplification (AOF2+AOR2), to modify the 3′ end of the gene, was ligated to isolate MC4 on a Sal I to Not I fragment, according to standard molecular biology techniques. The tcda coding region was sequenced and found to be complete, the resulting plasmid is called pDAB2035.

Construction of Plasmids pDAB2036, pDAB2037 and pDAB2038 for Bacterial Expression of tcdA

The tcdA coding region was cut from plasmid pDAB2035 with restriction enzymes Nco I and Xho I and gel purified. The fragment was ligated into the Nco I and Xho I sites of the expression vector pET15 to create plasmid pDAB2036. Additionally, pDAB2035 was cut with Nco I and Not I to release the tcdA coding region which was ligated into the Nco I and Not I sites of the expression vector pET28b to create plasmid pDAB2037. Finally, plasmid pDAB2035 was cut with Nco I and Stu I to release the tcdA coding region. This fragment was ligated into the expression vector Trc99a which was cut with Hind III followed by treatment with T4 DNA polymerase to blunt the ends. The vector was then cut with Nco I and ligated with the Nco I/Stu I cut tcdA fragment. The resulting plasmid is called pDAB2038.

Expression of tcdA from Plasmid pDAB2038

Plasmid pDAB2038 was transformed into BL21 cells and expressed as described above for plasmid pDAB2033 in Example 19.

Purification of tcdA from E. coli

The expression culture was centrifuged at 10,300 g for 30 min and the supernatant was collected. It was diluted with two volumes of H 2 O and applied at a flow rate of 7.5 ml/min to a poros 50 HQ (Perspective Systems, Mass.) column (1.6 cm×10 cm) which was pre-equilibrated with 10 mM sodium phosphate buffer, pH 7.0 (Buffer A). The column was washed with Buffer A until the optical density at 280 nm returned to baseline level. The proteins bound to the column were then eluted with 1M NaCl in Buffer A.

The fraction was loaded in 20 ml aliquots onto a gel filtration column, Sepharose CL-4B (2.6×100 cm), which was equilibrated with Buffer A. The protein was eluted in Buffer A at a flow rate of 0.75 mL/min. Fractions with a retention time between 260 minutes and 460 minutes were pooled and applied at 1 mL/min to a Mono Q 5/5 column which was equilibrated with 20 mM Tris-HCl, pH 7.0 (Buffer B). The column was washed with Buffer B until the optical density at 280 nm returned to baseline level. The proteins bound to the column were eluted with a linear gradient of 0 to 1 M NaCl in Buffer B at 1 mL/min for 30 min. One milliliter fractions were collected, serial diluted, and subjected to SCR bioassay. Fractions eluted out between 0.1 and 0.3 M NaCl were found to have the highest insecticidal activity. Western analysis of the active fractions using pAb TcdA ii -syn antibody and pAb Tcd iii -syn antibody indicated the presence of peptides TcdA ii and TcdA iii .

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn

1 5 10

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Trp

1 5 10

Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala

1 5 10 15

Leu Val Ala

Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn

1 5 10

Ala Gly Asp Thr Ala Asn Ile Gly Asp

1 5

Leu Gly Gly Ala Ala Thr Leu Leu Asp Leu Leu Leu Pro Gln Ile

1 5 10 15

Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu

1 5 10

Met Asn Leu Ala Ser Pro Leu Ile Ser

1 5

Met Ile Asn Leu Asp Ile Asn Glu Gln Asn Lys Ile Met Val Val Ser

1 5 10 15

Ala Ala Lys Asp Val Lys Phe Gly Ser Asp Ala Arg Val Lys Met Leu

1 5 10 15

Arg Gly Val Asn

20

ATG CAA AAC TCA TTA TCA AGC ACT ATC GAT ACT ATT TGT CAG AAA CTG 48

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu

1 5 10 15

CAA TTA ACT TGT CCG GCG GAA ATT GCT TTG TAT CCC TTT GAT ACT TTC 96

Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe

20 25 30

CGG GAA AAA ACT CGG GGA ATG GTT AAT TGG GGG GAA GCA AAA CGG ATT 144

Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile

35 40 45

TAT GAA ATT GCA CAA GCG GAA CAG GAT AGA AAC CTA CTT CAT GAA AAA 192

Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys

50 55 60

CGT ATT TTT GCC TAT GCT AAT CCG CTG CTG AAA AAC GCT GTT CGG TTG 240

Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu

65 70 75 80

GGT ACC CGG CAA ATG TTG GGT TTT ATA CAA GGT TAT AGT GAT CTG TTT 288

Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe

85 90 95

GGT AAT CGT GCT GAT AAC TAT GCC GCG CCG GGC TCG GTT GCA TCG ATG 336

Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met

100 105 110

TTC TCA CCG GCG GCT TAT TTG ACG GAA TTG TAC CGT GAA GCC AAA AAC 384

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn

115 120 125

TTG CAT GAC AGC AGC TCA ATT TAT TAC CTA GAT AAA CGT CGC CCG GAT 432

Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp

130 135 140

TTA GCA AGC TTA ATG CTC AGC CAG AAA AAT ATG GAT GAG GAA ATT TCA 480

Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser

145 150 155 160

ACG CTG GCT CTC TCT AAT GAA TTG TGC CTT GCC GGG ATC GAA ACA AAA 528

Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys

165 170 175

ACA GGA AAA TCA CAA GAT GAA GTG ATG GAT ATG TTG TCA ACT TAT CGT 576

Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg

180 185 190

TTA AGT GGA GAG ACA CCT TAT CAT CAC GCT TAT GAA ACT GTT CGT GAA 624

Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu

195 200 205

ATC GTT CAT GAA CGT GAT CCA GGA TTT CGT CAT TTG TCA CAG GCA CCC 672

Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro

210 215 220

ATT GTT GCT GCT AAG CTC GAT CCT GTG ACT TTG TTG GGT ATT AGC TCC 720

Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser

225 230 235 240

CAT ATT TCG CCA GAA CTG TAT AAC TTG CTG ATT GAG GAG ATC CCG GAA 768

His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu

245 250 255

AAA GAT GAA GCC GCG CTT GAT ACG CTT TAT AAA ACA AAC TTT GGC GAT 816

Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp

260 265 270

ATT ACT ACT GCT CAG TTA ATG TCC CCA AGT TAT CTG GCC CGG TAT TAT 864

Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr

275 280 285

GGC GTC TCA CCG GAA GAT ATT GCC TAC GTG ACG ACT TCA TTA TCA CAT 912

Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His

290 295 300

GTT GGA TAT AGC AGT GAT ATT CTG GTT ATT CCG TTG GTC GAT GGT GTG 960

Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val

305 310 315 320

GGT AAG ATG GAA GTA GTT CGT GTT ACC CGA ACA CCA TCG GAT AAT TAT 1008

Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr

325 330 335

ACC AGT CAG ACG AAT TAT ATT GAG CTG TAT CCA CAG GGT GGC GAC AAT 1056

Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn

340 345 350

TAT TTG ATC AAA TAC AAT CTA AGC AAT AGT TTT GGT TTG GAT GAT TTT 1104

Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe

355 360 365

TAT CTG CAA TAT AAA GAT GGT TCC GCT GAT TGG ACT GAG ATT GCC CAT 1152

Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His

370 375 380

AAT CCC TAT CCT GAT ATG GTC ATA AAT CAA AAG TAT GAA TCA CAG GCG 1200

Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala

385 390 395 400

ACA ATC AAA CGT AGT GAC TCT GAC AAT ATA CTC AGT ATA GGG TTA CAA 1248

Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln

405 410 415

AGA TGG CAT AGC GGT AGT TAT AAT TTT GCC GCC GCC AAT TTT AAA ATT 1296

Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile

420 425 430

GAC CAA TAC TCC CCG AAA GCT TTC CTG CTT AAA ATG AAT AAG GCT ATT 1344

Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile

435 440 445

CGG TTG CTC AAA GCT ACC GGC CTC TCT TTT GCT ACG TTG GAG CGT ATT 1392

Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile

450 455 460

GTT GAT AGT GTT AAT AGC ACC AAA TCC ATC ACG GTT GAG GTA TTA AAC 1440

Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn

465 470 475 480

AAG GTT TAT CGG GTA AAA TTC TAT ATT GAT CGT TAT GGC ATC AGT GAA 1488

Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu

485 490 495

GAG ACA GCC GCT ATT TTG GCT AAT ATT AAT ATC TCT CAG CAA GCT GTT 1536

Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val

500 505 510

GGC AAT CAG CTT AGC CAG TTT GAG CAA CTA TTT AAT CAC CCG CCG CTC 1584

Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu

515 520 525

AAT GGT ATT CGC TAT GAA ATC AGT GAG GAC AAC TCC AAA CAT CTT CCT 1632

Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro

530 535 540

AAT CCT GAT CTG AAC CTT AAA CCA GAC AGT ACC GGT GAT GAT CAA CGC 1680

Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg

545 550 555 560

AAG GCG GTT TTA AAA CGC GCG TTT CAG GTT AAC GCC AGT GAG TTG TAT 1728

Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr

565 570 575

CAG ATG TTA TTG ATC ACT GAT CGT AAA GAA GAC GGT GTT ATC AAA AAT 1776

Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn

580 585 590

AAC TTA GAG AAT TTG TCT GAT CTG TAT TTG GTT AGT TTG CTG GCC CAG 1824

Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln

595 600 605

ATT CAT AAC CTG ACT ATT GCT GAA TTG AAC ATT TTG TTG GTG ATT TGT 1872

Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys

610 615 620

GGC TAT GGC GAC ACC AAC ATT TAT CAG ATT ACC GAC GAT AAT TTA GCC 1920

Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala

625 630 635 640

AAA ATA GTG GAA ACA TTG TTG TGG ATC ACT CAA TGG TTG AAG ACC CAA 1968

Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln

645 650 655

AAA TGG ACA GTT ACC GAC CTG TTT CTG ATG ACC ACG GCC ACT TAC AGC 2016

Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser

660 665 670

ACC ACT TTA ACG CCA GAA ATT AGC AAT CTG ACG GCT ACG TTG TCT TCA 2064

Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser

675 680 685

ACT TTG CAT GGC AAA GAG AGT CTG ATT GGG GAA GAT CTG AAA AGA GCA 2112

Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala

690 695 700

ATG GCG CCT TGC TTC ACT TCG GCT TTG CAT TTG ACT TCT CAA GAA GTT 2160

Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val

705 710 715 720

GCG TAT GAC CTG CTG TTG TGG ATA GAC CAG ATT CAA CCG GCA CAA ATA 2208

Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile

725 730 735

ACT GTT GAT GGG TTT TGG GAA GAA GTG CAA ACA ACA CCA ACC AGC TTG 2256

Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu

740 745 750

AAG GTG ATT ACC TTT GCT CAG GTG CTG GCA CAA TTG AGC CTG ATC TAT 2304

Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr

755 760 765

CGT CGT ATT GGG TTA AGT GAA ACG GAA CTG TCA CTG ATC GTG ACT CAA 2352

Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln

770 775 780

TCT TCT CTG CTA GTG GCA GGC AAA AGC ATA CTG GAT CAC GGT CTG TTA 2400

Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu

785 790 795 800

ACC CTG ATG GCC TTG GAA GGT TTT CAT ACC TGG GTT AAT GGC TTG GGG 2448

Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly

805 810 815

CAA CAT GCC TCC TTG ATA TTG GCG GCG TTG AAA GAC GGA GCC TTG ACA 2496

Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr

820 825 830

GTT ACC GAT GTA GCA CAA GCT ATG AAT AAG GAG GAA TCT CTC CTA CAA 2544

Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln

835 840 845

ATG GCA GCT AAT CAG GTG GAG AAG GAT CTA ACA AAA CTG ACC AGT TGG 2592

Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp

850 855 860

ACA CAG ATT GAC GCT ATT CTG CAA TGG TTA CAG ATG TCT TCG GCC TTG 2640

Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu

865 870 875 880

GCG GTT TCT CCA CTG GAT CTG GCA GGG ATG ATG GCC CTG AAA TAT GGG 2688

Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly

885 890 895

ATA GAT CAT AAC TAT GCT GCC TGG CAA GCT GCG GCG GCT GCG CTG ATG 2736

Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met

900 905 910

GCT GAT CAT GCT AAT CAG GCA CAG AAA AAA CTG GAT GAG ACG TTC AGT 2784

Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser

915 920 925

AAG GCA TTA TGT AAC TAT TAT ATT AAT GCT GTT GTC GAT AGT GCT GCT 2832

Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala

930 935 940

GGA GTA CGT GAT CGT AAC GGT TTA TAT ACC TAT TTG CTG ATT GAT AAT 2880

Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn

945 950 955 960

CAG GTT TCT GCC GAT GTG ATC ACT TCA CGT ATT GCA GAA GCT ATC GCC 2928

Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala

965 970 975

GGT ATT CAA CTG TAC GTT AAC CGG GCT TTA AAC CGA GAT GAA GGT CAG 2976

Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln

980 985 990

CTT GCA TCG GAC GTT AGT ACC CGT CAG TTC TTC ACT GAC TGG GAA CGT 3024

Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg

995 1000 1005

TAC AAT AAA CGT TAC AGT ACT TGG GCT GGT GTC TCT GAA CTG GTC TAT 3072

Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr

1010 1015 1020

TAT CCA GAA AAC TAT GTT GAT CCC ACT CAG CGC ATT GGG CAA ACC AAA 3120

Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys

1025 1030 1035 1040

ATG ATG GAT GCG CTG TTG CAA TCC ATC AAC CAG AGC CAG CTA AAT GCG 3168

Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala

1045 1050 1055

GAT ACG GTG GAA GAT GCT TTC AAA ACT TAT TTG ACC AGC TTT GAG CAG 3216

Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln

1060 1065 1070

GTA GCA AAT CTG AAA GTA ATT AGT GCT TAC CAC GAT AAT GTG AAT GTG 3264

Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val

1075 1080 1085

GAT CAA GGA TTA ACT TAT TTT ATC GGT ATC GAC CAA GCA GCT CCG GGT 3312

Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly

1090 1095 1100

ACG TAT TAC TGG CGT AGT GTT GAT CAC AGC AAA TGT GAA AAT GGC AAG 3360

Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys

1105 1110 1115 1120

TTT GCC GCT AAT GCT TGG GGT GAG TGG AAT AAA ATT ACC TGT GCT GTC 3408

Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val

1125 1130 1135

AAT CCT TGG AAA AAT ATC ATC CGT CCG GTT GTT TAT ATG TCC CGC TTA 3456

Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu

1140 1145 1150

TAT CTG CTA TGG CTG GAG CAG CAA TCA AAG AAA AGT GAT GAT GGT AAA 3504

Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys

1155 1160 1165

ACC ACG ATT TAT CAA TAT AAC TTA AAA CTG GCT CAT ATT CGT TAC GAC 3552

Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp

1170 1175 1180

GGT AGT TGG AAT ACA CCA TTT ACT TTT GAT GTG ACA GAA AAG GTA AAA 3600

Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys

1185 1190 1195 1200

AAT TAC ACG TCG AGT ACT GAT GCT GCT GAA TCT TTA GGG TTG TAT TGT 3648

Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys

1205 1210 1215

ACT GGT TAT CAA GGG GAA GAC ACT CTA TTA GTT ATG TTC TAT TCG ATG 3696

Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met

1220 1225 1230

CAG AGT AGT TAT AGC TCC TAT ACC GAT AAT AAT GCG CCG GTC ACT GGG 3744

Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly

1235 1240 1245

CTA TAT ATT TTC GCT GAT ATG TCA TCA GAC AAT ATG ACG AAT GCA CAA 3792

Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln

1250 1255 1260

GCA ACT AAC TAT TGG AAT AAC AGT TAT CCG CAA TTT GAT ACT GTG ATG 3840

Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met

1265 1270 1275 1280

GCA GAT CCG GAT AGC GAC AAT AAA AAA GTC ATA ACC AGA AGA GTT AAT 3888

Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn

1285 1290 1295

AAC CGT TAT GCG GAG GAT TAT GAA ATT CCT TCC TCT GTG ACA AGT AAC 3936

Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn

1300 1305 1310

AGT AAT TAT TCT TGG GGT GAT CAC AGT TTA ACC ATG CTT TAT GGT GGT 3984

Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly

1315 1320 1325

AGT GTT CCT AAT ATT ACT TTT GAA TCG GCG GCA GAA GAT TTA AGG CTA 4032

Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu

1330 1335 1340

TCT ACC AAT ATG GCA TTG AGT ATT ATT CAT AAT GGA TAT GCG GGA ACC 4080

Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr

1345 1350 1355 1360

CGC CGT ATA CAA TGT AAT CTT ATG AAA CAA TAC GCT TCA TTA GGT GAT 4128

Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp

1365 1370 1375

AAA TTT ATA ATT TAT GAT TCA TCA TTT GAT GAT GCA AAC CGT TTT AAT 4176

Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn

1380 1385 1390

CTG GTG CCA TTG TTT AAA TTC GGA AAA GAC GAG AAC TCA GAT GAT AGT 4224

Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser

1395 1400 1405

ATT TGT ATA TAT AAT GAA AAC CCT TCC TCT GAA GAT AAG AAG TGG TAT 4272

Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr

1410 1415 1420

TTT TCT TCG AAA GAT GAC AAT AAA ACA GCG GAT TAT AAT GGT GGA ACT 4320

Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr

1425 1430 1435 1440

CAA TGT ATA GAT GCT GGA ACC AGT AAC AAA GAT TTT TAT TAT AAT CTC 4368

Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu

1445 1450 1455

CAG GAG ATT GAA GTA ATT AGT GTT ACT GGT GGG TAT TGG TCG AGT TAT 4416

Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr

1460 1465 1470

AAA ATA TCC AAC CCG ATT AAT ATC AAT ACG GGC ATT GAT AGT GCT AAA 4464

Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1475 1480 1485

GTA AAA GTC ACC GTA AAA GCG GGT GGT GAC GAT CAA ATC TTT ACT GCT 4512

Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala

1490 1495 1500

GAT AAT AGT ACC TAT GTT CCT CAG CAA CCG GCA CCC AGT TTT GAG GAG 4560

Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu

1505 1510 1515 1520

ATG ATT TAT CAG TTC AAT AAC CTG ACA ATA GAT TGT AAG AAT TTA AAT 4608

Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn

1525 1530 1535

TTC ATC GAC AAT CAG GCA CAT ATT GAG ATT GAT TTC ACC GCT ACG GCA 4656

Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala

1540 1545 1550

CAA GAT GGC CGA TTC TTG GGT GCA GAA ACT TTT ATT ATC CCG GTA ACT 4704

Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr

1555 1560 1565

AAA AAA GTT CTC GGT ACT GAG AAC GTG ATT GCG TTA TAT AGC GAA AAT 4752

Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn

1570 1575 1580

AAC GGT GTT CAA TAT ATG CAA ATT GGC GCA TAT CGT ACC CGT TTG AAT 4800

Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn

1585 1590 1595 1600

ACG TTA TTC GCT CAA CAG TTG GTT AGC CGT GCT AAT CGT GGC ATT GAT 4848

Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp

1605 1610 1615

GCA GTG CTC AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAA TTA GGA 4896

Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly

1620 1625 1630

GCG GGC ACA TAT GTG CAG CTT GTG TTG GAT AAA TAT GAT GAG TCT ATT 4944

Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile

1635 1640 1645

CAT GGC ACT AAT AAA AGC TTT GCT ATT GAA TAT GTT GAT ATA TTT AAA 4992

His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys

1650 1655 1660

GAG AAC GAT AGT TTT GTG ATT TAT CAA GGA GAA CTT AGC GAA ACA AGT 5040

Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser

1665 1670 1675 1680

CAA ACT GTT GTG AAA GTT TTC TTA TCC TAT TTT ATA GAG GCG ACT GGA 5088

Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly

1685 1690 1695

AAT AAG AAC CAC TTA TGG GTA CGT GCT AAA TAC CAA AAG GAA ACG ACT 5136

Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr

1700 1705 1710

GAT AAG ATC TTG TTC GAC CGT ACT GAT GAG AAA GAT CCG CAC GGT TGG 5184

Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp

1715 1720 1725

TTT CTC AGC GAC GAT CAC AAG ACC TTT AGT GGT CTC TCT TCC GCA CAG 5232

Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln

1730 1735 1740

GCA TTA AAG AAC GAC AGT GAA CCG ATG GAT TTC TCT GGC GCC AAT GCT 5280

Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala

1745 1750 1755 1760

CTC TAT TTC TGG GAA CTG TTC TAT TAC ACG CCG ATG ATG ATG GCT CAT 5328

Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His

1765 1770 1775

CGT TTG TTG CAG GAA CAG AAT TTT GAT GCG GCG AAC CAT TGG TTC CGT 5376

Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg

1780 1785 1790

TAT GTC TGG AGT CCA TCC GGT TAT ATC GTT GAT GGT AAA ATT GCT ATC 5424

Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile

1795 1800 1805

TAC CAC TGG AAC GTG CGA CCG CTG GAA GAA GAC ACC AGT TGG AAT GCA 5472

Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala

1810 1815 1820

CAA CAA CTG GAC TCC ACC GAT CCA GAT GCT GTA GCC CAA GAT GAT CCG 5520

Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro

1825 1830 1835 1840

ATG CAC TAC AAG GTG GCT ACC TTT ATG GCG ACG TTG GAT CTG CTA ATG 5568

Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met

1845 1850 1855

GCC CGT GGT GAT GCT GCT TAC CGC CAG TTA GAG CGT GAT ACG TTG GCT 5616

Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala

1860 1865 1870

GAA GCT AAA ATG TGG TAT ACA CAG GCG CTT AAT CTG TTG GGT GAT GAG 5664

Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu

1875 1880 1885

CCA CAA GTG ATG CTG AGT ACG ACT TGG GCT AAT CCA ACA TTG GGT AAT 5712

Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn

1890 1895 1900

GCT GCT TCA AAA ACC ACA CAG CAG GTT CGT CAG CAA GTG CTT ACC CAG 5760

Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln

1905 1910 1915 1920

TTG CGT CTC AAT AGC AGG GTA AAA ACC CCG TTG CTA GGA ACA GCC AAT 5808

Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn

1925 1930 1935

TCC CTG ACC GCT TTA TTC CTG CCG CAG GAA AAT AGC AAG CTC AAA GGC 5856

Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly

1940 1945 1950

TAC TGG CGG ACA CTG GCG CAG CGT ATG TTT AAT TTA CGT CAT AAT CTG 5904

Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu

1955 1960 1965

TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG TAT GCT AAA CCG GCT 5952

Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala

1970 1975 1980

GAT CCA AAA GCT TTA CTG AGT GCG GCG GTT TCA GCT TCT CAA GGG GGA 6000

Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly

1985 1990 1995 2000

GCC GAC TTG CCG AAG GCG CCG CTG ACT ATT CAC CGC TTC CCT CAA ATG 6048

Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met

2005 2010 2015

CTA GAA GGG GCA CGG GGC TTG GTT AAC CAG CTT ATA CAG TTC GGT AGT 6096

Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser

2020 2025 2030

TCA CTA TTG GGG TAC AGT GAG CGT CAG GAT GCG GAA GCT ATG AGT CAA 6144

Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln

2035 2040 2045

CTA CTG CAA ACC CAA GCC AGC GAG TTA ATA CTG ACC AGT ATT CGT ATG 6192

Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met

2050 2055 2060

CAG GAT AAC CAA TTG GCA GAG CTG GAT TCG GAA AAA ACC GCC TTG CAA 6240

Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln

2065 2070 2075 2080

GTC TCT TTA GCT GGA GTG CAA CAA CGG TTT GAC AGC TAT AGC CAA CTG 6288

Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu

2085 2090 2095

TAT GAG GAG AAC ATC AAC GCA GGT GAG CAG CGA GCG CTG GCG TTA CGC 6336

Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg

2100 2105 2110

TCA GAA TCT GCT ATT GAG TCT CAG GGA GCG CAG ATT TCC CGT ATG GCA 6384

Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala

2115 2120 2125

GGC GCG GGT GTT GAT ATG GCA CCA AAT ATC TTC GGC CTG GCT GAT GGC 6432

Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly

2130 2135 2140

GGC ATG CAT TAT GGT GCT ATT GCC TAT GCC ATC GCT GAC GGT ATT GAG 6480

Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu

2145 2150 2155 2160

TTG AGT GCT TCT GCC AAG ATG GTT GAT GCG GAG AAA GTT GCT CAG TCG 6528

Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser

2165 2170 2175

GAA ATA TAT CGC CGT CGC CGT CAA GAA TGG AAA ATT CAG CGT GAC AAC 6576

Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn

2180 2185 2190

GCA CAA GCG GAG ATT AAC CAG TTA AAC GCG CAA CTG GAA TCA CTG TCT 6624

Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser

2195 2200 2205

ATT CGC CGT GAA GCC GCT GAA ATG CAA AAA GAG TAC CTG AAA ACC CAG 6672

Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln

2210 2215 2220

CAA GCT CAG GCG CAG GCA CAA CTT ACT TTC TTA AGA AGC AAA TTC AGT 6720

Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser

2225 2230 2235 2240

AAT CAA GCG TTA TAT AGT TGG TTA CGA GGG CGT TTG TCA GGT ATT TAT 6768

Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr

2245 2250 2255

TTC CAG TTC TAT GAC TTG GCC GTA TCA CGT TGC CTG ATG GCA GAG CAA 6816

Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln

2260 2265 2270

TCC TAT CAA TGG GAA GCT AAT GAT AAT TCC ATT AGC TTT GTC AAA CCG 6864

Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro

2275 2280 2285

GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG TGT GGA GAA GCT TTG 6912

Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu

2290 2295 2300

ATA CAA AAT CTG GCA CAA ATG GAA GAG GCA TAT CTG AAA TGG GAA TCT 6960

Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser

2305 2310 2315 2320

CGC GCT TTG GAA GTA GAA CGC ACG GTT TCA TTG GCA GTG GTT TAT GAT 7008

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp

2325 2330 2335

TCA CTG GAA GGT AAT GAT CGT TTT AAT TTA GCG GAA CAA ATA CCT GCA 7056

Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala

2340 2345 2350

TTA TTG GAT AAG GGG GAG GGA ACA GCA GGA ACT AAA GAA AAT GGG TTA 7104

Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu

2355 2360 2365

TCA TTG GCT AAT GCT ATC CTG TCA GCT TCG GTC AAA TTG TCC GAC TTG 7152

Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu

2370 2375 2380

AAA CTG GGA ACG GAT TAT CCA GAC AGT ATC GTT GGT AGC AAC AAG GTT 7200

Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val

2385 2390 2395 2400

CGT CGT ATT AAG CAA ATC AGT GTT TCG CTA CCT GCA TTG GTT GGG CCT 7248

Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro

2405 2410 2415

TAT CAG GAT GTT CAG GCT ATG CTC AGC TAT GGT GGC AGT ACT CAA TTG 7296

Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu

2420 2425 2430

CCG AAA GGT TGT TCA GCG TTG GCT GTG TCT CAT GGT ACC AAT GAT AGT 7344

Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser

2435 2440 2445

GGT CAG TTC CAG TTG GAT TTC AAT GAC GGC AAA TAC CTG CCA TTT GAA 7392

Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu

2450 2455 2460

GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT CTT CAA TTT CCG AAT 7440

Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn

2465 2470 2475 2480

GCT ACC GAC AAG CAG AAA GCA ATA TTG CAA ACT ATG AGC GAT ATT ATT 7488

Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile

2485 2490 2495

TTG CAT ATT CGT TAT ACC ATC CGT TAA 7515

Leu His Ile Arg Tyr Thr Ile Arg

2500

2504 amino acids

amino acid

linear

protein

12

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu

1 5 10 15

Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe

20 25 30

Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile

35 40 45

Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys

50 55 60

Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu

65 70 75 80

Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe

85 90 95

Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met

100 105 110

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn

115 120 125

Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp

130 135 140

Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser

145 150 155 160

Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys

165 170 175

Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg

180 185 190

Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu

195 200 205

Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro

210 215 220

Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser

225 230 235 240

His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu

245 250 255

Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp

260 265 270

Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr

275 280 285

Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His

290 295 300

Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val

305 310 315 320

Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr

325 330 335

Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn

340 345 350

Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe

355 360 365

Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His

370 375 380

Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala

385 390 395 400

Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln

405 410 415

Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile

420 425 430

Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile

435 440 445

Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile

450 455 460

Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn

465 470 475 480

Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu

485 490 495

Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val

500 505 510

Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu

515 520 525

Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro

530 535 540

Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg

545 550 555 560

Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr

565 570 575

Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn

580 585 590

Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln

595 600 605

Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys

610 615 620

Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala

625 630 635 640

Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln

645 650 655

Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser

660 665 670

Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser

675 680 685

Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala

690 695 700

Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val

705 710 715 720

Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile

725 730 735

Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu

740 745 750

Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr

755 760 765

Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln

770 775 780

Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu

785 790 795 800

Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly

805 810 815

Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr

820 825 830

Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln

835 840 845

Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp

850 855 860

Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu

865 870 875 880

Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly

885 890 895

Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met

900 905 910

Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser

915 920 925

Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala

930 935 940

Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn

945 950 955 960

Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala

965 970 975

Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln

980 985 990

Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg

995 1000 1005

Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr

1010 1015 1020

Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys

1025 1030 1035 1040

Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala

1045 1050 1055

Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln

1060 1065 1070

Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val

1075 1080 1085

Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly

1090 1095 1100

Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys

1105 1110 1115 1120

Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val

1125 1130 1135

Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu

1140 1145 1150

Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys

1155 1160 1165

Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp

1170 1175 1180

Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys

1185 1190 1195 1200

Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys

1205 1210 1215

Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met

1220 1225 1230

Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly

1235 1240 1245

Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln

1250 1255 1260

Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met

1265 1270 1275 1280

Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn

1285 1290 1295

Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn

1300 1305 1310

Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly

1315 1320 1325

Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu

1330 1335 1340

Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr

1345 1350 1355 1360

Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp

1365 1370 1375

Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn

1380 1385 1390

Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser

1395 1400 1405

Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr

1410 1415 1420

Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr

1425 1430 1435 1440

Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu

1445 1450 1455

Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr

1460 1465 1470

Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1475 1480 1485

Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala

1490 1495 1500

Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu

1505 1510 1515 1520

Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn

1525 1530 1535

Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala

1540 1545 1550

Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr

1555 1560 1565

Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn

1570 1575 1580

Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn

1585 1590 1595 1600

Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp

1605 1610 1615

Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly

1620 1625 1630

Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile

1635 1640 1645

His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys

1650 1655 1660

Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser

1665 1670 1675 1680

Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly

1685 1690 1695

Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr

1700 1705 1710

Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp

1715 1720 1725

Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln

1730 1735 1740

Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala

1745 1750 1755 1760

Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His

1765 1770 1775

Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg

1780 1785 1790

Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile

1795 1800 1805

Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala

1810 1815 1820

Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro

1825 1830 1835 1840

Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met

1845 1850 1855

Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala

1860 1865 1870

Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu

1875 1880 1885

Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn

1890 1895 1900

Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln

1905 1910 1915 1920

Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn

1925 1930 1935

Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly

1940 1945 1950

Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu

1955 1960 1965

Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala

1970 1975 1980

Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly

1985 1990 1995 2000

Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met

2005 2010 2015

Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser

2020 2025 2030

Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln

2035 2040 2045

Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met

2050 2055 2060

Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln

2065 2070 2075 2080

Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu

2085 2090 2095

Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg

2100 2105 2110

Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala

2115 2120 2125

Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly

2130 2135 2140

Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu

2145 2150 2155 2160

Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser

2165 2170 2175

Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn

2180 2185 2190

Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser

2195 2200 2205

Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln

2210 2215 2220

Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser

2225 2230 2235 2240

Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr

2245 2250 2255

Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln

2260 2265 2270

Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro

2275 2280 2285

Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu

2290 2295 2300

Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser

2305 2310 2315 2320

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp

2325 2330 2335

Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala

2340 2345 2350

Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu

2355 2360 2365

Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu

2370 2375 2380

Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val

2385 2390 2395 2400

Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro

2405 2410 2415

Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu

2420 2425 2430

Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser

2435 2440 2445

Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu

2450 2455 2460

Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn

2465 2470 2475 2480

Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile

2485 2490 2495

Leu His Ile Arg Tyr Thr Ile Arg

2500

12 amino acids

amino acid

single

linear

peptide

13

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Xaa Ala

1 5 10

12 amino acids

amino acid

single

linear

peptide

14

Met Gln Asn Ser Gln Thr Phe Ser Val Gly Glu Leu

1 5 10

14 amino acids

amino acid

single

linear

peptide

15

Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr

1 5 10

5 amino acids

amino acid

single

linear

peptide

16

Met Gln Asn Ser Leu

1 5

10 amino acids

amino acid

single

linear

peptide

17

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe

1 5 10

16 amino acids

amino acid

single

linear

peptide

18

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1 5 10 15

21 amino acids

amino acid

single

linear

peptide

19

Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser

1 5 10 15

Leu Gln Leu Phe Ile

20

12 amino acids

amino acid

single

linear

peptide

20

Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro

1 5 10

26 amino acids

amino acid

single

linear

peptide

21

Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro

1 5 10 15

Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu

20 25

15 amino acids

amino acid

single

linear

peptide

22

Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1 5 10 15

13 amino acids

amino acid

single

linear

peptide

23

Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys

1 5 10

22 amino acids

amino acid

single

linear

peptide

24

Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly

1 5 10 15

Val Gln Tyr Met Gln Ile

20

AGTAGCCCAA AACTTAAGTG CCGCAATCAG CAATCGTCAG TAACCGGATA AAGAAGGAAT 60

TGATT ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA GCG CGC 107

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg

1 5 10

CGT GAT GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC GCA GAT 155

Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp

15 20 25 30

TTA AAA GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT CTG TTG 203

Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu

35 40 45

CTG GAT ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG TCC GAA 251

Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu

50 55 60

GCG ATT GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG GGC TAT 299

Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr

65 70 75

GAC GGC ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT GAA CAG 347

Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln

80 85 90

TTT TTA TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT TGG GCT 395

Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala

95 100 105 110

GGC AAG GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT CCA ACA 443

Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr

115 120 125

TTG CGA TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA GGT ATT 491

Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile

130 135 140

TCT CAA GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA CGT GAT 539

Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp

145 150 155

TAT CTA ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT ACT GCC 587

Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala

160 165 170

TGC CAA GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT ACA CAG 635

Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln

175 180 185 190

AAT GCA CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC ACT GAT 683

Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp

195 200 205

GGC GGT AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA ATT AAT 731

Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn

210 215 220

GCC GGG ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC TGG GAA 779

Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu

225 230 235

AAT AAC AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA GAT AAA 827

Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys

240 245 250

ATA GAT TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT TAT AGC 875

Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser

255 260 265 270

TGG GCA TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC TAC AAT 923

Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn

275 280 285

AGA GTT GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT TCA CAA 971

Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln

290 295 300

TAT GGT TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT GTA CTT 1019

Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu

305 310 315

ATT TTT CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG ACG TTA 1067

Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu

320 325 330

TGT TAT GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA GGA AGT 1115

Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser

335 340 345 350

GCA AAT TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC ATG TGT 1163

Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys

355 360 365

CAT GGA CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA CTC TCT 1211

His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser

370 375 380

ATT AAT ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA GAT GGA 1259

Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly

385 390 395

AAA CAA TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC CTC CCT 1307

Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro

400 405 410

AAT TAT GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT TCA CTA 1355

Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu

415 420 425 430

CTT AAT TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG GTT GAT 1403

Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp

435 440 445

AAT TTC AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC TTC CAT 1451

Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His

450 455 460

ATT CCG TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT TAC GAA 1499

Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu

465 470 475

GAC GCG GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT TAT CGC 1547

Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg

480 485 490

GAT GCT AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT TAT TGG 1595

Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp

495 500 505 510

AAT GTG ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA CAG CCC 1643

Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro

515 520 525

GCC ACC ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG CAT TAC 1691

Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr

530 535 540

AAG CTG GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC CGA GGC 1739

Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly

545 550 555

GAC AGC GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA GCC AAA 1787

Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys

560 565 570

ATG TAC TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT GAT ATC 1835

Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile

575 580 585 590

CAT ACC ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA GCT GGC 1883

His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly

595 600 605

GCT ATT GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG ACG TTC 1931

Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe

610 615 620

GCT GCC TGG CTA AGC GCA GGC GAT ACC GCA AAT ATT GGC GAC GGT GAT 1979

Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp

625 630 635

TTC TTG CCA CCG TAC AAC GAT GTA CTA CTC GGT TAC TGG GAT AAA CTT 2027

Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu

640 645 650

GAG TTA CGC CTA TAC AAC CTG CGC CAC AAT CTG AGT CTG GAT GGT CAA 2075

Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln

655 660 665 670

CCG CTA AAT CTG CCA CTG TAT GCC ACG CCG GTA GAC CCG AAA ACC CTG 2123

Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu

675 680 685

CAA CGC CAG CAA GCC GGA GGG GAC GGT ACA GGC AGT AGT CCG GCT GGT 2171

Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly

690 695 700

GGT CAA GGC AGT GTT CAG GGC TGG CGC TAT CCG TTA TTG GTA GAA CGC 2219

Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg

705 710 715

GCC CGC TCT GCC GTG AGT TTG TTG ACT CAG TTC GGC AAC AGC TTA CAA 2267

Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln

720 725 730

ACA ACG TTA GAA CAT CAG GAT AAT GAA AAA ATG ACG ATA CTG TTG CAG 2315

Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln

735 740 745 750

ACT CAA CAG GAA GCC ATC CTG AAA CAT CAG CAC GAT ATA CAA CAA AAT 2363

Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn

755 760 765

AAT CTA AAA GGA TTA CAA CAC AGC CTG ACC GCA TTA CAG GCT AGC CGT 2411

Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg

770 775 780

GAT GGC GAC ACA TTG CGG CAA AAA CAT TAC AGC GAC CTG ATT AAC GGT 2459

Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly

785 790 795

GGT CTA TCT GCG GCA GAA ATC GCC GGT CTG ACA CTA CGC AGC ACC GCC 2507

Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala

800 805 810

ATG ATT ACC AAT GGC GTT GCA ACG GGA TTG CTG ATT GCC GGC GGA ATC 2555

Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile

815 820 825 830

GCC AAC GCG GTA CCT AAC GTC TTC GGG CTG GCT AAC GGT GGA TCG GAA 2603

Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu

835 840 845

TGG GGA GCG CCA TTA ATT GGC TCC GGG CAA GCA ACC CAA GTT GGC GCC 2651

Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala

850 855 860

GGC ATC CAG GAT CAG AGC GCG GGC ATT TCA GAA GTG ACA GCA GGC TAT 2699

Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr

865 870 875

CAG CGT CGT CAG GAA GAA TGG GCA TTG CAA CGG GAT ATT GCT GAT AAC 2747

Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn

880 885 890

GAA ATA ACC CAA CTG GAT GCC CAG ATA CAA AGC CTG CAA GAG CAA ATC 2795

Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile

895 900 905 910

ACG ATG GCA CAA AAA CAG ATC ACG CTC TCT GAA ACC GAA CAA GCG AAT 2843

Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn

915 920 925

GCC CAA GCG ATT TAT GAC CTG CAA ACC ACT CGT TTT ACC GGG CAG GCA 2891

Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala

930 935 940

CTG TAT AAC TGG ATG GCC GGT CGT CTC TCC GCG CTC TAT TAC CAA ATG 2939

Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met

945 950 955

TAT GAT TCC ACT CTG CCA ATC TGT CTC CAG CCA AAA GCC GCA TTA GTA 2987

Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val

960 965 970

CAG GAA TTA GGC GAG AAA GAG AGC GAC AGT CTT TTC CAG GTT CCG GTG 3035

Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val

975 980 985 990

TGG AAT GAT CTG TGG CAA GGG CTG TTA GCA GGA GAA GGT TTA AGT TCA 3083

Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser

995 1000 1005

GAG CTA CAG AAA CTG GAT GCC ATC TGG CTT GCA CGT GGT GGT ATT GGG 3131

Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly

1010 1015 1020

CTA GAA GCC ATC CGC ACC GTG TCG CTG GAT ACC CTG TTT GGC ACA GGG 3179

Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly

1025 1030 1035

ACG TTA AGT GAA AAT ATC AAT AAA GTG CTT AAC GGG GAA ACG GTA TCT 3227

Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser

1040 1045 1050

CCA TCC GGT GGC GTC ACT CTG GCG CTG ACA GGG GAT ATC TTC CAA GCA 3275

Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala

1055 1060 1065 1070

ACA CTG GAT TTG AGT CAG CTA GGT TTG GAT AAC TCT TAC AAC TTG GGT 3323

Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly

1075 1080 1085

AAC GAG AAG AAA CGT CGT ATT AAA CGT ATC GCC GTC ACC CTG CCA ACA 3371

Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr

1090 1095 1100

CTT CTG GGG CCA TAT CAA GAT CTT GAA GCC ACA CTG GTA ATG GGT GCG 3419

Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala

1105 1110 1115

GAA ATC GCC GCC TTA TCA CAC GGT GTG AAT GAC GGA GGC CGG TTT GTT 3467

Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val

1120 1125 1130

ACC GAC TTT AAC GAC AGC CGT TTT CTG CCT TTT GAA GGT CGA GAT GCA 3515

Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala

1135 1140 1145 1150

ACA ACC GGC ACA CTG GAG CTC AAT ATT TTC CAT GCG GGT AAA GAG GGA 3563

Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly

1155 1160 1165

ACG CAA CAC GAG TTG GTC GCG AAT CTG AGT GAC ATC ATT GTG CAT CTG 3611

Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu

1170 1175 1180

AAT TAC ATC ATT CGA GAC GCG TAA ATTTCTTTTC TTTGTCGATT ACAGGTCCCT 3665

Asn Tyr Ile Ile Arg Asp Ala *

1185 1190

ATCAGGGGCC TGTTATTAAG GAGTACTTTA TGCAGGATTC ACCAGAAGTA TCGATTACAA 3725

CGCTGTCACT TCCCAAAGGT GGCGGTGCTA TCAATGGCAT GGGAGAAGCA CTGAATGCTG 3785

CCGGCCCTGA TGGAATGGCC TCCCTATCTC TGCCATTACC CCTTTCGACC GGCAGAGGGA 3845

CGGCTCCTGG ATTATCGCTG ATTTACAGCA ACAGTGCAGG TAATGGGCCT TTCGGCATCG 3905

GCTGGCAATG CGGTGTTATG TCCATTAGCC GACGCACCCA ACATGGCATT CCACAATACG 3965

GTAATGACGA CACGTTCCTA TCCCCACAAG GCGAGGTCAT GAATATCGCC CTGAATGACC 4025

AAGGGCAACC TGATATCCGT CAAGACGTTA AAACGCTGCA AGGCGTTACC TTGCCAATTT 4085

CCTATACCGT GACCCGCTAT CAAGCCCGCC AGATCCTGGA TTTCAGTAAA ATCGAATACT 4145

GGCAACCTGC CTCCGGTCAA GAAGGACGCG CTTTCTGGCT GATATCGACA CCGGACGGGC 4205

ATCTACACAT CTTAGGGAAA ACCGCGCAGG CTTGTCTGGC AAATCCGCAA AATGACCAAC 4265

AAATCGCCCA GTGGTTGCTG GAAGAAACTG TGACGCCAGC CGGTGAACAT GTCAGCTATC 4325

AATATCGAGC CGAAGATGAA GCCCATTGTG ACGACAATGA AAAAACCGCT CATCCCAATG 4385

TTACCGCACA GCGCTATCTG GTACAGGTGA ACTACAGGCA ACATCAAACC ACAAGCCAGC 4445

CTGTTCGTAC TGGATAACGC ACCTCCCGCA CCGGAAGAGT GGCTGTTTCA TCTGGTCTTT 4505

GACCACGGTG AGCGCGTACC TCACTTCATA CCGTGCCAAC ATGGGATGCA GGTACAGCGC 4565

AATGGTCTGT ACGCCCGGAT ATCTTCTCTC GCTATGAATA TGGTTTTGAA GTGCGTACTC 4625

GCCGCTTATG TCAACAAGTG CTGATGTTTC ACCGCACCGC GCTCATGGCC GGAGAAGCCA 4685

GTACCAATGA CGCCCCGGAA CTGGTTGGAC GCTTAATACT GGAATATGAC AAAAACGCCA 4745

GCGTCACCAC GTTGATTACC ATCCGTCAAT TAAGCCATGA ATCGGACGGG AGGCCAGTCA 4805

CCCAGCCACC ACTAGAACTA GCCTGGCAAC GGTTTGATCT GGAGAAAATC CCGACATGGC 4865

AACGCTTTGA CGCACTAGAT AATTTTAACT CGCAGCAACG TTATCAACTG GTTGATCTGC 4925

GGGGAGAAGG GTTGCCAGGT ATGCTGTATC AAGATCGAGG CGCTTGGTGG TATAAAGCTC 4985

CGCAACGTCA GGAAGACGGA GACAGCAATG CCGTCACTTA CGACAAAATC GCCCCACTGC 5045

CTACCCTACC CAATTTGCAG GATAATGCCT CATTGATGGA TATCAACGGA GACGGCCAAC 5105

TGGATTGGGT TGTTACCGCC TCCGGTATTC GCGGATACCA TAGTCAGCAA CCCGATGGAA 5165

AGTGGACGCA CTTTACGCCA ATCAATGCCT TGCCCGTGGA ATATTTTCAT CCAAGCATCC 5225

AGTTCGCTGA CCTTACCGGG GCAGGCTTAT CTGATTTAGT GTTGATCGGG CCGAAAAGCG 5285

TGCGTCTATA TGCCAACCAG CGAAACGGCT GGCGTAAAGG AGAAGATGTC CCCCAATCCA 5345

CAGGTATCAC CCTGCCTGTC ACAGGGACCG ATGCCCGCAA ACTGGTGGCT TTCAGTGATA 5405

TGCTCGGTTC CGGTCAACAA CATCTGGTGG AAATCAAGGG TAATCGCGTC ACCTGTTGGC 5465

CGAATCTAGG GCATGGCCGT TTCGGTCAAC CACTAACTCT GTCAGGATTT AGCCAGCCCG 5525

AAAATAGCTT CAATCCCGAA CGGCTGTTTC TGGCGGATAT CGACGGCTCC GGCACCACCG 5585

ACCTTATCTA TGCGCAATCC GGCTCTTTGC TCATTTATCT CAACCAAAGT GGTAATCAGT 5645

TTGATGCCCC GTTGACATTA GCGTTGCCAG AAGGCGTACA ATTTGACAAC ACTTGCCAAC 5705

TTCAAGTCGC CGATATTCAG GGATTAGGGA TAGCCAGCTT GATTCTGACT GTGCCACATA 5765

TCGCGCCACA TCACTGGCGT TGTGACCTGT CACTGACCAA ACCCTGGTTG TTGAATGTAA 5825

TGAACAATAA CCGGGGCGCA CATCACACGC TACATTATCG TAGTTCCGCG CAATTCTGGT 5885

TGGATGAAAA ATTACAGCTC ACCAAAGCAG GCAAATCTCC GGCTTGTTAT CTGCCGTTTC 5945

CAATGCATTT GCTATGGTAT ACCGAAATTC AGGATGAAAT CAGCGGCAAC CGGCTCACCA 6005

GTGAAGTCAA CTACAGCCAC GGCGTCTGGG ATGGTAAAGA GCGGGAATTC 6055

1189 amino acids

amino acid

linear

protein

26

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu

625 630 635 640

Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu

645 650 655

Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu

660 665 670

Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg

675 680 685

Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln

690 695 700

Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg

705 710 715 720

Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr

725 730 735

Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln

740 745 750

Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu

755 760 765

Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly

770 775 780

Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu

785 790 795 800

Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile

805 810 815

Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn

820 825 830

Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly

835 840 845

Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile

850 855 860

Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg

865 870 875 880

Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile

885 890 895

Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met

900 905 910

Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln

915 920 925

Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr

930 935 940

Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp

945 950 955 960

Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu

965 970 975

Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn

980 985 990

Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu

995 1000 1005

Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu

1010 1015 1020

Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu

1025 1030 1035 1040

Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser

1045 1050 1055

Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu

1060 1065 1070

Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu

1075 1080 1085

Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu

1090 1095 1100

Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile

1105 1110 1115 1120

Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp

1125 1130 1135

Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr

1140 1145 1150

Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln

1155 1160 1165

His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr

1170 1175 1180

Ile Ile Arg Asp Ala

1185

ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA GCG CGC CGT GAT 48

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC GCA GAT TTA AAA 96

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT CTG TTG CTG GAT 144

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG TCC GAA GCG ATT 192

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG GGC TAT GAC GGC 240

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT GAA CAG TTT TTA 288

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT TGG GCT GGC AAG 336

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT CCA ACA TTG CGA 384

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA GGT ATT TCT CAA 432

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA CGT GAT TAT CTA 480

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT ACT GCC TGC CAA 528

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT ACA CAG AAT GCA 576

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC ACT GAT GGC GGT 624

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA ATT AAT GCC GGG 672

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC TGG GAA AAT AAC 720

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA GAT AAA ATA GAT 768

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT TAT AGC TGG GCA 816

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC TAC AAT AGA GTT 864

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT TCA CAA TAT GGT 912

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT GTA CTT ATT TTT 960

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG ACG TTA TGT TAT 1008

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA GGA AGT GCA AAT 1056

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC ATG TGT CAT GGA 1104

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA CTC TCT ATT AAT 1152

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA GAT GGA AAA CAA 1200

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC CTC CCT AAT TAT 1248

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT TCA CTA CTT AAT 1296

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG GTT GAT AAT TTC 1344

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC TTC CAT ATT CCG 1392

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT TAC GAA GAC GCG 1440

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT TAT CGC GAT GCT 1488

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT TAT TGG AAT GTG 1536

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA CAG CCC GCC ACC 1584

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG CAT TAC AAG CTG 1632

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC CGA GGC GAC AGC 1680

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA GCC AAA ATG TAC 1728

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT GAT ATC CAT ACC 1776

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA GCT GGC GCT ATT 1824

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG ACG TTC GCT GCC 1872

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

TGG CTA AGC 1881

Trp Leu Ser

625

627 amino acids

amino acid

linear

protein

28

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

Trp Leu Ser

625

GCA GGC GAT ACC GCA AAT ATT GGC GAC GGT GAT TTC TTG CCA CCG TAC 48

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr

1 5 10 15

AAC GAT GTA CTA CTC GGT TAC TGG GAT AAA CTT GAG TTA CGC CTA TAC 96

Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr

20 25 30

AAC CTG CGC CAC AAT CTG AGT CTG GAT GGT CAA CCG CTA AAT CTG CCA 144

Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro

35 40 45

CTG TAT GCC ACG CCG GTA GAC CCG AAA ACC CTG CAA CGC CAG CAA GCC 192

Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala

50 55 60

GGA GGG GAC GGT ACA GGC AGT AGT CCG GCT GGT GGT CAA GGC AGT GTT 240

Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val

65 70 75 80

CAG GGC TGG CGC TAT CCG TTA TTG GTA GAA CGC GCC CGC TCT GCC GTG 288

Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val

85 90 95

AGT TTG TTG ACT CAG TTC GGC AAC AGC TTA CAA ACA ACG TTA GAA CAT 336

Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His

100 105 110

CAG GAT AAT GAA AAA ATG ACG ATA CTG TTG CAG ACT CAA CAG GAA GCC 384

Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala

115 120 125

ATC CTG AAA CAT CAG CAC GAT ATA CAA CAA AAT AAT CTA AAA GGA TTA 432

Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu

130 135 140

CAA CAC AGC CTG ACC GCA TTA CAG GCT AGC CGT GAT GGC GAC ACA TTG 480

Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu

145 150 155 160

CGG CAA AAA CAT TAC AGC GAC CTG ATT AAC GGT GGT CTA TCT GCG GCA 528

Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala

165 170 175

GAA ATC GCC GGT CTG ACA CTA CGC AGC ACC GCC ATG ATT ACC AAT GGC 576

Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly

180 185 190

GTT GCA ACG GGA TTG CTG ATT GCC GGC GGA ATC GCC AAC GCG GTA CCT 624

Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro

195 200 205

AAC GTC TTC GGG CTG GCT AAC GGT GGA TCG GAA TGG GGA GCG CCA TTA 672

Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu

210 215 220

ATT GGC TCC GGG CAA GCA ACC CAA GTT GGC GCC GGC ATC CAG GAT CAG 720

Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln

225 230 235 240

AGC GCG GGC ATT TCA GAA GTG ACA GCA GGC TAT CAG CGT CGT CAG GAA 768

Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu

245 250 255

GAA TGG GCA TTG CAA CGG GAT ATT GCT GAT AAC GAA ATA ACC CAA CTG 816

Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu

260 265 270

GAT GCC CAG ATA CAA AGC CTG CAA GAG CAA ATC ACG ATG GCA CAA AAA 864

Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys

275 280 285

CAG ATC ACG CTC TCT GAA ACC GAA CAA GCG AAT GCC CAA GCG ATT TAT 912

Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr

290 295 300

GAC CTG CAA ACC ACT CGT TTT ACC GGG CAG GCA CTG TAT AAC TGG ATG 960

Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met

305 310 315 320

GCC GGT CGT CTC TCC GCG CTC TAT TAC CAA ATG TAT GAT TCC ACT CTG 1008

Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu

325 330 335

CCA ATC TGT CTC CAG CCA AAA GCC GCA TTA GTA CAG GAA TTA GGC GAG 1056

Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu

340 345 350

AAA GAG AGC GAC AGT CTT TTC CAG GTT CCG GTG TGG AAT GAT CTG TGG 1104

Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp

355 360 365

CAA GGG CTG TTA GCA GGA GAA GGT TTA AGT TCA GAG CTA CAG AAA CTG 1152

Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu

370 375 380

GAT GCC ATC TGG CTT GCA CGT GGT GGT ATT GGG CTA GAA GCC ATC CGC 1200

Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg

385 390 395 400

ACC GTG TCG CTG GAT ACC CTG TTT GGC ACA GGG ACG TTA AGT GAA AAT 1248

Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn

405 410 415

ATC AAT AAA GTG CTT AAC GGG GAA ACG GTA TCT CCA TCC GGT GGC GTC 1296

Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val

420 425 430

ACT CTG GCG CTG ACA GGG GAT ATC TTC CAA GCA ACA CTG GAT TTG AGT 1344

Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser

435 440 445

CAG CTA GGT TTG GAT AAC TCT TAC AAC TTG GGT AAC GAG AAG AAA CGT 1392

Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg

450 455 460

CGT ATT AAA CGT ATC GCC GTC ACC CTG CCA ACA CTT CTG GGG CCA TAT 1440

Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr

465 470 475 480

CAA GAT CTT GAA GCC ACA CTG GTA ATG GGT GCG GAA ATC GCC GCC TTA 1488

Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu

485 490 495

TCA CAC GGT GTG AAT GAC GGA GGC CGG TTT GTT ACC GAC TTT AAC GAC 1536

Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp

500 505 510

AGC CGT TTT CTG CCT TTT GAA GGT CGA GAT GCA ACA ACC GGC ACA CTG 1584

Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu

515 520 525

GAG CTC AAT ATT TTC CAT GCG GGT AAA GAG GGA ACG CAA CAC GAG TTG 1632

Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu

530 535 540

GTC GCG AAT CTG AGT GAC ATC ATT GTG CAT CTG AAT TAC ATC ATT CGA 1680

Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg

545 550 555 560

GAC GCG TAA 1689

Asp Ala

562 amino acids

amino acid

linear

protein

30

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr

1 5 10 15

Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr

20 25 30

Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro

35 40 45

Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala

50 55 60

Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val

65 70 75 80

Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val

85 90 95

Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His

100 105 110

Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala

115 120 125

Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu

130 135 140

Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu

145 150 155 160

Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala

165 170 175

Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly

180 185 190

Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro

195 200 205

Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu

210 215 220

Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln

225 230 235 240

Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu

245 250 255

Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu

260 265 270

Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys

275 280 285

Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr

290 295 300

Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met

305 310 315 320

Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu

325 330 335

Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu

340 345 350

Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp

355 360 365

Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu

370 375 380

Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg

385 390 395 400

Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn

405 410 415

Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val

420 425 430

Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser

435 440 445

Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg

450 455 460

Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr

465 470 475 480

Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu

485 490 495

Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp

500 505 510

Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu

515 520 525

Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu

530 535 540

Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg

545 550 555 560

Asp Ala

ATG CAG GAT TCA CCA GAA GTA TCG ATT ACA ACG CTG TCA CTT CCC AAA 48

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys

1 5 10 15

GGT GGC GGT GCT ATC AAT GGC ATG GGA GAA GCA CTG AAT GCT GCC GGC 96

Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly

20 25 30

CCT GAT GGA ATG GCC TCC CTA TCT CTG CCA TTA CCC CTT TCG ACC GGC 144

Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly

35 40 45

AGA GGG ACG GCT CCT GGA TTA TCG CTG ATT TAC AGC AAC AGT GCA GGT 192

Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly

50 55 60

AAT GGG CCT TTC GGC ATC GGC TGG CAA TGC GGT GTT ATG TCC ATT AGC 240

Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser

65 70 75 80

CGA CGC ACC CAA CAT GGC ATT CCA CAA TAC GGT AAT GAC GAC ACG TTC 288

Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe

85 90 95

CTA TCC CCA CAA GGC GAG GTC ATG AAT ATC GCC CTG AAT GAC CAA GGG 336

Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly

100 105 110

CAA CCT GAT ATC CGT CAA GAC GTT AAA ACG CTG CAA GGC GTT ACC TTG 384

Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu

115 120 125

CCA ATT TCC TAT ACC GTG ACC CGC TAT CAA GCC CGC CAG ATC CTG GAT 432

Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp

130 135 140

TTC AGT AAA ATC GAA TAC TGG CAA CCT GCC TCC GGT CAA GAA GGA CGC 480

Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg

145 150 155 160

GCT TTC TGG CTG ATA TCG ACA CCG GAC GGG CAT CTA CAC ATC TTA GGG 528

Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly

165 170 175

AAA ACC GCG CAG GCT TGT CTG GCA AAT CCG CAA AAT GAC CAA CAA ATC 576

Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile

180 185 190

GCC CAG TGG TTG CTG GAA GAA ACT GTG ACG CCA GCC GGT GAA CAT GTC 624

Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val

195 200 205

AGC TAT CAA TAT CGA GCC GAA GAT GAA GCC CAT TGT GAC GAC AAT GAA 672

Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu

210 215 220

AAA ACC GCT CAT CCC AAT GTT ACC GCA CAG CGC TAT CTG GTA CAG GTG 720

Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val

225 230 235 240

AAC TAC GGC AAC ATC AAA CCA CAA GCC AGC CTG TTC GTA CTG GAT AAC 768

Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn

245 250 255

GCA CCT CCC GCA CCG GAA GAG TGG CTG TTT CAT CTG GTC TTT GAC CAC 816

Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His

260 265 270

GGT GAG CGC GAT ACC TCA CTT CAT ACC GTG CCA ACA TGG GAT GCA GGT 864

Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly

275 280 285

ACA GCG CAA TGG TCT GTA CGC CCG GAT ATC TTC TCT CGC TAT GAA TAT 912

Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr

290 295 300

GGT TTT GAA GTG CGT ACT CGC CGC TTA TGT CAA CAA GTG CTG ATG TTT 960

Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe

305 310 315 320

CAC CGC ACC GCG CTC ATG GCC GGA GAA GCC AGT ACC AAT GAC GCC CCG 1008

His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro

325 330 335

GAA CTG GTT GGA CGC TTA ATA CTG GAA TAT GAC AAA AAC GCC AGC GTC 1056

Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val

340 345 350

ACC ACG TTG ATT ACC ATC CGT CAA TTA AGC CAT GAA TCG GAC GGG AGG 1104

Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg

355 360 365

CCA GTC ACC CAG CCA CCA CTA GAA CTA GCC TGG CAA CGG TTT GAT CTG 1152

Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu

370 375 380

GAG AAA ATC CCG ACA TGG CAA CGC TTT GAC GCA CTA GAT AAT TTT AAC 1200

Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn

385 390 395 400

TCG CAG CAA CGT TAT CAA CTG GTT GAT CTG CGG GGA GAA GGG TTG CCA 1248

Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro

405 410 415

GGT ATG CTG TAT CAA GAT CGA GGC GCT TGG TGG TAT AAA GCT CCG CAA 1296

Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln

420 425 430

CGT CAG GAA GAC GGA GAC AGC AAT GCC GTC ACT TAC GAC AAA ATC GCC 1344

Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala

435 440 445

CCA CTG CCT ACC CTA CCC AAT TTG CAG GAT AAT GCC TCA TTG ATG GAT 1392

Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp

450 455 460

ATC AAC GGA GAC GGC CAA CTG GAT TGG GTT GTT ACC GCC TCC GGT ATT 1440

Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile

465 470 475 480

CGC GGA TAC CAT AGT CAG CAA CCC GAT GGA AAG TGG ACG CAC TTT ACG 1488

Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr

485 490 495

CCA ATC AAT GCC TTG CCC GTG GAA TAT TTT CAT CCA AGC ATC CAG TTC 1536

Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe

500 505 510

GCT GAC CTT ACC GGG GCA GGC TTA TCT GAT TTA GTG TTG ATC GGG CCG 1584

Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro

515 520 525

AAA AGC GTG CGT CTA TAT GCC AAC CAG CGA AAC GGC TGG CGT AAA GGA 1632

Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly

530 535 540

GAA GAT GTC CCC CAA TCC ACA GGT ATC ACC CTG CCT GTC ACA GGG ACC 1680

Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr

545 550 555 560

GAT GCC CGC AAA CTG GTG GCT TTC AGT GAT ATG CTC GGT TCC GGT CAA 1728

Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln

565 570 575

CAA CAT CTG GTG GAA ATC AAG GGT AAT CGC GTC ACC TGT TGG CCG AAT 1776

Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn

580 585 590

CTA GGG CAT GGC CGT TTC GGT CAA CCA CTA ACT CTG TCA GGA TTT AGC 1824

Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser

595 600 605

CAG CCC GAA AAT AGC TTC AAT CCC GAA CGG CTG TTT CTG GCG GAT ATC 1872

Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile

610 615 620

GAC GGC TCC GGC ACC ACC GAC CTT ATC TAT GCG CAA TCC GGC TCT TTG 1920

Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu

625 630 635 640

CTC ATT TAT CTC AAC CAA AGT GGT AAT CAG TTT GAT GCC CCG TTG ACA 1968

Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr

645 650 655

TTA GCG TTG CCA GAA GGC GTA CAA TTT GAC AAC ACT TGC CAA CTT CAA 2016

Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln

660 665 670

GTC GCC GAT ATT CAG GGA TTA GGG ATA GCC AGC TTG ATT CTG ACT GTG 2064

Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val

675 680 685

CCA CAT ATC GCG CCA CAT CAC TGG CGT TGT GAC CTG TCA CTG ACC AAA 2112

Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys

690 695 700

CCC TGG TTG TTG AAT GTA ATG AAC AAT AAC CGG GGC GCA CAT CAC ACG 2160

Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr

705 710 715 720

CTA CAT TAT CGT AGT TCC GCG CAA TTC TGG TTG GAT GAA AAA TTA CAG 2208

Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln

725 730 735

CTC ACC AAA GCA GGC AAA TCT CCG GCT TGT TAT CTG CCG TTT CCA ATG 2256

Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met

740 745 750

CAT TTG CTA TGG TAT ACC GAA ATT CAG GAT GAA ATC AGC GGC AAC CGG 2304

His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg

755 760 765

CTC ACC AGT GAA GTC AAC TAC AGC CAC GGC GTC TGG GAT GGT AAA GAG 2352

Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu

770 775 780

CGG GAA TTC AGA GGA TTT GGC TGC ATC AAA CAG ACA GAT ACC ACA ACG 2400

Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr

785 790 795 800

TTT TCT CAC GGC ACC GCC CCC GAA CAG GCG GCA CCG TCG CTG AGT ATT 2448

Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile

805 810 815

AGC TGG TTT GCC ACC GGC ATG GAT GAA GTA GAC AGC CAA TTA GCT ACG 2496

Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr

820 825 830

GAA TAT TGG CAG GCA GAC ACG CAA GCT TAT AGC GGA TTT GAA ACC CGT 2544

Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg

835 840 845

TAT ACC GTC TGG GAT CAC ACC AAC CAG ACA GAC CAA GCA TTT ACC CCC 2592

Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro

850 855 860

AAT GAG ACA CAA CGT AAC TGG CTG ACG CGA GCG CTT AAA GGC CAA CTG 2640

Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu

865 870 875 880

CTA CGC ACT GAG CTC TAC GGT CTG GAC GGA ACA GAT AAG CAA ACA GTG 2688

Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val

885 890 895

CCT TAT ACC GTC AGT GAA TCG CGC TAT CAG GTA CGC TCT ATT CCC GTA 2736

Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val

900 905 910

AAT AAA GAA ACT GAA TTA TCT GCC TGG GTG ACT GCT ATT GAA AAT CGC 2784

Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg

915 920 925

AGC TAC CAC TAT GAA CGT ATC ATC ACT GAC CCA CAG TTC AGC CAG AGT 2832

Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser

930 935 940

ATC AAG TTG CAA CAC GAT ATC TTT GGT CAA TCA CTG CAA AGT GTC GAT 2880

Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp

945 950 955 960

ATT GCC TGG CCG CGC CGC GAA AAA CCA GCA GTG AAT CCC TAC CCG CCT 2928

Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro

965 970 975

ACC CTG CCG GAA ACG CTA TTT GAC AGC AGC TAT GAT GAT CAA CAA CAA 2976

Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln

980 985 990

CTA TTA CGT CTG GTG AGA CAA AAA AAT AGC TGG CAT CAC CTG ACT GAT 3024

Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp

995 1000 1005

GGG GAA AAC TGG CGA TTA GGT TTA CCG AAT GCA CAA CGC CGT GAT GTT 3072

Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val

1010 1015 1020

TAT ACT TAT GAC CGG AGC AAA ATT CCA ACC GAA GGG ATT TCC CTT GAA 3120

Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu

1025 1030 1035 1040

ATC TTG CTG AAA GAT GAT GGC CTG CTA GCA GAT GAA AAA GCG GCC GTT 3168

Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val

1045 1050 1055

TAT CTG GGA CAA CAA CAG ACG TTT TAC ACC GCC GGT CAA GCG GAA GTC 3216

Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val

1060 1065 1070

ACT CTA GAA AAA CCC ACG TTA CAA GCA CTG GTC GCG TTC CAA GAA ACC 3264

Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr

1075 1080 1085

GCC ATG ATG GAC GAT ACC TCA TTA CAG GCG TAT GAA GGC GTG ATT GAA 3312

Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu

1090 1095 1100

GAG CAA GAG TTG AAT ACC GCG CTG ACA CAG GCC GGT TAT CAG CAA GTC 3360

Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val

1105 1110 1115 1120

GCG CGG TTG TTT AAT ACC AGA TCA GAA AGC CCG GTA TGG GCG GCA CGG 3408

Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg

1125 1130 1135

CAA GGT TAT ACC GAT TAC GGT GAC GCC GCA CAG TTC TGG CGG CCT CAG 3456

Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln

1140 1145 1150

GCT CAG CGT AAC TCG TTG CTG ACA GGG AAA ACC ACA CTG ACC TGG GAT 3504

Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp

1155 1160 1165

ACC CAT CAT TGT GTA ATA ATA CAG ACT CAA GAT GCC GCT GGA TTA ACG 3552

Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr

1170 1175 1180

ACG CAA GCC CAT TAC GAT TAT CGT TTC CTT ACA CCG GTA CAA CTG ACA 3600

Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr

1185 1190 1195 1200

GAT ATT AAT GAT AAT CAA CAT ATT GTG ACT CTG GAC GCG CTA GGT CGC 3648

Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg

1205 1210 1215

GTA ACC ACC AGC CGG TTC TGG GGC ACA GAG GCA GGA CAA GCC GCA GGC 3696

Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly

1220 1225 1230

TAT TCC AAC CAG CCC TTC ACA CCA CCG GAC TCC GTA GAT AAA GCG CTG 3744

Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu

1235 1240 1245

GCA TTA ACC GGC GCA CTC CCT GTT GCC CAA TGT TTA GTC TAT GCC GTT 3792

Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val

1250 1255 1260

GAT AGC TGG ATG CCG TCG TTA TCT TTG TCT CAG CTT TCT CAG TCA CAA 3840

Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln

1265 1270 1275 1280

GAA GAG GCA GAA GCG CTA TGG GCG CAA CTG CGT GCC GCT CAT ATG ATT 3888

Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile

1285 1290 1295

ACC GAA GAT GGG AAA GTG TGT GCG TTA AGC GGG AAA CGA GGA ACA AGC 3936

Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser

1300 1305 1310

CAT CAG AAC CTG ACG ATT CAA CTT ATT TCG CTA TTG GCA AGT ATT CCC 3984

His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro

1315 1320 1325

CGT TTA CCG CCA CAT GTA CTG GGG ATC ACC ACT GAT CGC TAT GAT AGC 4032

Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser

1330 1335 1340

GAT CCG CAA CAG CAG CAC CAA CAG ACG GTG AGC TTT AGT GAC GGT TTT 4080

Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe

1345 1350 1355 1360

GGC CGG TTA CTC CAG AGT TCA GCT CGT CAT GAG TCA GGT GAT GCC TGG 4128

Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp

1365 1370 1375

CAA CGT AAA GAG GAT GGC GGG CTG GTC GTG GAT GCA AAT GGC GTT CTG 4176

Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu

1380 1385 1390

GTC AGT GCC CCT ACA GAC ACC CGA TGG GCC GTT TCC GGT CGC ACA GAA 4224

Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu

1395 1400 1405

TAT GAC GAC AAA GGC CAA CCT GTG CGT ACT TAT CAA CCC TAT TTT CTA 4272

Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu

1410 1415 1420

AAT GAC TGG CGT TAC GTT AGT GAT GAC AGC GCA CGA GAT GAC CTG TTT 4320

Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe

1425 1430 1435 1440

GCC GAT ACC CAC CTT TAT GAT CCA TTG GGA CGG GAA TAC AAA GTC ATC 4368

Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile

1445 1450 1455

ACT GCT AAG AAA TAT TTG CGA GAA AAG CTG TAC ACC CCG TGG TTT ATT 4416

Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile

1460 1465 1470

GTC AGT GAG GAT GAA AAC GAT ACA GCA TCA AGA ACC CCA TAG 4458

Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro

1475 1480 1485

1485 amino acids

amino acid

linear

protein

32

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys

1 5 10 15

Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly

20 25 30

Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly

35 40 45

Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly

50 55 60

Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser

65 70 75 80

Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe

85 90 95

Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly

100 105 110

Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu

115 120 125

Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp

130 135 140

Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg

145 150 155 160

Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly

165 170 175

Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile

180 185 190

Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val

195 200 205

Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu

210 215 220

Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val

225 230 235 240

Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn

245 250 255

Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His

260 265 270

Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly

275 280 285

Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr

290 295 300

Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe

305 310 315 320

His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro

325 330 335

Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val

340 345 350

Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg

355 360 365

Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu

370 375 380

Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn

385 390 395 400

Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro

405 410 415

Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln

420 425 430

Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala

435 440 445

Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp

450 455 460

Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile

465 470 475 480

Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr

485 490 495

Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe

500 505 510

Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro

515 520 525

Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly

530 535 540

Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr

545 550 555 560

Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln

565 570 575

Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn

580 585 590

Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser

595 600 605

Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile

610 615 620

Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu

625 630 635 640

Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr

645 650 655

Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln

660 665 670

Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val

675 680 685

Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys

690 695 700

Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr

705 710 715 720

Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln

725 730 735

Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met

740 745 750

His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg

755 760 765

Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu

770 775 780

Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr

785 790 795 800

Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile

805 810 815

Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr

820 825 830

Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg

835 840 845

Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro

850 855 860

Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu

865 870 875 880

Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val

885 890 895

Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val

900 905 910

Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg

915 920 925

Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser

930 935 940

Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp

945 950 955 960

Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro

965 970 975

Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln

980 985 990

Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp

995 1000 1005

Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val

1010 1015 1020

Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu

1025 1030 1035 1040

Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val

1045 1050 1055

Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val

1060 1065 1070

Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr

1075 1080 1085

Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu

1090 1095 1100

Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val

1105 1110 1115 1120

Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg

1125 1130 1135

Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln

1140 1145 1150

Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp

1155 1160 1165

Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr

1170 1175 1180

Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr

1185 1190 1195 1200

Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg

1205 1210 1215

Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly

1220 1225 1230

Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu

1235 1240 1245

Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val

1250 1255 1260

Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln

1265 1270 1275 1280

Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile

1285 1290 1295

Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser

1300 1305 1310

His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro

1315 1320 1325

Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser

1330 1335 1340

Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe

1345 1350 1355 1360

Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp

1365 1370 1375

Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu

1380 1385 1390

Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu

1395 1400 1405

Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu

1410 1415 1420

Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe

1425 1430 1435 1440

Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile

1445 1450 1455

Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile

1460 1465 1470

Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro

1475 1480 1485

3288 base pairs

nucleic acid

double

linear

DNA (genomic)

33

ATG GTG ACT GTT ATG CAA AAT AAA ATA TCA TTT TTA TCA GGT ACA TCC 48

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser

1 5 10 15

GAA CAG CCC CTG CTT GAC GCC GGT TAT CAA AAC GTA TTT GAT ATC GCA 96

Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala

20 25 30

TCA ATC AGC CGG GCT ACT TTC GTT CAA TCC GTT CCC ACC CTG CCC GTT 144

Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val

35 40 45

AAA GAG GCT CAT ACC GTC TAT CGT CAG GCG CGG CAA CGT GCG GAA AAT 192

Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn

50 55 60

CTG AAA TCC CTC TAC CGA GCC TGG CAA TTG CGT CAG GAG CCG GTT ATT 240

Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile

65 70 75 80

AAA GGG CTG GCT AAA CTT AAC CTA CAA TCC AAC GTT TCT GTG CTT CAA 288

Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln

85 90 95

GAT GCT TTG GTA GAG AAT ATT GGC GGT GAT GGG GAT TTC AGC GAT TTA 336

Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu

100 105 110

ATG AAC CGT GCC AGT CAA TAT GCT GAC GCT GCC TCT ATT CAA TCC CTA 384

Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu

115 120 125

TTT TCA CCG GGC CGT TAT GCT TCC GCA CTC TAC AGA GTT GCT AAA GAT 432

Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp

130 135 140

CTG CAT AAA TCA GAT TCC AGT TTG CAT ATT GAT AAT CGC CGC GCT GAT 480

Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp

145 150 155 160

CTG AAG GAT CTG ATA TTA AGC GAA ACG ACG ATG AAT AAA GAG GTC ACT 528

Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr

165 170 175

TCC CTT GAT ATC TTG TTG GAT GTG CTA CAA AAA GGC GGT AAA GAT ATT 576

Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile

180 185 190

ACT GAG CTG TCC GGC GCA TTC TTC CCA ATG ACG TTA CCT TAT GAC GAT 624

Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp

195 200 205

CAT CTG TCG CAA ATC GAT TCC GCT TTA TCG GCA CAA GCC AGA ACG CTG 672

His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu

210 215 220

AAC GGT GTG TGG AAT ACT TTG ACA GAT ACC ACG GCA CAA GCG GTT TCA 720

Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser

225 230 235 240

GAA CAA ACC AGT AAT ACG AAT ACA CGC AAA CTG TTC GCT GCC CAA GAT 768

Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp

245 250 255

GGT AAT CAA GAT ACA TTT TTT TCC GGA AAC ACT TTT TAT TTC AAA GCG 816

Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala

260 265 270

GTG GGA TTC AGC GGG CAA CCT ATG GTT TAC CTG TCA CAG TAC ACC AGC 864

Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser

275 280 285

GGG AAC GGC ATT GTC GGC GCA CAA TTG ATT GCA GGT AAT CCA GAC CAA 912

Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln

290 295 300

GCC GCC GCC GCA ATA GTC GCA CCG TTG AAA CTC ACT TGG TCA ATG GCA 960

Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala

305 310 315 320

AAA CAG TGT TAC TAC CTC GTC GCT CCC GAT GGT ACA ACG ATG GGA GAC 1008

Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp

325 330 335

GGT AAT GTT CTG ACC GGC TGT TTC TTA AGA GGC AAC AGC CCA ACT AAC 1056

Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn

340 345 350

CCG GAT AAA GAC GGT ATT TTT GCT CAG GTA GCC AAC AAA TCA GGC AGT 1104

Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser

355 360 365

ACT CAG CCT TTG CCA AGC TTC CAT CTG CCG GTC ACA CTG GAA CAC AGC 1152

Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser

370 375 380

GAG AAT AAA GAT CAG TAC TAT CTG AAA ACA GAG CAG GGT TAT ATC ACG 1200

Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr

385 390 395 400

GTA GAT AGT TCC GGA CAG TCA AAT TGG AAA AAC GCG CTG GTT ATC AAT 1248

Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn

405 410 415

GGG ACA AAA GAC AAG GGG CTG TTA TTA ACC TTT TGC AGC GAT AGC TCA 1296

Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser

420 425 430

GGC ACT CCG ACA AAC CCT GAT GAT GTG ATT CCT CCC GCT ATC AAT GAT 1344

Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp

435 440 445

ATT CCA TCG CCG CCA GCC CGC GAA ACA CTG TCA CTG ACG CCG GTC AGT 1392

Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser

450 455 460

TAT CAA TTG ATG ACC AAT CCG GCA CCG ACA GAA GAT GAT ATT ACC AAC 1440

Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn

465 470 475 480

CAT TAT GGT TTT AAC GGC GCT AGC TTA CGG GCT TCT CCA TTG TCA ACC 1488

His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr

485 490 495

AGC GAG TTG ACC AGC AAA CTG AAT TCT ATC GAT ACT TTC TGT GAG AAG 1536

Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys

500 505 510

ACC CGG TTA AGC TTC AAT CAG TTA ATG GAT TTG ACC GCT CAG CAA TCT 1584

Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser

515 520 525

TAC AGT CAA AGC AGC ATT GAT GCG AAA GCA GCC AGC CGC TAT GTT CGT 1632

Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg

530 535 540

TTT GGG GAA ACC ACC CCA ACC CGC GTC AAT GTC TAC GGT GCC GCT TAT 1680

Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr

545 550 555 560

CTG AAC AGC ACA CTG GCA GAC GCG GCT GAT GGT CAA TAT CTG TGG ATT 1728

Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile

565 570 575

CAG ACT GAT GGC AAG AGC CTA AAT TTC ACT GAC GAT ACG GTA GTC GCC 1776

Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala

580 585 590

TTA GCC GGT CGC GCT GAA AAG CTG GTA CGT TTA TCA TCC CAG ACC GGG 1824

Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly

595 600 605

CTA TCA TTT GAA GAA TTG GAC TGG CTG ATT GCC AAT GCC AGT CGT AGT 1872

Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser

610 615 620

GTG CCG GAC CAC CAC GAC AAA ATT GTG CTG GAT AAG CCG GTC CTT GAA 1920

Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu

625 630 635 640

GCA CTG GCA GAG TAT GTC AGC CTA AAA CAG CGC TAT GGG CTT GAT GCC 1968

Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala

645 650 655

AAT ACC TTT GCG ACC TTC ATT AGT GCA GTA AAT CCT TAT ACG CCA GAT 2016

Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp

660 665 670

CAG ACA CCC AGT TTC TAT GAA ACC GCT TTC CGC TCT GCC GAC GGT AAT 2064

Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn

675 680 685

CAT GTC ATT GCG CTA GGT ACA GAG GTG AAA TAT GCA GAA AAT GAG CAG 2112

His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln

690 695 700

GAT GAG TTA GCC GCC ATA TGC TGC AAA GCA TTG GGT GTC ACC AGT GAT 2160

Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp

705 710 715 720

GAA CTG CTC CGT ATT GGT CGC TAT TGC TTC GGT AAT GCA GGC AGT TTT 2208

Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe

725 730 735

ACC TTG GAT GAA TAT ACC GCC AGT CAG TTG TAT CGC TTC GGC GCC ATT 2256

Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile

740 745 750

CCC CGT TTG TTT GGG CTG ACA TTT GCC CAA GCC GAA ATT TTA TGG CGT 2304

Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg

755 760 765

CTG ATG GAA GGC GGA AAA GAT ATC TTA TTG CAA CAG TTA GGT CAG GCA 2352

Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala

770 775 780

AAA TCC CTG CAA CCA CTG GCT ATT TTA CGC CGT ACC GAG CAG GTG CTG 2400

Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu

785 790 795 800

GAT TGG ATG TCG TCC GTA AAT CTA AGT CTG ACT TAT CTG CAA GGG ATG 2448

Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met

805 810 815

GTA AGT ACG CAA TGG AGC GGT ACC GCC ACC GCT GAG ATG TTC AAT TTC 2496

Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe

820 825 830

TTG GAA AAC GTT TGT GAC AGC GTG AAT AGT CAA GCT GCC ACT AAA GAA 2544

Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu

835 840 845

ACA ATG GAT TCG GCG TTA CAG CAG AAA GTG CTG CGG GCG CTA AGC GCC 2592

Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala

850 855 860

GGT TTC GGC ATT AAG AGC AAT GTG ATG GGT ATC GTC ACC TTC TGG CTG 2640

Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu

865 870 875 880

GAG AAA ATC ACA ATC GGT AGT GAT AAT CCT TTT ACA TTG GCA AAC TAC 2688

Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr

885 890 895

TGG CAT GAT ATT CAA ACC CTG TTT AGC CAT GAC AAT GCC ACG TTA GAG 2736

Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu

900 905 910

TCC TTA CAA ACC GAC ACT TCT CTG GTA ATT GCT ACT CAG CAA CTT AGC 2784

Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser

915 920 925

CAG CTA GTG TTA ATT GTG AAA TGG CTG AGC CTG ACC GAG CAG GAT CTG 2832

Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu

930 935 940

CAA TTA CTG ACA ACC TAT CCC GAA CGT TTA ATC AAC GGC ATC ACG AAT 2880

Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn

945 950 955 960

GTT CCT GTA CCC AAT CCG GAG CTA TTA CTC ACG CTA TCA CGT TTT AAG 2928

Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys

965 970 975

CAG TGG GAA ACT CAA GTC ACC GTT TCC CGT GAT GAA GCG ATG CGC TGT 2976

Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys

980 985 990

TTC GAT CAA TTA AAT GCC AAT GAT ATG ACG ACT GAA AAT GCA GGT TCA 3024

Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser

995 1000 1005

CTG ATC GCC ACA TTG TAT GAG ATG GAT AAA GGT ACG GGA GCG CAA GTT 3072

Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val

1010 1015 1020

AAT ACC TTG CTA TTA GGT GAA AAT AAC TGG CCG AAA AGT TTT ACC TCT 3120

Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser

1025 1030 1035 1040

CTC TGG CAA CTT CTG ACC TGG TTA CGC GTC GGG CAA AGA CTG AAT GTC 3168

Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val

1045 1050 1055

GGT AGT ACC ACT CTG GGC AAT CTG TTG TCC ATG ATG CAA GCA GAC CCT 3216

Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro

1060 1065 1070

GCT GCC GAG AGT AGC GCT TTA TTG GCA TCA GTA GCC CAA AAC TTA AGT 3264

Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser

1075 1080 1085

GCC GCA ATC AGC AAT CGT CAG TAA 3288

Ala Ala Ile Ser Asn Arg Gln

1090 1095

1095 amino acids

amino acids

linear

protein

34

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser

1 5 10 15

Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala

20 25 30

Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val

35 40 45

Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn

50 55 60

Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile

65 70 75 80

Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln

85 90 95

Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu

100 105 110

Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu

115 120 125

Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp

130 135 140

Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp

145 150 155 160

Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr

165 170 175

Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile

180 185 190

Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp

195 200 205

His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu

210 215 220

Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser

225 230 235 240

Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp

245 250 255

Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala

260 265 270

Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser

275 280 285

Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln

290 295 300

Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala

305 310 315 320

Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp

325 330 335

Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn

340 345 350

Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser

355 360 365

Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser

370 375 380

Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr

385 390 395 400

Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn

405 410 415

Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser

420 425 430

Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp

435 440 445

Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser

450 455 460

Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn

465 470 475 480

His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr

485 490 495

Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys

500 505 510

Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser

515 520 525

Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg

530 535 540

Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr

545 550 555 560

Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile

565 570 575

Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala

580 585 590

Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly

595 600 605

Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser

610 615 620

Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu

625 630 635 640

Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala

645 650 655

Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp

660 665 670

Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn

675 680 685

His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln

690 695 700

Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp

705 710 715 720

Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe

725 730 735

Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile

740 745 750

Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg

755 760 765

Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala

770 775 780

Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu

785 790 795 800

Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met

805 810 815

Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe

820 825 830

Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu

835 840 845

Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala

850 855 860

Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu

865 870 875 880

Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr

885 890 895

Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu

900 905 910

Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser

915 920 925

Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu

930 935 940

Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn

945 950 955 960

Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys

965 970 975

Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys

980 985 990

Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser

995 1000 1005

Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val

1010 1015 1020

Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser

1025 1030 1035 1040

Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val

1045 1050 1055

Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro

1060 1065 1070

Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser

1075 1080 1085

Ala Ala Ile Ser Asn Arg Gln

1090 1095

603 amino acids

amino acid

linear

protein

35

Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr

1 5 10 15

Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr

20 25 30

Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser

35 40 45

Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr

50 55 60

Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln

65 70 75 80

Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp

85 90 95

Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser

100 105 110

Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn

115 120 125

Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys

130 135 140

Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr

145 150 155 160

Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro

165 170 175

Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser

180 185 190

Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala

195 200 205

Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly

210 215 220

Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn

225 230 235 240

Ala Gly Arg Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg

245 250 255

Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu

260 265 270

Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln

275 280 285

Xaa Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr

290 295 300

Glu Gln Val Leu Asp Trp Met Ser Pro Val Asn Leu Ser Leu Thr Tyr

305 310 315 320

Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu

325 330 335

Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala

340 345 350

Xaa Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg

355 360 365

Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val

370 375 380

Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Arg Asp Asn Pro Phe Thr

385 390 395 400

Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn

405 410 415

Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr

420 425 430

Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Val Ser Leu Thr

435 440 445

Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn

450 455 460

Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu

465 470 475 480

Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu

485 490 495

Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu

500 505 510

Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr

515 520 525

Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys

530 535 540

Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln

545 550 555 560

Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met

565 570 575

Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala

580 585 590

Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln

595 600

2557 base pairs

nucleic acid

double

linear

DNA (genomic)

36

GAATTCGGCT TGCGTTTAAT ATTGATGATG TCTCGCTCTT CCGCCTGCTT AAAATTACCG 60

ACCATGATAA TAAAGATGGA AAAATTAAAA ATAACCTAAA GAATCTTTCC AATTTATATA 120

TTGGAAAATT ACTGGCAGAT ATTCATCAAT TAACCATTGA TGAACTGGAT TTATTACTGA 180

TTGCCGTAGG TGAAGGAAAA ACTAATTTAT CCGCTATCAG TGATAAGCAA TTGGCTACCC 240

TGATCAGAAA ACTCAATACT ATTACCAGCT GGCTACATAC ACAGAAGTGG AGTGTATTCC 300

AGCTATTTAT CATGACCTCC ACCAGCTATA ACAAAACGCT AACGCCTGAA ATTAAGAATT 360

TGCTGGATAC CGTCTACCAC GGTTTACAAG GTTTTGATAA AGACAAAGCA GATTTGCTAC 420

ATGTCATGGC GCCCTATATT GCGGCCACCT TGCAATTATC ATCGGAAAAT GTCGCCCACT 480

CGGTACTCCT TTGGGCAGAT AAGTTACAGC CCGGCGACGG CGCAATGACA GCAGAGGGAN 540

TCTGGGACTG GTTGAATACT AAGTATACGC CGGGTTCATC GGAAGCCGTA GAAACGCAGG 600

AACATATCGT TCAGTATTGT CAGGCTCTGG CACAATTGGA AATGGTTTAC CATTCCACCG 660

GCATCAACGA AAACGCCTTC CGTCTATTTG TGACAAAACC AGAGATGTTT GGCGCTGCAA 720

CTGGAGCAGC GCCCGCGCAT GATGCCCTTT CACTGATTAT GCTGACACGT TTTGCGGATT 780

GGGTGAACGC ACTAGGCGAA AAAGCGTCCT CGGTGCTAGC GGCATTTGAA GCTAACTCGT 840

TAACGGCAGA ACAACTGGCT GATGCCATGA ATCTTGATGC TAATTTGCTG TTGCAAGCCA 900

GTATTCAAGC ACAAAATCAT CAACATCTTC CCCCAGTAAC TCCAGAAAAT GCGTTCTCCT 960

GTTGGACATC TATCAATACT ATCCTGCAAT GGGTTAATGT CGCACAACAA TTGAAATGTC 1020

GCCCCACAGG GCGTTTCCGC TTTGGTCGGG CTGGATTATA TTCAATCAAT GAAAGAGACA 1080

CCGACCTATG CCCAGTGGGA AAACGCGGCA GGCGTATTAA CCGCCGGGTT GAATTCAACA 1140

ACAGGCTAAT ACATTACAAC GCTTTTCTGG ATGAATCTCG CAGTGCCGCA TTAAGCACCT 1200

ACTATATCCG TCAAGTCGCC AAGGCAGCGG CGGCTATTAA AAGCCGTGAT GACTTGTATC 1260

AATACTTACT GATTGATAAT CAGGTTTCTG CGGCAATAAA AACCACCCGG ATCGCCGAAG 1320

CCATTGCCAG TATTCAACTG TACGTCAACC GGGCATTGGA AAATGTGGAA GAAAATGCCA 1380

ATTCGGGGGT TATCAGCCGC CAATTCTTTA TCGACTGGGA CAAATACAAT AAACGCTACA 1440

GCACTTGGGC GGGTGTTTCT CAATTAGTTT ACTACCCGGA AAACTATATT GATCCGACCA 1500

TGCGTATCGG ACAAACCAAA ATGATGGACG CATTACTGCA ATCCGTCAGC CAAAGCCAAT 1560

TAAACGCCGA TACCGTCGAA GATGCCTTTA TGTCTTATCT GACATCGTTT GAACAAGTGG 1620

CTAATCTTAA AGTTATTAGC GCATATCACG ATAATATTAA TAACGATCAA GGGCTGACCT 1680

ATTTTATCGG ACTCAGTGAA ACTGATGCCG GTGAATATTA TTGGCGCAGT GTCGATCACA 1740

GTAAATTCAA CGACGGTAAA TTCGCGGCTA ATGCCTGGAG TGAATGGCAT AAAATTGATT 1800

GTCCAATTAA CCCTTATAAA AGCACTATCC GTCCAGTGAT ATATAAATCC CGCCTGTATC 1860

TGCTCTGGTT GGAACAAAAG GAGATCACCA AACAGACAGG AAATAGTAAA GATGGCTATC 1920

AAACTGAAAC GGATTATCGT TATGAACTAA AATTGGCGCA TATCCGCTAT GATGGCACTT 1980

GGAATACGCC AATCACCTTT GATGTCAATA AAAAAATATC CGAGCTAAAA CTGGAAAAAA 2040

ATAGAGCGCC CGGACTCTAT TGTGCCGGTT ATCAAGGTGA AGATACGTTG CTGGTGATGT 2100

TTTATAACCA ACAAGACACA CTAGATAGTT ATAAAAACGC TTCAATGCAA GGACTATATA 2160

TCTTTGCTGA TATGGCATCC AAAGATATGA CCCCAGAACA GAGCAATGTT TATCGGGATA 2220

ATAGCTATCA ACAATTTGAT ACCAATAATG TCAGAAGAGT GAATAACCGC TATGCAGAGG 2280

ATTATGAGAT TCCTTCTTCG GTAAGTAGCC GTAAAGACTA TGGTTGGGGA GATTATTACC 2340

TCAGCATGGT ATATAACGGA GATATTCCAA CTATCAATTA CAAAGCCGCA TCAAGTGATT 2400

TAAAAATTTA TATTTCACCA AAATTAAGAA TTATTCATAA TGGATATGAA GGACAGAAGC 2460

GCAATCAATG CAATTTGATG AATAAATATG GCAAACTAGG TGATAAATTT ATTGTGTATA 2520

CCAGCCTGGG CGTTAATCCG AATAATAAGC CGAATTC 2557

845 amino acids

amino acids

linear

protein (partial)

37

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr

1 5 10 15

Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu

20 25 30

Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr

35 40 45

Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr

50 55 60

Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys

65 70 75 80

Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe

85 90 95

Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro

100 105 110

Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe

115 120 125

Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala

130 135 140

Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu

145 150 155 160

Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Gly

165 170 175

Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala

180 185 190

Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln

195 200 205

Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg

210 215 220

Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala

225 230 235 240

Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp

245 250 255

Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe

260 265 270

Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu

275 280 285

Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln

290 295 300

His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser

305 310 315 320

Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Lys Cys

325 330 335

Arg Pro Thr Gly Arg Phe Arg Phe Gly Arg Ala Gly Leu Tyr Ser Ile

340 345 350

Asn Glu Arg Asp Thr Asp Leu Cys Pro Val Gly Lys Arg Gly Arg Arg

355 360 365

Ile Asn Arg Arg Val Glu Phe Asn Asn Arg Leu Ile His Tyr Asn Ala

370 375 380

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

385 390 395 400

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

405 410 415

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

420 425 430

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

435 440 445

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

450 455 460

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

465 470 475 480

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

485 490 495

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

500 505 510

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

515 520 525

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

530 535 540

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

545 550 555 560

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

565 570 575

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

580 585 590

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

595 600 605

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

610 615 620

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

625 630 635 640

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

645 650 655

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

660 665 670

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

675 680 685

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

690 695 700

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

705 710 715 720

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

725 730 735

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

740 745 750

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

755 760 765

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

770 775 780

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

785 790 795 800

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

805 810 815

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

820 825 830

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn

835 840 845

Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly

1 5 10 15

Lys

Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala

1 5 10 15

Ile Ser Pro Ala Lys

20

Ala Asn Ser Leu Tyr Ala Leu Phe Leu Pro Gln

1 5 10

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln

1 5 10

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr

1 5 10 15

Ala Gly Leu Glu

Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys

1 5 10

Asp Asp Ser Gly Asp Asp Asp Lys Val Thr Asn Thr Asp Ile His

1 5 10 15

Arg

Asp Val Xaa Gly Ser Glu Lys Ala Asn Glu Lys Leu Lys

1 5 10

7551 base pairs

nucleic acid

double

linear

DNA (genomic)

46

ATG AAC GAG TCT GTA AAA GAG ATA CCT GAT GTA TTA AAA AGC CAG TGT 48

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys

1 5 10 15

GGT TTT AAT TGT CTG ACA GAT ATT AGC CAC AGC TCT TTT AAT GAA TTT 96

Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe

20 25 30

CGC CAG CAA GTA TCT GAG CAC CTC TCC TGG TCC GAA ACA CAC GAC TTA 144

Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu

35 40 45

TAT CAT GAT GCA CAA CAG GCA CAA AAG GAT AAT CGC CTG TAT GAA GCG 192

Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala

50 55 60

CGT ATT CTC AAA CGC GCC AAT CCC CAA TTA CAA AAT GCG GTG CAT CTT 240

Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu

65 70 75 80

GCC ATT CTC GCT CCC AAT GCT GAA CTG ATA GGC TAT AAC AAT CAA TTT 288

Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe

85 90 95

AGC GGT AGA GCC AGT CAA TAT GTT GCG CCG GGT ACC GTT TCT TCC ATG 336

Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met

100 105 110

TTC TCC CCC GCC GCT TAT TTG ACT GAA CTT TAT CGT GAA GCA CGC AAT 384

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn

115 120 125

TTA CAC GCA AGT GAC TCC GTT TAT TAT CTG GAT ACC CGC CGC CCA GAT 432

Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp

130 135 140

CTC AAA TCA ATG GCG CTC AGT CAG CAA AAT ATG GAT ATA GAA TTA TCC 480

Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser

145 150 155 160

ACA CTC TCT TTG TCC AAT GAG CTG TTA TTG GAA AGC ATT AAA ACT GAA 528

Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu

165 170 175

TCT AAA CTG GAA AAC TAT ACT AAA GTG ATG GAA ATG CTC TCC ACT TTC 576

Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe

180 185 190

CGT CCT TCC GGC GCA ACG CCT TAT CAT GAT GCT TAT GAA AAT GTG CGT 624

Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg

195 200 205

GAA GTT ATC CAG CTA CAA GAT CCT GGA CTT GAG CAA CTC AAT GCA TCA 672

Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser

210 215 220

CCG GCA ATT GCC GGG TTG ATG CAT CAA GCC TCC CTA TTG GGT ATT AAC 720

Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn

225 230 235 240

GCT TCA ATC TCG CCT GAG CTA TTT AAT ATT CTG ACG GAG GAG ATT ACC 768

Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr

245 250 255

GAA GGT AAT GCT GAG GAA CTT TAT AAG AAA AAT TTT GGT AAT ATC GAA 816

Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu

260 265 270

CCG GCC TCA TTG GCT ATG CCG GAA TAC CTT AAA CGT TAT TAT AAT TTA 864

Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu

275 280 285

AGC GAT GAA GAA CTT AGT CAG TTT ATT GGT AAA GCC AGC AAT TTT GGT 912

Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly

290 295 300

CAA CAG GAA TAT AGT AAT AAC CAA CTT ATT ACT CCG GTA GTC AAC AGC 960

Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser

305 310 315 320

AGT GAT GGC ACG GTT AAG GTA TAT CGG ATC ACC CGC GAA TAT ACA ACC 1008

Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr

325 330 335

AAT GCT TAT CAA ATG GAT GTG GAG CTA TTT CCC TTC GGT GGT GAG AAT 1056

Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn

340 345 350

TAT CGG TTA GAT TAT AAA TTC AAA AAT TTT TAT AAT GCC TCT TAT TTA 1104

Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu

355 360 365

TCC ATC AAG TTA AAT GAT AAA AGA GAA CTT GTT CGA ACT GAA GGC GCT 1152

Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala

370 375 380

CCT CAA GTC AAT ATA GAA TAC TCC GCA AAT ATC ACA TTA AAT ACC GCT 1200

Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala

385 390 395 400

GAT ATC AGT CAA CCT TTT GAA ATT GGC CTG ACA CGA GTA CTT CCT TCC 1248

Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser

405 410 415

GGT TCT TGG GCA TAT GCC GCC GCA AAA TTT ACC GTT GAA GAG TAT AAC 1296

Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn

420 425 430

CAA TAC TCT TTT CTG CTA AAA CTT AAC AAG GCT ATT CGT CTA TCA CGT 1344

Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg

435 440 445

GCG ACA GAA TTG TCA CCC ACG ATT CTG GAA GGC ATT GTG CGC AGT GTT 1392

Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val

450 455 460

AAT CTA CAA CTG GAT ATC AAC ACA GAC GTA TTA GGT AAA GTT TTT CTG 1440

Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu

465 470 475 480

ACT AAA TAT TAT ATG CAG CGT TAT GCT ATT CAT GCT GAA ACT GCC CTG 1488

Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu

485 490 495

ATA CTA TGC AAC GCG CCT ATT TCA CAA CGT TCA TAT GAT AAT CAA CCT 1536

Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro

500 505 510

AGC CAA TTT GAT CGC CTG TTT AAT ACG CCA TTA CTG AAC GGA CAA TAT 1584

Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr

515 520 525

TTT TCT ACC GGC GAT GAG GAG ATT GAT TTA AAT TCA GGT AGC ACC GGC 1632

Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly

530 535 540

GAT TGG CGA AAA ACC ATA CTT AAG CGT GCA TTT AAT ATT GAT GAT GTC 1680

Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val

545 550 555 560

TCG CTC TTC CGC CTG CTT AAA ATT ACC GAC CAT GAT AAT AAA GAT GGA 1728

Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly

565 570 575

AAA ATT AAA AAT AAC CTA AAG AAT CTT TCC AAT TTA TAT ATT GGA AAA 1776

Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys

580 585 590

TTA CTG GCA GAT ATT CAT CAA TTA ACC ATT GAT GAA CTG GAT TTA TTA 1824

Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu

595 600 605

CTG ATT GCC GTA GGT GAA GGA AAA ACT AAT TTA TCC GCT ATC AGT GAT 1872

Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp

610 615 620

AAG CAA TTG GCT ACC CTG ATC AGA AAA CTC AAT ACT ATT ACC AGC TGG 1920

Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp

625 630 635 640

CTA CAT ACA CAG AAG TGG AGT GTA TTC CAG CTA TTT ATC ATG ACC TCC 1968

Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser

645 650 655

ACC AGC TAT AAC AAA ACG CTA ACG CCT GAA ATT AAG AAT TTG CTG GAT 2016

Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp

660 665 670

ACC GTC TAC CAC GGT TTA CAA GGT TTT GAT AAA GAC AAA GCA GAT TTG 2064

Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu

675 680 685

CTA CAT GTC ATG GCG CCC TAT ATT GCG GCC ACC TTG CAA TTA TCA TCG 2112

Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser

690 695 700

GAA AAT GTC GCC CAC TCG GTA CTC CTT TGG GCA GAT AAG TTA CAG CCC 2160

Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro

705 710 715 720

GGC GAC GGC GCA ATG ACA GCA GAA AAA TTC TGG GAC TGG TTG AAT ACT 2208

Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr

725 730 735

AAG TAT ACG CCG GGT TCA TCG GAA GCC GTA GAA ACG CAG GAA CAT ATC 2256

Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile

740 745 750

GTT CAG TAT TGT CAG GCT CTG GCA CAA TTG GAA ATG GTT TAC CAT TCC 2304

Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser

755 760 765

ACC GGC ATC AAC GAA AAC GCC TTC CGT CTA TTT GTG ACA AAA CCA GAG 2352

Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu

770 775 780

ATG TTT GGC GCT GCA ACT GGA GCA GCG CCC GCG CAT GAT GCC CTT TCA 2400

Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser

785 790 795 800

CTG ATT ATG CTG ACA CGT TTT GCG GAT TGG GTG AAC GCA CTA GGC GAA 2448

Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu

805 810 815

AAA GCG TCC TCG GTG CTA GCG GCA TTT GAA GCT AAC TCG TTA ACG GCA 2496

Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala

820 825 830

GAA CAA CTG GCT GAT GCC ATG AAT CTT GAT GCT AAT TTG CTG TTG CAA 2544

Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln

835 840 845

GCC AGT ATT CAA GCA CAA AAT CAT CAA CAT CTT CCC CCA GTA ACT CCA 2592

Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro

850 855 860

GAA AAT GCG TTC TCC TGT TGG ACA TCT ATC AAT ACT ATC CTG CAA TGG 2640

Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp

865 870 875 880

GTT AAT GTC GCA CAA CAA TTG AAT GTC GCC CCA CAG GGC GTT TCC GCT 2688

Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala

885 890 895

TTG GTC GGG CTG GAT TAT ATT CAA TCA ATG AAA GAG ACA CCG ACC TAT 2736

Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr

900 905 910

GCC CAG TGG GAA AAC GCG GCA GGC GTA TTA ACC GCC GGG TTG AAT TCA 2784

Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser

915 920 925

CAA CAG GCT AAT ACA TTA CAC GCT TTT CTG GAT GAA TCT CGC AGT GCC 2832

Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala

930 935 940

GCA TTA AGC ACC TAC TAT ATC CGT CAA GTC GCC AAG GCA GCG GCG GCT 2880

Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala

945 950 955 960

ATT AAA AGC CGT GAT GAC TTG TAT CAA TAC TTA CTG ATT GAT AAT CAG 2928

Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln

965 970 975

GTT TCT GCG GCA ATA AAA ACC ACC CGG ATC GCC GAA GCC ATT GCC AGT 2976

Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser

980 985 990

ATT CAA CTG TAC GTC AAC CGG GCA TTG GAA AAT GTG GAA GAA AAT GCC 3024

Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala

995 1000 1005

AAT TCG GGG GTT ATC AGC CGC CAA TTC TTT ATC GAC TGG GAC AAA TAC 3072

Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr

1010 1015 1020

AAT AAA CGC TAC AGC ACT TGG GCG GGT GTT TCT CAA TTA GTT TAC TAC 3120

Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr

1025 1030 1035 1040

CCG GAA AAC TAT ATT GAT CCG ACC ATG CGT ATC GGA CAA ACC AAA ATG 3168

Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met

1045 1050 1055

ATG GAC GCA TTA CTG CAA TCC GTC AGC CAA AGC CAA TTA AAC GCC GAT 3216

Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp

1060 1065 1070

ACC GTC GAA GAT GCC TTT ATG TCT TAT CTG ACA TCG TTT GAA CAA GTG 3264

Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val

1075 1080 1085

GCT AAT CTT AAA GTT ATT AGC GCA TAT CAC GAT AAT ATT AAT AAC GAT 3312

Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp

1090 1095 1100

CAA GGG CTG ACC TAT TTT ATC GGA CTC AGT GAA ACT GAT GCC GGT GAA 3360

Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu

1105 1110 1115 1120

TAT TAT TGG CGC AGT GTC GAT CAC AGT AAA TTC AAC GAC GGT AAA TTC 3408

Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe

1125 1130 1135

GCG GCT AAT GCC TGG AGT GAA TGG CAT AAA ATT GAT TGT CCA ATT AAC 3456

Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn

1140 1145 1150

CCT TAT AAA AGC ACT ATC CGT CCA GTG ATA TAT AAA TCC CGC CTG TAT 3504

Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr

1155 1160 1165

CTG CTC TGG TTG GAA CAA AAG GAG ATC ACC AAA CAG ACA GGA AAT AGT 3552

Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser

1170 1175 1180

AAA GAT GGC TAT CAA ACT GAA ACG GAT TAT CGT TAT GAA CTA AAA TTG 3600

Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu

1185 1190 1195 1200

GCG CAT ATC CGC TAT GAT GGC ACT TGG AAT ACG CCA ATC ACC TTT GAT 3648

Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp

1205 1210 1215

GTC AAT AAA AAA ATA TCC GAG CTA AAA CTG GAA AAA AAT AGA GCG CCC 3696

Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro

1220 1225 1230

GGA CTC TAT TGT GCC GGT TAT CAA GGT GAA GAT ACG TTG CTG GTG ATG 3744

Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met

1235 1240 1245

TTT TAT AAC CAA CAA GAC ACA CTA GAT AGT TAT AAA AAC GCT TCA ATG 3792

Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met

1250 1255 1260

CAA GGA CTA TAT ATC TTT GCT GAT ATG GCA TCC AAA GAT ATG ACC CCA 3840

Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro

1265 1270 1275 1280

GAA CAG AGC AAT GTT TAT CGG GAT AAT AGC TAT CAA CAA TTT GAT ACC 3888

Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr

1285 1290 1295

AAT AAT GTC AGA AGA GTG AAT AAC CGC TAT GCA GAG GAT TAT GAG ATT 3936

Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile

1300 1305 1310

CCT TCC TCG GTA AGT AGC CGT AAA GAC TAT GGT TGG GGA GAT TAT TAC 3984

Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr

1315 1320 1325

CTC AGC ATG GTA TAT AAC GGA GAT ATT CCA ACT ATC AAT TAC AAA GCC 4032

Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala

1330 1335 1340

GCA TCA AGT GAT TTA AAA ATC TAT ATC TCA CCA AAA TTA AGA ATT ATT 4080

Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile

1345 1350 1355 1360

CAT AAT GGA TAT GAA GGA CAG AAG CGC AAT CAA TGC AAT CTG ATG AAT 4128

His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn

1365 1370 1375

AAA TAT GGC AAA CTA GGT GAT AAA TTT ATT GTT TAT ACT AGC TTG GGG 4176

Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly

1380 1385 1390

GTC AAT CCA AAT AAC TCG TCA AAT AAG CTC ATG TTT TAC CCC GTC TAT 4224

Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr

1395 1400 1405

CAA TAT AGC GGA AAC ACC AGT GGA CTC AAT CAA GGG AGA CTA CTA TTC 4272

Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe

1410 1415 1420

CAC CGT GAC ACC ACT TAT CCA TCT AAA GTA GAA GCT TGG ATT CCT GGA 4320

His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly

1425 1430 1435 1440

GCA AAA CGT TCT CTA ACC AAC CAA AAT GCC GCC ATT GGT GAT GAT TAT 4368

Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr

1445 1450 1455

GCT ACA GAC TCT CTG AAT AAA CCG GAT GAT CTT AAG CAA TAT ATC TTT 4416

Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe

1460 1465 1470

ATG ACT GAC AGT AAA GGG ACT GCT ACT GAT GTC TCA GGC CCA GTA GAG 4464

Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu

1475 1480 1485

ATT AAT ACT GCA ATT TCT CCA GCA AAA GTT CAG ATA ATA GTC AAA GCG 4512

Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala

1490 1495 1500

GGT GGC AAG GAG CAA ACT TTT ACC GCA GAT AAA GAT GTC TCC ATT CAG 4560

Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln

1505 1510 1515 1520

CCA TCA CCT AGC TTT GAT GAA ATG AAT TAT CAA TTT AAT GCC CTT GAA 4608

Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu

1525 1530 1535

ATA GAC GGT TCT GGT CTG AAT TTT ATT AAC AAC TCA GCC AGT ATT GAT 4656

Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp

1540 1545 1550

GTT ACT TTT ACC GCA TTT GCG GAG GAT GGC CGC AAA CTG GGT TAT GAA 4704

Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu

1555 1560 1565

AGT TTC AGT ATT CCT GTT ACC CTC AAG GTA AGT ACC GAT AAT GCC CTG 4752

Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu

1570 1575 1580

ACC CTG CAC CAT AAT GAA AAT GGT GCG CAA TAT ATG CAA TGG CAA TCC 4800

Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser

1585 1590 1595 1600

TAT CGT ACC CGC CTG AAT ACT CTA TTT GCC CGC CAG TTG GTT GCA CGC 4848

Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg

1605 1610 1615

GCC ACC ACC GGA ATC GAT ACA ATT CTG AGT ATG GAA ACT CAG AAT ATT 4896

Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile

1620 1625 1630

CAG GAA CCG CAG TTA GGC AAA GGT TTC TAT GCT ACG TTC GTG ATA CCT 4944

Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro

1635 1640 1645

CCC TAT AAC CTA TCA ACT CAT GGT GAT GAA CGT TGG TTT AAG CTT TAT 4992

Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr

1650 1655 1660

ATC AAA CAT GTT GTT GAT AAT AAT TCA CAT ATT ATC TAT TCA GGC CAG 5040

Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln

1665 1670 1675 1680

CTA ACA GAT ACA AAT ATA AAC ATC ACA TTA TTT ATT CCT CTT GAT GAT 5088

Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp

1685 1690 1695

GTC CCA TTG AAT CAA GAT TAT CAC GCC AAG GTT TAT ATG ACC TTC AAG 5136

Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys

1700 1705 1710

AAA TCA CCA TCA GAT GGT ACC TGG TGG GGC CCT CAC TTT GTT AGA GAT 5184

Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp

1715 1720 1725

GAT AAA GGA ATA GTA ACA ATA AAC CCT AAA TCC ATT TTG ACC CAT TTT 5232

Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe

1730 1735 1740

GAG AGC GTC AAT GTC CTG AAT AAT ATT AGT AGC GAA CCA ATG GAT TTC 5280

Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe

1745 1750 1755 1760

AGC GGC GCT AAC AGC CTC TAT TTC TGG GAA CTG TTC TAC TAT ACC CCG 5328

Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro

1765 1770 1775

ATG CTG GTT GCT CAA CGT TTG CTG CAT GAA CAG AAC TTC GAT GAA GCC 5376

Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala

1780 1785 1790

AAC CGT TGG CTG AAA TAT GTC TGG AGT CCA TCC GGT TAT ATT GTC CAC 5424

Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His

1795 1800 1805

GGC CAG ATT CAG AAC TAC CAG TGG AAC GTC CGC CCG TTA CTG GAA GAC 5472

Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp

1810 1815 1820

ACC AGT TGG AAC AGT GAT CCT TTG GAT TCC GTC GAT CCT GAC GCG GTA 5520

Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val

1825 1830 1835 1840

GCA CAG CAC GAT CCA ATG CAC TAC AAA GTT TCA ACT TTT ATG CGT ACC 5568

Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr

1845 1850 1855

TTG GAT CTA TTG ATA GCA CGC GGC GAC CAT GCT TAT CGC CAA CTG GAA 5616

Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu

1860 1865 1870

CGA GAT ACA CTC AAC GAA GCG AAG ATG TGG TAT ATG CAA GCG CTG CAT 5664

Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His

1875 1880 1885

CTA TTA GGT GAC AAA CCT TAT CTA CCG CTG AGT ACG ACA TGG AGT GAT 5712

Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp

1890 1895 1900

CCA CGA CTA GAC AGA GCC GCG GAT ATC ACT ACC CAA AAT GCT CAC GAC 5760

Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp

1905 1910 1915 1920

AGC GCA ATA GTC GCT CTG CGG CAG AAT ATA CCT ACA CCG GCA CCT TTA 5808

Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu

1925 1930 1935

TCA TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC 5856

Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile

1940 1945 1950

AAT GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC 5904

Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr

1955 1960 1965

AAT CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA 5952

Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro

1970 1975 1980

ATC TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT 6000

Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val

1985 1990 1995 2000

GCC ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG 6048

Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu

2005 2010 2015

TGG CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG 6096

Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln

2020 2025 2030

CTC ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC 6144

Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp

2035 2040 2045

GCG GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA 6192

Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile

2050 2055 2060

TTG ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC 6240

Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala

2065 2070 2075 2080

GAG AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT 6288

Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe

2085 2090 2095

GAT AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC 6336

Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn

2100 2105 2110

CAA GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT 6384

Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val

2115 2120 2125

CAG GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC 6432

Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile

2130 2135 2140

TTC GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG 6480

Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala

2145 2150 2155 2160

ACA GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG 6528

Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala

2165 2170 2175

GAT AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG 6576

Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp

2180 2185 2190

GAG ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT 6624

Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala

2195 2200 2205

CAG CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA 6672

Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys

2210 2215 2220

ACC AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC 6720

Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe

2225 2230 2235 2240

CTG CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT 6768

Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly

2245 2250 2255

CGA CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT 6816

Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg

2260 2265 2270

TGC CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT 6864

Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser

2275 2280 2285

GCC CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG 6912

Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu

2290 2295 2300

CTT GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT 6960

Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala

2305 2310 2315 2320

CAT CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG 7008

His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser

2325 2330 2335

CTG GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC 7056

Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser

2340 2345 2350

CTG GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC 7104

Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala

2355 2360 2365

GGC AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA 7152

Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys

2370 2375 2380

ACC TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA 7200

Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu

2385 2390 2395 2400

GAT TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC 7248

Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser

2405 2410 2415

GTC ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA 7296

Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile

2420 2425 2430

TTG TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG 7344

Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu

2435 2440 2445

GCA GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC 7392

Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe

2450 2455 2460

AAC GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC 7440

Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly

2465 2470 2475 2480

ACG CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA 7488

Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys

2485 2490 2495

CAA GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC 7536

Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg

2500 2505 2510

TAC ACC ATT AAA TAA 7551

Tyr Thr Ile Lys

2515

2516 amino acids

amino acids

linear

protein

47

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys

1 5 10 15

Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe

20 25 30

Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu

35 40 45

Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala

50 55 60

Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu

65 70 75 80

Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe

85 90 95

Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met

100 105 110

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn

115 120 125

Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp

130 135 140

Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser

145 150 155 160

Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu

165 170 175

Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe

180 185 190

Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg

195 200 205

Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser

210 215 220

Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn

225 230 235 240

Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr

245 250 255

Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu

260 265 270

Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu

275 280 285

Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly

290 295 300

Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser

305 310 315 320

Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr

325 330 335

Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn

340 345 350

Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu

355 360 365

Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala

370 375 380

Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala

385 390 395 400

Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser

405 410 415

Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn

420 425 430

Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg

435 440 445

Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val

450 455 460

Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu

465 470 475 480

Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu

485 490 495

Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro

500 505 510

Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr

515 520 525

Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly

530 535 540

Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val

545 550 555 560

Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly

565 570 575

Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys

580 585 590

Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu

595 600 605

Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp

610 615 620

Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp

625 630 635 640

Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser

645 650 655

Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp

660 665 670

Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu

675 680 685

Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser

690 695 700

Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro

705 710 715 720

Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr

725 730 735

Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile

740 745 750

Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser

755 760 765

Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu

770 775 780

Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser

785 790 795 800

Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu

805 810 815

Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala

820 825 830

Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln

835 840 845

Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro

850 855 860

Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp

865 870 875 880

Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala

885 890 895

Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr

900 905 910

Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser

915 920 925

Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala

930 935 940

Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala

945 950 955 960

Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln

965 970 975

Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser

980 985 990

Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala

995 1000 1005

Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr

1010 1015 1020

Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr

1025 1030 1035 1040

Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met

1045 1050 1055

Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp

1060 1065 1070

Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val

1075 1080 1085

Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp

1090 1095 1100

Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu

1105 1110 1115 1120

Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe

1125 1130 1135

Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn

1140 1145 1150

Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr

1155 1160 1165

Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser

1170 1175 1180

Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu

1185 1190 1195 1200

Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp

1205 1210 1215

Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro

1220 1225 1230

Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met

1235 1240 1245

Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met

1250 1255 1260

Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro

1265 1270 1275 1280

Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr

1285 1290 1295

Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile

1300 1305 1310

Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr

1315 1320 1325

Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala

1330 1335 1340

Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile

1345 1350 1355 1360

His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn

1365 1370 1375

Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly

1380 1385 1390

Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr

1395 1400 1405

Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe

1410 1415 1420

His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly

1425 1430 1435 1440

Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr

1445 1450 1455

Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe

1460 1465 1470

Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu

1475 1480 1485

Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala

1490 1495 1500

Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln

1505 1510 1515 1520

Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu

1525 1530 1535

Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp

1540 1545 1550

Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu

1555 1560 1565

Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu

1570 1575 1580

Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser

1585 1590 1595 1600

Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg

1605 1610 1615

Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile

1620 1625 1630

Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro

1635 1640 1645

Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr

1650 1655 1660

Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln

1665 1670 1675 1680

Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp

1685 1690 1695

Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys

1700 1705 1710

Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp

1715 1720 1725

Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe

1730 1735 1740

Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe

1745 1750 1755 1760

Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro

1765 1770 1775

Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala

1780 1785 1790

Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His

1795 1800 1805

Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp

1810 1815 1820

Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val

1825 1830 1835 1840

Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr

1845 1850 1855

Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu

1860 1865 1870

Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His

1875 1880 1885

Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp

1890 1895 1900

Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp

1905 1910 1915 1920

Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu

1925 1930 1935

Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile

1940 1945 1950

Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr

1955 1960 1965

Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro

1970 1975 1980

Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val

1985 1990 1995 2000

Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu

2005 2010 2015

Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln

2020 2025 2030

Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp

2035 2040 2045

Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile

2050 2055 2060

Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala

2065 2070 2075 2080

Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe

2085 2090 2095

Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn

2100 2105 2110

Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val

2115 2120 2125

Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile

2130 2135 2140

Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala

2145 2150 2155 2160

Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala

2165 2170 2175

Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp

2180 2185 2190

Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala

2195 2200 2205

Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys

2210 2215 2220

Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe

2225 2230 2235 2240

Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly

2245 2250 2255

Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg

2260 2265 2270

Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser

2275 2280 2285

Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu

2290 2295 2300

Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala

2305 2310 2315 2320

His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser

2325 2330 2335

Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser

2340 2345 2350

Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala

2355 2360 2365

Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys

2370 2375 2380

Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu

2385 2390 2395 2400

Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser

2405 2410 2415

Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile

2420 2425 2430

Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu

2435 2440 2445

Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe

2450 2455 2460

Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly

2465 2470 2475 2480

Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys

2485 2490 2495

Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg

2500 2505 2510

Tyr Thr Ile Lys

2515

5547 base pairs

nucleic acid

double

linear

DNA (genomic)

48

CTG ATA GGC TAT AAC AAT CAA TTT AGC GGT AGA GCC AGT CAA TAT GTT 48

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val

1 5 10 15

GCG CCG GGT ACC GTT TCT TCC ATG TTC TCC CCC GCC GCT TAT TTG ACT 96

Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr

20 25 30

GAA CTT TAT CGT GAA GCA CGC AAT TTA CAC GCA AGT GAC TCC GTT TAT 144

Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr

35 40 45

TAT CTG GAT ACC CGC CGC CCA GAT CTC AAA TCA ATG GCG CTC AGT CAG 192

Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln

50 55 60

CAA AAT ATG GAT ATA GAA TTA TCC ACA CTC TCT TTG TCC AAT GAG CTG 240

Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu

65 70 75 80

TTA TTG GAA AGC ATT AAA ACT GAA TCT AAA CTG GAA AAC TAT ACT AAA 288

Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys

85 90 95

GTG ATG GAA ATG CTC TCC ACT TTC CGT CCT TCC GGC GCA ACG CCT TAT 336

Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr

100 105 110

CAT GAT GCT TAT GAA AAT GTG CGT GAA GTT ATC CAG CTA CAA GAT CCT 384

His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro

115 120 125

GGA CTT GAG CAA CTC AAT GCA TCA CCG GCA ATT GCC GGG TTG ATG CAT 432

Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His

130 135 140

CAA GCC TCC CTA TTG GGT ATT AAC GCT TCA ATC TCG CCT GAG CTA TTT 480

Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe

145 150 155 160

AAT ATT CTG ACG GAG GAG ATT ACC GAA GGT AAT GCT GAG GAA CTT TAT 528

Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr

165 170 175

AAG AAA AAT TTT GGT AAT ATC GAA CCG GCC TCA TTG GCT ATG CCG GAA 576

Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu

180 185 190

TAC CTT AAA CGT TAT TAT AAT TTA AGC GAT GAA GAA CTT AGT CAG TTT 624

Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe

195 200 205

ATT GGT AAA GCC AGC AAT TTT GGT CAA CAG GAA TAT AGT AAT AAC CAA 672

Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln

210 215 220

CTT ATT ACT CCG GTA GTC AAC AGC AGT GAT GGC ACG GTT AAG GTA TAT 720

Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr

225 230 235 240

CGG ATC ACC CGC GAA TAT ACA ACC AAT GCT TAT CAA ATG GAT GTG GAG 768

Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu

245 250 255

CTA TTT CCC TTC GGT GGT GAG AAT TAT CGG TTA GAT TAT AAA TTC AAA 816

Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys

260 265 270

AAT TTT TAT AAT GCC TCT TAT TTA TCC ATC AAG TTA AAT GAT AAA AGA 864

Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg

275 280 285

GAA CTT GTT CGA ACT GAA GGC GCT CCT CAA GTC AAT ATA GAA TAC TCC 912

Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser

290 295 300

GCA AAT ATC ACA TTA AAT ACC GCT GAT ATC AGT CAA CCT TTT GAA ATT 960

Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile

305 310 315 320

GGC CTG ACA CGA GTA CTT CCT TCC GGT TCT TGG GCA TAT GCC GCC GCA 1008

Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala

325 330 335

AAA TTT ACC GTT GAA GAG TAT AAC CAA TAC TCT TTT CTG CTA AAA CTT 1056

Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu

340 345 350

AAC AAG GCT ATT CGT CTA TCA CGT GCG ACA GAA TTG TCA CCC ACG ATT 1104

Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile

355 360 365

CTG GAA GGC ATT GTG CGC AGT GTT AAT CTA CAA CTG GAT ATC AAC ACA 1152

Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr

370 375 380

GAC GTA TTA GGT AAA GTT TTT CTG ACT AAA TAT TAT ATG CAG CGT TAT 1200

Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr

385 390 395 400

GCT ATT CAT GCT GAA ACT GCC CTG ATA CTA TGC AAC GCG CCT ATT TCA 1248

Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser

405 410 415

CAA CGT TCA TAT GAT AAT CAA CCT AGC CAA TTT GAT CGC CTG TTT AAT 1296

Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn

420 425 430

ACG CCA TTA CTG AAC GGA CAA TAT TTT TCT ACC GGC GAT GAG GAG ATT 1344

Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile

435 440 445

GAT TTA AAT TCA GGT AGC ACC GGC GAT TGG CGA AAA ACC ATA CTT AAG 1392

Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys

450 455 460

CGT GCA TTT AAT ATT GAT GAT GTC TCG CTC TTC CGC CTG CTT AAA ATT 1440

Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile

465 470 475 480

ACC GAC CAT GAT AAT AAA GAT GGA AAA ATT AAA AAT AAC CTA AAG AAT 1488

Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn

485 490 495

CTT TCC AAT TTA TAT ATT GGA AAA TTA CTG GCA GAT ATT CAT CAA TTA 1536

Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu

500 505 510

ACC ATT GAT GAA CTG GAT TTA TTA CTG ATT GCC GTA GGT GAA GGA AAA 1584

Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys

515 520 525

ACT AAT TTA TCC GCT ATC AGT GAT AAG CAA TTG GCT ACC CTG ATC AGA 1632

Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg

530 535 540

AAA CTC AAT ACT ATT ACC AGC TGG CTA CAT ACA CAG AAG TGG AGT GTA 1680

Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val

545 550 555 560

TTC CAG CTA TTT ATC ATG ACC TCC ACC AGC TAT AAC AAA ACG CTA ACG 1728

Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr

565 570 575

CCT GAA ATT AAG AAT TTG CTG GAT ACC GTC TAC CAC GGT TTA CAA GGT 1776

Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly

580 585 590

TTT GAT AAA GAC AAA GCA GAT TTG CTA CAT GTC ATG GCG CCC TAT ATT 1824

Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile

595 600 605

GCG GCC ACC TTG CAA TTA TCA TCG GAA AAT GTC GCC CAC TCG GTA CTC 1872

Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu

610 615 620

CTT TGG GCA GAT AAG TTA CAG CCC GGC GAC GGC GCA ATG ACA GCA GAA 1920

Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu

625 630 635 640

AAA TTC TGG GAC TGG TTG AAT ACT AAG TAT ACG CCG GGT TCA TCG GAA 1968

Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu

645 650 655

GCC GTA GAA ACG CAG GAA CAT ATC GTT CAG TAT TGT CAG GCT CTG GCA 2016

Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala

660 665 670

CAA TTG GAA ATG GTT TAC CAT TCC ACC GGC ATC AAC GAA AAC GCC TTC 2064

Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe

675 680 685

CGT CTA TTT GTG ACA AAA CCA GAG ATG TTT GGC GCT GCA ACT GGA GCA 2112

Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala

690 695 700

GCG CCC GCG CAT GAT GCC CTT TCA CTG ATT ATG CTG ACA CGT TTT GCG 2160

Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala

705 710 715 720

GAT TGG GTG AAC GCA CTA GGC GAA AAA GCG TCC TCG GTG CTA GCG GCA 2208

Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala

725 730 735

TTT GAA GCT AAC TCG TTA ACG GCA GAA CAA CTG GCT GAT GCC ATG AAT 2256

Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn

740 745 750

CTT GAT GCT AAT TTG CTG TTG CAA GCC AGT ATT CAA GCA CAA AAT CAT 2304

Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His

755 760 765

CAA CAT CTT CCC CCA GTA ACT CCA GAA AAT GCG TTC TCC TGT TGG ACA 2352

Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr

770 775 780

TCT ATC AAT ACT ATC CTG CAA TGG GTT AAT GTC GCA CAA CAA TTG AAT 2400

Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn

785 790 795 800

GTC GCC CCA CAG GGC GTT TCC GCT TTG GTC GGG CTG GAT TAT ATT CAA 2448

Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln

805 810 815

TCA ATG AAA GAG ACA CCG ACC TAT GCC CAG TGG GAA AAC GCG GCA GGC 2496

Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly

820 825 830

GTA TTA ACC GCC GGG TTG AAT TCA CAA CAG GCT AAT ACA TTA CAC GCT 2544

Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala

835 840 845

TTT CTG GAT GAA TCT CGC AGT GCC GCA TTA AGC ACC TAC TAT ATC CGT 2592

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

850 855 860

CAA GTC GCC AAG GCA GCG GCG GCT ATT AAA AGC CGT GAT GAC TTG TAT 2640

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

865 870 875 880

CAA TAC TTA CTG ATT GAT AAT CAG GTT TCT GCG GCA ATA AAA ACC ACC 2688

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

885 890 895

CGG ATC GCC GAA GCC ATT GCC AGT ATT CAA CTG TAC GTC AAC CGG GCA 2736

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

900 905 910

TTG GAA AAT GTG GAA GAA AAT GCC AAT TCG GGG GTT ATC AGC CGC CAA 2784

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

915 920 925

TTC TTT ATC GAC TGG GAC AAA TAC AAT AAA CGC TAC AGC ACT TGG GCG 2832

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

930 935 940

GGT GTT TCT CAA TTA GTT TAC TAC CCG GAA AAC TAT ATT GAT CCG ACC 2880

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

945 950 955 960

ATG CGT ATC GGA CAA ACC AAA ATG ATG GAC GCA TTA CTG CAA TCC GTC 2928

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

965 970 975

AGC CAA AGC CAA TTA AAC GCC GAT ACC GTC GAA GAT GCC TTT ATG TCT 2976

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

980 985 990

TAT CTG ACA TCG TTT GAA CAA GTG GCT AAT CTT AAA GTT ATT AGC GCA 3024

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

995 1000 1005

TAT CAC GAT AAT ATT AAT AAC GAT CAA GGG CTG ACC TAT TTT ATC GGA 3072

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

1010 1015 1020

CTC AGT GAA ACT GAT GCC GGT GAA TAT TAT TGG CGC AGT GTC GAT CAC 3120

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

1025 1030 1035 1040

AGT AAA TTC AAC GAC GGT AAA TTC GCG GCT AAT GCC TGG AGT GAA TGG 3168

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

1045 1050 1055

CAT AAA ATT GAT TGT CCA ATT AAC CCT TAT AAA AGC ACT ATC CGT CCA 3216

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

1060 1065 1070

GTG ATA TAT AAA TCC CGC CTG TAT CTG CTC TGG TTG GAA CAA AAG GAG 3264

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

1075 1080 1085

ATC ACC AAA CAG ACA GGA AAT AGT AAA GAT GGC TAT CAA ACT GAA ACG 3312

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

1090 1095 1100

GAT TAT CGT TAT GAA CTA AAA TTG GCG CAT ATC CGC TAT GAT GGC ACT 3360

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

1105 1110 1115 1120

TGG AAT ACG CCA ATC ACC TTT GAT GTC AAT AAA AAA ATA TCC GAG CTA 3408

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

1125 1130 1135

AAA CTG GAA AAA AAT AGA GCG CCC GGA CTC TAT TGT GCC GGT TAT CAA 3456

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

1140 1145 1150

GGT GAA GAT ACG TTG CTG GTG ATG TTT TAT AAC CAA CAA GAC ACA CTA 3504

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

1155 1160 1165

GAT AGT TAT AAA AAC GCT TCA ATG CAA GGA CTA TAT ATC TTT GCT GAT 3552

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

1170 1175 1180

ATG GCA TCC AAA GAT ATG ACC CCA GAA CAG AGC AAT GTT TAT CGG GAT 3600

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

1185 1190 1195 1200

AAT AGC TAT CAA CAA TTT GAT ACC AAT AAT GTC AGA AGA GTG AAT AAC 3648

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

1205 1210 1215

CGC TAT GCA GAG GAT TAT GAG ATT CCT TCC TCG GTA AGT AGC CGT AAA 3696

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

1220 1225 1230

GAC TAT GGT TGG GGA GAT TAT TAC CTC AGC ATG GTA TAT AAC GGA GAT 3744

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

1235 1240 1245

ATT CCA ACT ATC AAT TAC AAA GCC GCA TCA AGT GAT TTA AAA ATC TAT 3792

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

1250 1255 1260

ATC TCA CCA AAA TTA AGA ATT ATT CAT AAT GGA TAT GAA GGA CAG AAG 3840

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

1265 1270 1275 1280

CGC AAT CAA TGC AAT CTG ATG AAT AAA TAT GGC AAA CTA GGT GAT AAA 3888

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

1285 1290 1295

TTT ATT GTT TAT ACT AGC TTG GGG GTC AAT CCA AAT AAC TCG TCA AAT 3936

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1300 1305 1310

AAG CTC ATG TTT TAC CCC GTC TAT CAA TAT AGC GGA AAC ACC AGT GGA 3984

Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly

1315 1320 1325

CTC AAT CAA GGG AGA CTA CTA TTC CAC CGT GAC ACC ACT TAT CCA TCT 4032

Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser

1330 1335 1340

AAA GTA GAA GCT TGG ATT CCT GGA GCA AAA CGT TCT CTA ACC AAC CAA 4080

Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln

1345 1350 1355 1360

AAT GCC GCC ATT GGT GAT GAT TAT GCT ACA GAC TCT CTG AAT AAA CCG 4128

Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro

1365 1370 1375

GAT GAT CTT AAG CAA TAT ATC TTT ATG ACT GAC AGT AAA GGG ACT GCT 4176

Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala

1380 1385 1390

ACT GAT GTC TCA GGC CCA GTA GAG ATT AAT ACT GCA ATT TCT CCA GCA 4224

Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala

1395 1400 1405

AAA GTT CAG ATA ATA GTC AAA GCG GGT GGC AAG GAG CAA ACT TTT ACC 4272

Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr

1410 1415 1420

GCA GAT AAA GAT GTC TCC ATT CAG CCA TCA CCT AGC TTT GAT GAA ATG 4320

Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met

1425 1430 1435 1440

AAT TAT CAA TTT AAT GCC CTT GAA ATA GAC GGT TCT GGT CTG AAT TTT 4368

Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe

1445 1450 1455

ATT AAC AAC TCA GCC AGT ATT GAT GTT ACT TTT ACC GCA TTT GCG GAG 4416

Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu

1460 1465 1470

GAT GGC CGC AAA CTG GGT TAT GAA AGT TTC AGT ATT CCT GTT ACC CTC 4464

Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu

1475 1480 1485

AAG GTA AGT ACC GAT AAT GCC CTG ACC CTG CAC CAT AAT GAA AAT GGT 4512

Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly

1490 1495 1500

GCG CAA TAT ATG CAA TGG CAA TCC TAT CGT ACC CGC CTG AAT ACT CTA 4560

Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu

1505 1510 1515 1520

TTT GCC CGC CAG TTG GTT GCA CGC GCC ACC ACC GGA ATC GAT ACA ATT 4608

Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile

1525 1530 1535

CTG AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAG TTA GGC AAA GGT 4656

Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly

1540 1545 1550

TTC TAT GCT ACG TTC GTG ATA CCT CCC TAT AAC CTA TCA ACT CAT GGT 4704

Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly

1555 1560 1565

GAT GAA CGT TGG TTT AAG CTT TAT ATC AAA CAT GTT GTT GAT AAT AAT 4752

Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn

1570 1575 1580

TCA CAT ATT ATC TAT TCA GGC CAG CTA ACA GAT ACA AAT ATA AAC ATC 4800

Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile

1585 1590 1595 1600

ACA TTA TTT ATT CCT CTT GAT GAT GTC CCA TTG AAT CAA GAT TAT CAC 4848

Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His

1605 1610 1615

GCC AAG GTT TAT ATG ACC TTC AAG AAA TCA CCA TCA GAT GGT ACC TGG 4896

Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp

1620 1625 1630

TGG GGC CCT CAC TTT GTT AGA GAT GAT AAA GGA ATA GTA ACA ATA AAC 4944

Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn

1635 1640 1645

CCT AAA TCC ATT TTG ACC CAT TTT GAG AGC GTC AAT GTC CTG AAT AAT 4992

Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn

1650 1655 1660

ATT AGT AGC GAA CCA ATG GAT TTC AGC GGC GCT AAC AGC CTC TAT TTC 5040

Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe

1665 1670 1675 1680

TGG GAA CTG TTC TAC TAT ACC CCG ATG CTG GTT GCT CAA CGT TTG CTG 5088

Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu

1685 1690 1695

CAT GAA CAG AAC TTC GAT GAA GCC AAC CGT TGG CTG AAA TAT GTC TGG 5136

His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp

1700 1705 1710

AGT CCA TCC GGT TAT ATT GTC CAC GGC CAG ATT CAG AAC TAC CAG TGG 5184

Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp

1715 1720 1725

AAC GTC CGC CCG TTA CTG GAA GAC ACC AGT TGG AAC AGT GAT CCT TTG 5232

Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu

1730 1735 1740

GAT TCC GTC GAT CCT GAC GCG GTA GCA CAG CAC GAT CCA ATG CAC TAC 5280

Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr

1745 1750 1755 1760

AAA GTT TCA ACT TTT ATG CGT ACC TTG GAT CTA TTG ATA GCA CGC GGC 5328

Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly

1765 1770 1775

GAC CAT GCT TAT CGC CAA CTG GAA CGA GAT ACA CTC AAC GAA GCG AAG 5376

Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys

1780 1785 1790

ATG TGG TAT ATG CAA GCG CTG CAT CTA TTA GGT GAC AAA CCT TAT CTA 5424

Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu

1795 1800 1805

CCG CTG AGT ACG ACA TGG AGT GAT CCA CGA CTA GAC AGA GCC GCG GAT 5472

Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp

1810 1815 1820

ATC ACT ACC CAA AAT GCT CAC GAC AGC GCA ATA GTC GCT CTG CGG CAG 5520

Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln

1825 1830 1835 1840

AAT ATA CCT ACA CCG GCA CCT TTA TCA 5547

Asn Ile Pro Thr Pro Ala Pro Leu Ser

1845

1849 amino acids

amino acids

linear

protein

49

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val

1 5 10 15

Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr

20 25 30

Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr

35 40 45

Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln

50 55 60

Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu

65 70 75 80

Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys

85 90 95

Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr

100 105 110

His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro

115 120 125

Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His

130 135 140

Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe

145 150 155 160

Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr

165 170 175

Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu

180 185 190

Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe

195 200 205

Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln

210 215 220

Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr

225 230 235 240

Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu

245 250 255

Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys

260 265 270

Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg

275 280 285

Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser

290 295 300

Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile

305 310 315 320

Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala

325 330 335

Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu

340 345 350

Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile

355 360 365

Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr

370 375 380

Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr

385 390 395 400

Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser

405 410 415

Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn

420 425 430

Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile

435 440 445

Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys

450 455 460

Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile

465 470 475 480

Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn

485 490 495

Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu

500 505 510

Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys

515 520 525

Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg

530 535 540

Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val

545 550 555 560

Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr

565 570 575

Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly

580 585 590

Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile

595 600 605

Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu

610 615 620

Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu

625 630 635 640

Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu

645 650 655

Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala

660 665 670

Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe

675 680 685

Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala

690 695 700

Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala

705 710 715 720

Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala

725 730 735

Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn

740 745 750

Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His

755 760 765

Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr

770 775 780

Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn

785 790 795 800

Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln

805 810 815

Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly

820 825 830

Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala

835 840 845

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

850 855 860

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

865 870 875 880

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

885 890 895

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

900 905 910

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

915 920 925

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

930 935 940

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

945 950 955 960

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

965 970 975

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

980 985 990

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

995 1000 1005

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

1010 1015 1020

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

1025 1030 1035 1040

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

1045 1050 1055

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

1060 1065 1070

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

1075 1080 1085

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

1090 1095 1100

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

1105 1110 1115 1120

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

1125 1130 1135

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

1140 1145 1150

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

1155 1160 1165

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

1170 1175 1180

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

1185 1190 1195 1200

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

1205 1210 1215

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

1220 1225 1230

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

1235 1240 1245

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

1250 1255 1260

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

1265 1270 1275 1280

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

1285 1290 1295

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1300 1305 1310

Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly

1315 1320 1325

Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser

1330 1335 1340

Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln

1345 1350 1355 1360

Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro

1365 1370 1375

Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala

1380 1385 1390

Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala

1395 1400 1405

Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr

1410 1415 1420

Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met

1425 1430 1435 1440

Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe

1445 1450 1455

Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu

1460 1465 1470

Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu

1475 1480 1485

Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly

1490 1495 1500

Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu

1505 1510 1515 1520

Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile

1525 1530 1535

Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly

1540 1545 1550

Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly

1555 1560 1565

Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn

1570 1575 1580

Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile

1585 1590 1595 1600

Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His

1605 1610 1615

Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp

1620 1625 1630

Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn

1635 1640 1645

Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn

1650 1655 1660

Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe

1665 1670 1675 1680

Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu

1685 1690 1695

His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp

1700 1705 1710

Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp

1715 1720 1725

Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu

1730 1735 1740

Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr

1745 1750 1755 1760

Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly

1765 1770 1775

Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys

1780 1785 1790

Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu

1795 1800 1805

Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp

1810 1815 1820

Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln

1825 1830 1835 1840

Asn Ile Pro Thr Pro Ala Pro Leu Ser

1845

1740 base pairs

nucleic acid

double

linear

DNA (genomic)

50

TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC AAT 48

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn

1 5 10 15

GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC AAT 96

Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn

20 25 30

CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA ATC 144

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile

35 40 45

TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT GCC 192

Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala

50 55 60

ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG TGG 240

Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp

65 70 75 80

CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG CTC 288

Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu

85 90 95

ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC GCG 336

Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala

100 105 110

GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA TTG 384

Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu

115 120 125

ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC GAG 432

Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu

130 135 140

AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT GAT 480

Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp

145 150 155 160

AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC CAA 528

Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln

165 170 175

GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT CAG 576

Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln

180 185 190

GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC TTC 624

Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe

195 200 205

GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG ACA 672

Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr

210 215 220

GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG GAT 720

Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp

225 230 235 240

AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG GAG 768

Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu

245 250 255

ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT CAG 816

Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln

260 265 270

CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA ACC 864

Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr

275 280 285

AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC CTG 912

Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu

290 295 300

CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT CGA 960

Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg

305 310 315 320

CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT TGC 1008

Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys

325 330 335

CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT GCC 1056

Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala

340 345 350

CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG CTT 1104

Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT CAT 1152

Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His

370 375 380

CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG CTG 1200

Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC CTG 1248

Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu

405 410 415

GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC GGC 1296

Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly

420 425 430

AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA ACC 1344

Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr

435 440 445

TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA GAT 1392

Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp

450 455 460

TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC GTC 1440

Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val

465 470 475 480

ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA TTG 1488

Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu

485 490 495

TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG GCA 1536

Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala

500 505 510

GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC AAC 1584

Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn

515 520 525

GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC ACG 1632

Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr

530 535 540

CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA CAA 1680

Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln

545 550 555 560

GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC TAC 1728

Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr

565 570 575

ACC ATT AAA TAA 1740

Thr Ile Lys

579 amino acids

amino acids

linear

protein

51

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn

1 5 10 15

Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn

20 25 30

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile

35 40 45

Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala

50 55 60

Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp

65 70 75 80

Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu

85 90 95

Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala

100 105 110

Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu

115 120 125

Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu

130 135 140

Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp

145 150 155 160

Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln

165 170 175

Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln

180 185 190

Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe

195 200 205

Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr

210 215 220

Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp

225 230 235 240

Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu

245 250 255

Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln

260 265 270

Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr

275 280 285

Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu

290 295 300

Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg

305 310 315 320

Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys

325 330 335

Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala

340 345 350

Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His

370 375 380

Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu

405 410 415

Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly

420 425 430

Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr

435 440 445

Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp

450 455 460

Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val

465 470 475 480

Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu

485 490 495

Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala

500 505 510

Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn

515 520 525

Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr

530 535 540

Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln

545 550 555 560

Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr

565 570 575

Thr Ile Lys

5532 base pairs

nucleic acid

double

linear

DNA (genomic)

52

TTT ATA CAA GGT TAT AGT GAT CTG TTT GGT AAT CGT GCT GAT AAC TAT 48

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr

1 5 10 15

GCC GCG CCG GGC TCG GTT GCA TCG ATG TTC TCA CCG GCG GCT TAT TTG 96

Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu

20 25 30

ACG GAA TTG TAC CGT GAA GCC AAA AAC TTG CAT GAC AGC AGC TCA ATT 144

Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile

35 40 45

TAT TAC CTA GAT AAA CGT CGC CCG GAT TTA GCA AGC TTA ATG CTC AGC 192

Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser

50 55 60

CAG AAA AAT ATG GAT GAG GAA ATT TCA ACG CTG GCT CTC TCT AAT GAA 240

Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu

65 70 75 80

TTG TGC CTT GCC GGG ATC GAA ACA AAA ACA GGA AAA TCA CAA GAT GAA 288

Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu

85 90 95

GTG ATG GAT ATG TTG TCA ACT TAT CGT TTA AGT GGA GAG ACA CCT TAT 336

Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr

100 105 110

CAT CAC GCT TAT GAA ACT GTT CGT GAA ATC GTT CAT GAA CGT GAT CCA 384

His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro

115 120 125

GGA TTT CGT CAT TTG TCA CAG GCA CCC ATT GTT GCT GCT AAG CTC GAT 432

Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp

130 135 140

CCT GTG ACT TTG TTG GGT ATT AGC TCC CAT ATT TCG CCA GAA CTG TAT 480

Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr

145 150 155 160

AAC TTG CTG ATT GAG GAG ATC CCG GAA AAA GAT GAA GCC GCG CTT GAT 528

Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp

165 170 175

ACG CTT TAT AAA ACA AAC TTT GGC GAT ATT ACT ACT GCT CAG TTA ATG 576

Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met

180 185 190

TCC CCA AGT TAT CTG GCC CGG TAT TAT GGC GTC TCA CCG GAA GAT ATT 624

Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile

195 200 205

GCC TAC GTG ACG ACT TCA TTA TCA CAT GTT GGA TAT AGC AGT GAT ATT 672

Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile

210 215 220

CTG GTT ATT CCG TTG GTC GAT GGT GTG GGT AAG ATG GAA GTA GTT CGT 720

Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg

225 230 235 240

GTT ACC CGA ACA CCA TCG GAT AAT TAT ACC AGT CAG ACG AAT TAT ATT 768

Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile

245 250 255

GAG CTG TAT CCA CAG GGT GGC GAC AAT TAT TTG ATC AAA TAC AAT CTA 816

Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu

260 265 270

AGC AAT AGT TTT GGT TTG GAT GAT TTT TAT CTG CAA TAT AAA GAT GGT 864

Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly

275 280 285

TCC GCT GAT TGG ACT GAG ATT GCC CAT AAT CCC TAT CCT GAT ATG GTC 912

Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val

290 295 300

ATA AAT CAA AAG TAT GAA TCA CAG GCG ACA ATC AAA CGT AGT GAC TCT 960

Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser

305 310 315 320

GAC AAT ATA CTC AGT ATA GGG TTA CAA AGA TGG CAT AGC GGT AGT TAT 1008

Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr

325 330 335

AAT TTT GCC GCC GCC AAT TTT AAA ATT GAC CAA TAC TCC CCG AAA GCT 1056

Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala

340 345 350

TTC CTG CTT AAA ATG AAT AAG GCT ATT CGG TTG CTC AAA GCT ACC GGC 1104

Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly

355 360 365

CTC TCT TTT GCT ACG TTG GAG CGT ATT GTT GAT AGT GTT AAT AGC ACC 1152

Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr

370 375 380

AAA TCC ATC ACG GTT GAG GTA TTA AAC AAG GTT TAT CGG GTA AAA TTC 1200

Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe

385 390 395 400

TAT ATT GAT CGT TAT GGC ATC AGT GAA GAG ACA GCC GCT ATT TTG GCT 1248

Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala

405 410 415

AAT ATT AAT ATC TCT CAG CAA GCT GTT GGC AAT CAG CTT AGC CAG TTT 1296

Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe

420 425 430

GAG CAA CTA TTT AAT CAC CCG CCG CTC AAT GGT ATT CGC TAT GAA ATC 1344

Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile

435 440 445

AGT GAG GAC AAC TCC AAA CAT CTT CCT AAT CCT GAT CTG AAC CTT AAA 1392

Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys

450 455 460

CCA GAC AGT ACC GGT GAT GAT CAA CGC AAG GCG GTT TTA AAA CGC GCG 1440

Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala

465 470 475 480

TTT CAG GTT AAC GCC AGT GAG TTG TAT CAG ATG TTA TTG ATC ACT GAT 1488

Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp

485 490 495

CGT AAA GAA GAC GGT GTT ATC AAA AAT AAC TTA GAG AAT TTG TCT GAT 1536

Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp

500 505 510

CTG TAT TTG GTT AGT TTG CTG GCC CAG ATT CAT AAC CTG ACT ATT GCT 1584

Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala

515 520 525

GAA TTG AAC ATT TTG TTG GTG ATT TGT GGC TAT GGC GAC ACC AAC ATT 1632

Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile

530 535 540

TAT CAG ATT ACC GAC GAT AAT TTA GCC AAA ATA GTG GAA ACA TTG TTG 1680

Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu

545 550 555 560

TGG ATC ACT CAA TGG TTG AAG ACC CAA AAA TGG ACA GTT ACC GAC CTG 1728

Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu

565 570 575

TTT CTG ATG ACC ACG GCC ACT TAC AGC ACC ACT TTA ACG CCA GAA ATT 1776

Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile

580 585 590

AGC AAT CTG ACG GCT ACG TTG TCT TCA ACT TTG CAT GGC AAA GAG AGT 1824

Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser

595 600 605

CTG ATT GGG GAA GAT CTG AAA AGA GCA ATG GCG CCT TGC TTC ACT TCG 1872

Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser

610 615 620

GCT TTG CAT TTG ACT TCT CAA GAA GTT GCG TAT GAC CTG CTG TTG TGG 1920

Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp

625 630 635 640

ATA GAC CAG ATT CAA CCG GCA CAA ATA ACT GTT GAT GGG TTT TGG GAA 1968

Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu

645 650 655

GAA GTG CAA ACA ACA CCA ACC AGC TTG AAG GTG ATT ACC TTT GCT CAG 2016

Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln

660 665 670

GTG CTG GCA CAA TTG AGC CTG ATC TAT CGT CGT ATT GGG TTA AGT GAA 2064

Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu

675 680 685

ACG GAA CTG TCA CTG ATC GTG ACT CAA TCT TCT CTG CTA GTG GCA GGC 2112

Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly

690 695 700

AAA AGC ATA CTG GAT CAC GGT CTG TTA ACC CTG ATG GCC TTG GAA GGT 2160

Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly

705 710 715 720

TTT CAT ACC TGG GTT AAT GGC TTG GGG CAA CAT GCC TCC TTG ATA TTG 2208

Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu

725 730 735

GCG GCG TTG AAA GAC GGA GCC TTG ACA GTT ACC GAT GTA GCA CAA GCT 2256

Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala

740 745 750

ATG AAT AAG GAG GAA TCT CTC CTA CAA ATG GCA GCT AAT CAG GTG GAG 2304

Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu

755 760 765

AAG GAT CTA ACA AAA CTG ACC AGT TGG ACA CAG ATT GAC GCT ATT CTG 2352

Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu

770 775 780

CAA TGG TTA CAG ATG TCT TCG GCC TTG GCG GTT TCT CCA CTG GAT CTG 2400

Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu

785 790 795 800

GCA GGG ATG ATG GCC CTG AAA TAT GGG ATA GAT CAT AAC TAT GCT GCC 2448

Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala

805 810 815

TGG CAA GCT GCG GCG GCT GCG CTG ATG GCT GAT CAT GCT AAT CAG GCA 2496

Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala

820 825 830

CAG AAA AAA CTG GAT GAG ACG TTC AGT AAG GCA TTA TGT AAC TAT TAT 2544

Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr

835 840 845

ATT AAT GCT GTT GTC GAT AGT GCT GCT GGA GTA CGT GAT CGT AAC GGT 2592

Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly

850 855 860

TTA TAT ACC TAT TTG CTG ATT GAT AAT CAG GTT TCT GCC GAT GTG ATC 2640

Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile

865 870 875 880

ACT TCA CGT ATT GCA GAA GCT ATC GCC GGT ATT CAA CTG TAC GTT AAC 2688

Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn

885 890 895

CGG GCT TTA AAC CGA GAT GAA GGT CAG CTT GCA TCG GAC GTT AGT ACC 2736

Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr

900 905 910

CGT CAG TTC TTC ACT GAC TGG GAA CGT TAC AAT AAA CGT TAC AGT ACT 2784

Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr

915 920 925

TGG GCT GGT GTC TCT GAA CTG GTC TAT TAT CCA GAA AAC TAT GTT GAT 2832

Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp

930 935 940

CCC ACT CAG CGC ATT GGG CAA ACC AAA ATG ATG GAT GCG CTG TTG CAA 2880

Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln

945 950 955 960

TCC ATC AAC CAG AGC CAG CTA AAT GCG GAT ACG GTG GAA GAT GCT TTC 2928

Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe

965 970 975

AAA ACT TAT TTG ACC AGC TTT GAG CAG GTA GCA AAT CTG AAA GTA ATT 2976

Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile

980 985 990

AGT GCT TAC CAC GAT AAT GTG AAT GTG GAT CAA GGA TTA ACT TAT TTT 3024

Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe

995 1000 1005

ATC GGT ATC GAC CAA GCA GCT CCG GGT ACG TAT TAC TGG CGT AGT GTT 3072

Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val

1010 1015 1020

GAT CAC AGC AAA TGT GAA AAT GGC AAG TTT GCC GCT AAT GCT TGG GGT 3120

Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly

1025 1030 1035 1040

GAG TGG AAT AAA ATT ACC TGT GCT GTC AAT CCT TGG AAA AAT ATC ATC 3168

Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile

1045 1050 1055

CGT CCG GTT GTT TAT ATG TCC CGC TTA TAT CTG CTA TGG CTG GAG CAG 3216

Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln

1060 1065 1070

CAA TCA AAG AAA AGT GAT GAT GGT AAA ACC ACG ATT TAT CAA TAT AAC 3264

Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn

1075 1080 1085

TTA AAA CTG GCT CAT ATT CGT TAC GAC GGT AGT TGG AAT ACA CCA TTT 3312

Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe

1090 1095 1100

ACT TTT GAT GTG ACA GAA AAG GTA AAA AAT TAC ACG TCG AGT ACT GAT 3360

Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp

1105 1110 1115 1120

GCT GCT GAA TCT TTA GGG TTG TAT TGT ACT GGT TAT CAA GGG GAA GAC 3408

Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp

1125 1130 1135

ACT CTA TTA GTT ATG TTC TAT TCG ATG CAG AGT AGT TAT AGC TCC TAT 3456

Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr

1140 1145 1150

ACC GAT AAT AAT GCG CCG GTC ACT GGG CTA TAT ATT TTC GCT GAT ATG 3504

Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met

1155 1160 1165

TCA TCA GAC AAT ATG ACG AAT GCA CAA GCA ACT AAC TAT TGG AAT AAC 3552

Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn

1170 1175 1180

AGT TAT CCG CAA TTT GAT ACT GTG ATG GCA GAT CCG GAT AGC GAC AAT 3600

Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn

1185 1190 1195 1200

AAA AAA GTC ATA ACC AGA AGA GTT AAT AAC CGT TAT GCG GAG GAT TAT 3648

Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr

1205 1210 1215

GAA ATT CCT TCC TCT GTG ACA AGT AAC AGT AAT TAT TCT TGG GGT GAT 3696

Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp

1220 1225 1230

CAC AGT TTA ACC ATG CTT TAT GGT GGT AGT GTT CCT AAT ATT ACT TTT 3744

His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe

1235 1240 1245

GAA TCG GCG GCA GAA GAT TTA AGG CTA TCT ACC AAT ATG GCA TTG AGT 3792

Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser

1250 1255 1260

ATT ATT CAT AAT GGA TAT GCG GGA ACC CGC CGT ATA CAA TGT AAT CTT 3840

Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu

1265 1270 1275 1280

ATG AAA CAA TAC GCT TCA TTA GGT GAT AAA TTT ATA ATT TAT GAT TCA 3888

Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser

1285 1290 1295

TCA TTT GAT GAT GCA AAC CGT TTT AAT CTG GTG CCA TTG TTT AAA TTC 3936

Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe

1300 1305 1310

GGA AAA GAC GAG AAC TCA GAT GAT AGT ATT TGT ATA TAT AAT GAA AAC 3984

Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn

1315 1320 1325

CCT TCC TCT GAA GAT AAG AAG TGG TAT TTT TCT TCG AAA GAT GAC AAT 4032

Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn

1330 1335 1340

AAA ACA GCG GAT TAT AAT GGT GGA ACT CAA TGT ATA GAT GCT GGA ACC 4080

Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr

1345 1350 1355 1360

AGT AAC AAA GAT TTT TAT TAT AAT CTC CAG GAG ATT GAA GTA ATT AGT 4128

Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser

1365 1370 1375

GTT ACT GGT GGG TAT TGG TCG AGT TAT AAA ATA TCC AAC CCG ATT AAT 4176

Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn

1380 1385 1390

ATC AAT ACG GGC ATT GAT AGT GCT AAA GTA AAA GTC ACC GTA AAA GCG 4224

Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala

1395 1400 1405

GGT GGT GAC GAT CAA ATC TTT ACT GCT GAT AAT AGT ACC TAT GTT CCT 4272

Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro

1410 1415 1420

CAG CAA CCG GCA CCC AGT TTT GAG GAG ATG ATT TAT CAG TTC AAT AAC 4320

Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn

1425 1430 1435 1440

CTG ACA ATA GAT TGT AAG AAT TTA AAT TTC ATC GAC AAT CAG GCA CAT 4368

Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His

1445 1450 1455

ATT GAG ATT GAT TTC ACC GCT ACG GCA CAA GAT GGC CGA TTC TTG GGT 4416

Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly

1460 1465 1470

GCA GAA ACT TTT ATT ATC CCG GTA ACT AAA AAA GTT CTC GGT ACT GAG 4464

Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu

1475 1480 1485

AAC GTG ATT GCG TTA TAT AGC GAA AAT AAC GGT GTT CAA TAT ATG CAA 4512

Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln

1490 1495 1500

ATT GGC GCA TAT CGT ACC CGT TTG AAT ACG TTA TTC GCT CAA CAG TTG 4560

Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu

1505 1510 1515 1520

GTT AGC CGT GCT AAT CGT GGC ATT GAT GCA GTG CTC AGT ATG GAA ACT 4608

Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr

1525 1530 1535

CAG AAT ATT CAG GAA CCG CAA TTA GGA GCG GGC ACA TAT GTG CAG CTT 4656

Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu

1540 1545 1550

GTG TTG GAT AAA TAT GAT GAG TCT ATT CAT GGC ACT AAT AAA AGC TTT 4704

Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe

1555 1560 1565

GCT ATT GAA TAT GTT GAT ATA TTT AAA GAG AAC GAT AGT TTT GTG ATT 4752

Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile

1570 1575 1580

TAT CAA GGA GAA CTT AGC GAA ACA AGT CAA ACT GTT GTG AAA GTT TTC 4800

Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe

1585 1590 1595 1600

TTA TCC TAT TTT ATA GAG GCG ACT GGA AAT AAG AAC CAC TTA TGG GTA 4848

Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val

1605 1610 1615

CGT GCT AAA TAC CAA AAG GAA ACG ACT GAT AAG ATC TTG TTC GAC CGT 4896

Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg

1620 1625 1630

ACT GAT GAG AAA GAT CCG CAC GGT TGG TTT CTC AGC GAC GAT CAC AAG 4944

Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys

1635 1640 1645

ACC TTT AGT GGT CTC TCT TCC GCA CAG GCA TTA AAG AAC GAC AGT GAA 4992

Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu

1650 1655 1660

CCG ATG GAT TTC TCT GGC GCC AAT GCT CTC TAT TTC TGG GAA CTG TTC 5040

Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe

1665 1670 1675 1680

TAT TAC ACG CCG ATG ATG ATG GCT CAT CGT TTG TTG CAG GAA CAG AAT 5088

Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn

1685 1690 1695

TTT GAT GCG GCG AAC CAT TGG TTC CGT TAT GTC TGG AGT CCA TCC GGT 5136

Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly

1700 1705 1710

TAT ATC GTT GAT GGT AAA ATT GCT ATC TAC CAC TGG AAC GTG CGA CCG 5184

Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro

1715 1720 1725

CTG GAA GAA GAC ACC AGT TGG AAT GCA CAA CAA CTG GAC TCC ACC GAT 5232

Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp

1730 1735 1740

CCA GAT GCT GTA GCC CAA GAT GAT CCG ATG CAC TAC AAG GTG GCT ACC 5280

Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr

1745 1750 1755 1760

TTT ATG GCG ACG TTG GAT CTG CTA ATG GCC CGT GGT GAT GCT GCT TAC 5328

Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr

1765 1770 1775

CGC CAG TTA GAG CGT GAT ACG TTG GCT GAA GCT AAA ATG TGG TAT ACA 5376

Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr

1780 1785 1790

CAG GCG CTT AAT CTG TTG GGT GAT GAG CCA CAA GTG ATG CTG AGT ACG 5424

Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr

1795 1800 1805

ACT TGG GCT AAT CCA ACA TTG GGT AAT GCT GCT TCA AAA ACC ACA CAG 5472

Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln

1810 1815 1820

CAG GTT CGT CAG CAA GTG CTT ACC CAG TTG CGT CTC AAT AGC AGG GTA 5520

Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val

1825 1830 1835 1840

AAA ACC CCG TTG 5532

Lys Thr Pro Leu

1844

1844 amino acids

amino acids

linear

protein

53

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr

1 5 10 15

Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu

20 25 30

Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile

35 40 45

Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser

50 55 60

Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu

65 70 75 80

Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu

85 90 95

Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr

100 105 110

His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro

115 120 125

Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp

130 135 140

Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr

145 150 155 160

Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp

165 170 175

Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met

180 185 190

Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile

195 200 205

Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile

210 215 220

Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg

225 230 235 240

Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile

245 250 255

Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu

260 265 270

Ser As