Isolation of biosynthesis genes for pseudo-oligosaccharides from Streptomyces glaucescens GLA.O, and their use

Synthesis and Sequences of the PCR Primers and Amplification of the Fragments from S. glaucescens GLA.O

The PCR was carried out under standard conditions using in each case 100 pmol of primer 1 and of primer 2 in 100 μl of reaction mixture

The samples are overlaid with 75 μl of mineral oil and the amplification is carried out using a Perkin Elmer TC1 DNA thermal cyler.

Parameters:

Table 1 lists the sequences of the degenerate primers which should be used for amplifying dTDP-glucose dehydratases from different streptomycetes .

DNA Sequences of the PCR Fragments Isolated From Streptomyces glaucescens GLA.O

The sequencing was performed by the dideoxy chain termination method of Sanger et al. (PNAS USA, 74: 5463-5467 (1977)). The reactions were carried out using the Auto Read Sequenzing Kit® from Pharmacia Biotech (Freiburg, Germany) in accordance with the manufacturer's instructions. An ALF DNA Sequencer® from Pharmacia Biotech (Freiburg, Germany) was used for separation and detection.

The subsequent cloning of the PCR fragments (Sure Clone Kit®, Pharmacia Biotech, Frieburg) into the E. coli vector pUC 18, and the sequencing of the fragment, provided support for the supposition that the fragment encoded a dTDP-glucose 4,6-dehydratase. However, 2 different genes were isolated which both exhibit high degrees of homology with dTDP-glucose 4,6-dehydratase but are not identical. In that which follows, the PCR fragments are designated acbD and HstrE.

The sequences of the isolated PCR fragments are shown in Table 2A and 2B and the homology comparison of the deduced amino acid sequences of HstrE and acbD is shown in Table 2C. The two proteins exhibit an identity of only 65%.

Southern Analysis Using Chromosomal DNA From Streptomyces glaucescens GLA.O and the Isolated and Labeled PCR Fragments

The cells were grown in R2YENG medium and harvested for the DNA isolation after 30 h. The chromosomal DNA was isolated from S. glaucescens GLA.O as described in Hopwood et al. (1985) Genetic manipulations of Streptomyces : a laboratory manual. The John Innes Foundation, Norwich UK).

A Southern blot analysis was carried out using the S. glaucescens GLA.O producer strain chromosomal DNA, which was digested with Pstl, BgIII and BamHI, using the labeled probes consisting of the acbD and HstrE PCR fragments. The two PCR fragments were labeled with digoxygenin in accordance with the manufacturer's (Boehringer Mannheim; Mannheim) instructions, and a digest of the Streptomyces glaucescens GLA.O producer strain chromosomal DNA was separated on an agarose gel. The DNA was transferred by capillary transfer to nylon membranes and DNA regions which were homologous with the labeled probes were subsequently visualized following hybridization.

The two genes label different DNA regions (FIG. 1 and FIG. 2 ), with the fragments which were labeled by HstrE having to be gene fragments from Streptomyces glaucescens GLA.O hydroxystreptomycin biosynthesis. While the DNA sequence is not published, the high degree of homology of the protein sequence deduced from HstrE with StrE (Pissowotzki et al. (1991) Mol. Gen. Genet. 231: 113-123) from Streptomyces griseus N2-3-11 streptomycin biosynthesis (82% identity) and the concordance of the HstrE-labeled DNA fragments (FIG. 1) with the published restriction map of the Streptomyces glaucescens GLA.O hydroxystreptomycin gene cluster (Retzlaff et al. (1993) Industrial Microorganisms. Basic and applied molecular genetics ASM, Washington D.C., USA) permits this conclusion. The fragments which were labeled by the acbD probe (FIG. 2) belong to a DNA region which has not previously been investigated. This region encodes the enzymes for biosynthesizing the Streptomyces glaucescens GLA.O pseudo-oligosaccharides.

Cloning the 6.8 kb Pstl Fragment

Inter alia, the acbD PCR fragment labels a 6.8 kB Pstl DNA fragment (FIG. 2 ). This DNA fragment was isolated as follows. The region of the gel was excised with a razor blade and the DNA was isolated from the gel using an isolation kit from Pharmacia Biotech and cloned into plasmid pUC19 which had been cut with the restriction enzyme Pstl (plasmid pacb1); this latter plasmid was then transformed into the E. coli strain DH5α. The individual clones were subcultured from these plates and a plasmid DNA isolation was carried out using these clones. A PCR amplification using the above-described primers 1 and 2 (Tab. 1) was carried out using the DNA from these clones (250). In this manner, the appropriate E. coli clone containing the 6.8 kb Pstl fragment was isolated.

Sequencing the Isolated 6.8 kb Pstl DNA Fragment

The DNA was digested with various restriction enzymes and individual DNA fragments were cloned into pUC19. The DNA sequence of the entire fragment, which is shown in Tab. 4 (SEQ ID NO.: 7), was then determined. The DNA sequence of the 6.8 kb Pstl fragment was only partially confirmed by supplementary sequencing of the opposing strand. Several open reading frames, encoding various proteins, were found (programs: CODONPREFERENCE and BlastX). A total of 6 coding regions was found, i.e. a gene having a high degree of homology with ATP-binding protein, acbA, an aminotransferase acbB, a dTDP-glucose synthase acbC, a dTDP-glucose dehydratase acbD, a regulatory gene having homologies with the Lacl protein family acbE, and a protein having similarities to sugar-binding proteins acbF. The sequences of the acbA and acbF genes were only determined in part. The homologies with other proteins from the databases, and the properties of the putative proteins, are summarized in Tab. 3. FIG. 3 shows, in summary form, a restriction map of the fragment, containing the most important restriction cleavage sites mentioned in the text, and the arrangement of the identified open reading frames.

Deletion of Genes acbBCD for Pseudo-Oligosaccharide Biosynthesis from the Streptomyces glaucescens GLA.O Chromosome

Evidence that the identified DNA fragment encoded pseudo-oligosaccharide biosynthesis genes was produced as follows. A 3.4 kb gene region (EcoR1/Sstl fragment b, FIG. 3) was replaced with the erythromycin resistance gene (1.6 kb) and cloned, together with flanking DNA regions from the 6.8 kb Pstl fragment (pacb1) into the temperature-sensitive plasmid pGM160. The plasmid was constructed as described in the following: the 2.2 kb EcoR1/HindIII fragment (c, FIG. 3) from plasmid pacb1 was cloned into pGEM7zf (Promega, Madison, Wis., USA; plasmid pacb2), and the 1 kb Sstl fragment from pacb1 (a, FIG. 3) was cloned into pUC19 (plasmid pacb3). A ligation was then carried out using the following fragments. The plasmid pGM160 (Muth et al. (1989) Mol. Gen Genet. 219:341-348) was cut with BamH/HindIII, the plasmid pacb2 was cut with Xbal/BamHI (c, FIG. 3 ), the plasmid pacb3 was cut with EcoRI/HindIII (a, FIG. 3 ), and the plasmid plJ4026 (Bibb et al. (1985) Gene 38:215-226) was cut with EcoRI/Xbal in order to isolate the 1.6 kb ermE resistance gene.

The fragments were ligated in a mixture and transformed into E. coli DH5α and selected on ampicillin. The resulting plasmid, i.e. pacb4, was isolated from E. coli DH5α, tested for its correctness by means of restriction digestion and then transferred by protoplast transformation into S. glaucescens GLA.O. The transformants were selected with thiostrepton at 27° C. in R2YENG agar. The transformants were subsequently incubated at the non-permissive temperature of 39° C. and integration of the plasmid into the genome by way of homologous recombination thereby instituted (selection with thiostrepton (25 μg/ml) and erythromycin (50 μg/ml)). Under these conditions, the only clones which can grow are those in which the plasmid has become integrated into the genome. The corresponding clones were isolated, caused to sporulate (medium 1, see below) and plated out on erythromycin-containing agar (medium 1). Individual clones were isolated once again from this plate and streaked out on both thiostrepton-containing medium and erythromycin-containing medium. The clones which were erythromycin-resistant but no longer thiostrepton-resistant were analyzed. In these clones, the acbBCD genes had been replaced with ermE. Several clones were examined and the strain S. glaucescens GLA.O Δacb was finally selected as the reference strain (erythromycin-resistant, thiostrepton-sensitive) for further investigation.

A further experiment examined whether the corresponding strain still produced acarbose. Some clones were grown and investigated for formation of the α-amylase inhibitor in a bioassay; however, no activity was found. The mutants were subsequently further characterized by means of Southern hybridization. Integration of the ermE gene had taken place at the predicted site. FIG. 4 shows a Southern hybridization which was carried out with the wild type and with the Streptomyces glaucescens GLA.O Δacb deletion mutant. The Sstl fragment from pacb3 was used as the probe. The chromosomal DNA was isolated from the wild type and mutant and digested with the enzymes Pstl and Pstl/HindIII. The fragment pattern obtained for the deletion mutant corresponds to the predicted recombination event. The wild type exhibits the unchanged 6.8 kb Pstl fragment, whereas the mutant exhibits a fragment which has been truncated by 1.8 kb (compare lanes 1 and 3 , FIG. 4 ). Integration of the ermE resistance gene additionally introduced an internal HindIII cleavage site into the Pstl fragment (compare lanes 2 and 4 , FIG. 4 ).

Inhibition of α-amylase by Acarbose

Using an enzymic test for detecting starch (TC-Starch, Boehringer-Mannheim, Cat. No. 297748), it was possible to demonstrate that the compound isolated from Streptomyces glaucescens GLA.O inhibits α-amylase. Test principle: starch is cleaved into D-glucose by amyloglucosidase. The glucose is then converted with hexokinase into glucose-6-phosphate and the latter is converted with glucose-6-phosphate dehydrogenase into D-gluconate-6-phosphate. This reaction produces NADPH, whose formation can be determined photometrically. Acarbose inhibits the α-amylase and thereby prevents the formation of D-glucose and ultimately the formation of NADPH as well.

Medium for Growing S. glaucescens GLA.O and Producing Acarbose

The fermentation was carried out, at 27° C. on an orbital shaker at 120 rpm, in 500 ml Erlenmeyer flasks which were fitted with side baffles and which contained 100 ml of medium 2. The fermentation was terminated after 2 or 3 days. The pseudo-oligosaccharides were detected in a plate diffusion test as described in Example 9. No α-amylase inhibitors were produced when medium 3 was used. This means that the production of the pseudo-oligosaccharides is inhibited by glucose. Other sugars, such as maltose and sucrose, or complex sugar sources (malt extract) can also come into consideration for producing pseudo-oligosaccharides using S. glaucescens GLA.O.

Biotest Using Mucor miehei

A suspension of spores of the strain Mucor miehei was poured into agar (medium 5) (10 5 spores/ml), and 10 ml of this mixture were in each case poured into Petri dishes. Paper test disks (6 mm diameter) were loaded with 10 μl of acarbose [lacuna] (1 mg/ml) or with a sample from an S. glaucescens culture and laid on the test plates. The plates were then incubated at 37° C. Inhibition halos appeared on the starch-containing medium 5. A plate which was prepared with glucose (medium 4) instead of starch was used as a control. On this medium, no inhibition halo formed around the filter disks loaded with active compound.

Transformation of S. glaucescens GLA.O

Protoplasts of the Streptomyces glaucescens strain were isolated as described in Motamedi and Hutchinson ((1987) PNAS USA 84: 4445-4449), and the isolated plasmid DNA was transferred into the cells by means of PEG transformation as explained in Hopwood et al. ((1985) Genetic manipulations of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich UK). The protoplasts were regenerated on R2YENG medium at 30° C. (Motamedi and Hutchinson (1987) PNAS USA 84: 4445-4449). After 18 h, the agar plates were overlaid with a thiostrepton-containing solution and incubated at 30° C. (final concentration of thiostrepton: 20 μg/ml).

Isolation of the Pseudo-Oligosaccharides from Streptomyces glaucescens GLA.O, HPLC Analysis and Mass Spectroscopy

Isolation

The culture broth was separated from the mycelium by filtration. The culture filtrate which has been obtained in this way is then loaded onto an XAD16 column, after which the column is washed with water and the active components are eluted with 30% methanol. The eluate was concentrated down to the aqueous phase and the latter was extracted with ethyl acetate in order to remove lipophilic impurities. The aqueous phase was then concentrated and the active components were further purified in 5% methanol using a biogel P2 column. The individual fractions are collected in a fraction collector. The individual fractions were analyzed by means of the Mucor miehei biotest. Active eluates were rechromatographed, for further purification, in 5% methanol on biogel P2. The material which was isolated in this way was investigated by HPLC and MS.

Southern Hybridization Using Actinoplanes sp. SE50/110 (ATCC31044)

The chromosomal DNA was isolated from the strain Actinoplanes sp. SE50/100 and digested with restriction enzymes (Pstl and BamHI). A Southern hybridization was then carried out using a probe which encompasses the coding region of the dTDP-glucose 4,6-dehydratase acbD from Streptomyces glaucescens GLA.O (fragment d, FIG. 3 ). The probe hybridizes with distinct bands from Actinoplanes sp. SE50/110 (FIG. 5, lanes 1 and 2 ). This provides the possibility of isolating the corresponding fragments from Actinoplanes sp. SE50/100 and other strain lines. Whether these DNA regions are in fact involved in the biosynthesis of acarbose remains to be demonstrated in subsequent investigations. Alternatively, the PCR primers 1 and 2 (Tab. 1) could also be used for amplifying the dTDP-glucose 4,6-dehydratase from Actinoplanes sp.

Legends:

FIG. 1 : Southern hybridization using S. glaucescens GLA.O. Lane 1 : Pstl, lane 2 : BamHI, lane 3 : BgIII. The labeled PCR fragment HstrE was used as the probe. Labeling of DNA fragments which are involved in the biosynthesis of hydroxy-streptomycin.

FIG. 2 : Southern hybridization using S. glaucescens GLA.O. Lane 1 : Pstl, lane 2 : BamHI, lane 3 : BgIII. The labeled PCR fragment acbD was used as the probe. Labeling of DNA fragments which are involved in the biosynthesis of the pseudo-oligosaccharides.

FIG. 3 : Restriction map of the 6.8 kb Pstl fragment from Streptomyces glaucescens GLA.O. Open reading frames and the direction in which each is transcribed are indicated by arrows. The fragments a, b, c and d identify DNA regions which are explained in more detail in the text.

FIG. 4 : Southern hybridization using Streptomyces glaucescens Δacb: lane 1 : Pstl, lane 2 : Pstl/HindIII, and Streptomyces glaucescens GLA.O lane 3 : Pstl, lane 4 : Pstl/HindIII. The labeled 1.0 kb Sstl fragment a (FIG. 3) was used as the probe.

FIG. 5 : Southern hybridization using Actinoplanes sp. SE50/100: lane 1 : Pstl, lane 2 : BamHI and Streptomyces glaucescens GLA.O lane 3 : Pstl. The labeled 1.0 kb SmaI/EcoRI fragment d (dTDP-glucose 4,6-hydratase, FIG. 3) was used as the probe. The arrows indicate the labeled DNA fragments (BamHI: 2.1 and 0.7 kb, Pstl: ˜11-12 kb)

Tab. 4: DNA sequence of the 6.8 kb Pstl fragment from Streptomyces glaucescens GLA.O (SEQ ID NO.: 7). The deduced amino acid sequences (SEQ ID NO.: 8-13) of the identified open reading frames are given under the DNA sequences. Start and stop codons and potential ribosome binding sites are underlined.

acbA: SEQ ID NO.: 8

acbB: SEQ ID NO.: 9

acbC: SEQ ID NO.: 10

acbD: SEQ ID NO.: 11

acbE: SEQ ID NO.: 12

acbF: SEQ ID NO.: 13

CSGGSGSSGC SGGSTTCATS GG 22

GGGWVCTGGY VSGGSCCGTA GTTG 24

546 base pairs

nucleic acid

single

linear

DNA (genomic)

3

CCCGGGCGGG GCGGGGTTCA TCGGCTCCGC CTACGTCCGC CGGCTCCTGT CGCCCGGGGC 60

CCCCGGCGGC GTCGCGGTGA CCGTCCTCGA CAAACTCACC TACGCCGGCA GCCTCGCCCG 120

CCTGCACGCG GTGCGTGACC ATCCCGGCCT CACCTTCGTC CAGGGCGACG TGTGCGACAC 180

CGCGCTCGTC GACACGCTGG CCGCGCGGCA CGACGACATC GTGCACTTCG CGGCCGAGTC 240

GCACGTCGAC CGCTCCATCA CCGACAGCGG TGCCTTCACC CGCACCAACG TGCTGGGCAC 300

CCAGGTCCTG CTCGACGCCG CGCTCCGCCA CGGTGTGCGC ACCCTCGTGC ACGTCTCCAC 360

CGACGAGGTG TACGGCTCCC TCCCGCACGG GGCCGCCGCG GAGAGCGACC CCCTGCTCCC 420

GACCTCGCCG TACGCGGCGT CGAAGGCGGC CTCGGACCTC ATGGCGCTCG CCCACCACCG 480

CACCCACGGC CTGGACGTCC GGGTGACCCG CTGTTCGAAC AACTACGGCC CGCACCAGTT 540

CCCGGG 546

541 base pairs

nucleic acid

single

linear

DNA (genomic)

4

CCCCGGGTGC TGGTAGGGGC CGTAGTTGTT GGAGCAGCGG GTGATGCGCA CGTCCAGGCC 60

GTGGCTGACG TGCATGGCCA GCGCGAGCAG GTCGCCCGAC GCCTTGGAGG TGGCATAGGG 120

GCTGTTGGGG CGCAGCGGCT CGTCCTCCGT CCACGACCCC GTCTCCAGCG AGCCGTAGAC 180

CTCGTCGGTG GACACCTGCA CGAAGGGGGC CACGCCGTGC CGCAGGGCCG CGTCGAGGAG 240

TGTCTGCGTG CCGCCGGCGT TGGTCCGCAC GAACGCGGCG GCATCGAGCA GCGAGCGGTC 300

CACGTGCGAC TCGGCGGCGA GGTGCACGAC CTGGTCCTGG CCGGCCATGA CCCGGTCGAC 360

CAGGTCCGCG TCGCAGATGT CGCCGTGGAC GAAGCGCAGC CGGGGGTGGT CGCGGACCGG 420

GTCGAGGTTG GCGAGGTTGC CGGCGTAGCT CAGGGCGTCG AGCACGGTGA CGACGGCGTC 480

GGGCGGCCCG TCCGGACCGA GGAGGGTGCG GACGTAGTGC GAGCCCATGA ACCCCGCCGC 540

C 541

180 amino acids

amino acid

<Unknown>

linear

protein

5

Ala Ala Gly Phe Met Gly Ser His Tyr Val Arg Thr Leu Leu Gly Pro

1 5 10 15

Asp Gly Pro Pro Asp Ala Val Val Thr Val Leu Asp Ala Leu Ser Tyr

20 25 30

Ala Gly Asn Leu Ala Asn Leu Asp Pro Val Arg Asp His Pro Arg Leu

35 40 45

Arg Phe Val His Gly Asp Ile Cys Asp Ala Asp Leu Val Asp Arg Val

50 55 60

Met Ala Gly Gln Asp Gln Val Val His Leu Ala Ala Glu Ser His Val

65 70 75 80

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

85 90 95

Gly Thr Gln Thr Leu Leu Asp Ala Ala Leu Arg His Gly Val Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Leu Asp Val Arg Ile Thr Arg Cys Ser Asn Asn Tyr Gly Pro Tyr

165 170 175

Gln His Pro Gly

180

181 amino acids

amino acid

<Unknown>

linear

protein

6

Pro Gly Gly Ala Gly Phe Ile Gly Ser Ala Tyr Val Arg Arg Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Thr Phe Val Gln Gly Asp Val Cys Asp Thr Ala Leu Val Asp

50 55 60

Thr Leu Ala Ala Arg His Asp Asp Ile Val His Phe Ala Ala Glu Ser

65 70 75 80

His Val Asp Arg Ser Ile Thr Asp Ser Gly Ala Phe Thr Arg Thr Asn

85 90 95

Val Leu Gly Thr Gln Val Leu Leu Asp Ala Ala Leu Arg His Gly Val

100 105 110

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

115 120 125

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

130 135 140

Ala Ala Ser Lys Ala Ala Ser Asp Leu Met Ala Leu Ala His His Arg

145 150 155 160

Thr His Gly Leu Asp Val Arg Val Thr Arg Cys Ser Asn Asn Tyr Gly

165 170 175

Pro His Gln Phe Pro

180

6854 base pairs

nucleic acid

single

linear

DNA (genomic)

7

CTGCAGGGTT CCCTGGTGCA CGACCCGCCC CTGGTCGACG ACCAGGGCGC TGTCGCAGAT 60

CGCGGCGATG TCGGCGATGT CGTGGCTGGT GAGCACCACG GTGGTGCCCA GTTCCCGGTG 120

GGCGCGGTTG ACCAGCCGGC GCACCGCGTC CTTCAGCACC ATGTCGAGGC CGATCGTGGG 180

CTCGTCCCAG AACAGCACGG CCGGGTCGTG CAGCAGGCTC GCCGCGATCT CGGCGCGCAT 240

GCGCTGTCCG AGGCTGAGCT GCCGCACGGG GGTGGACCCC AGCGCGTCGA TGTCGAGGAG 300

GTCCCGGAAC AGGGCGAGGT TGCGCCGGTA GACCGGTCCG GGGATGTCGT AGATGCGGCG 360

CAGGATGCGG AAGGAGTCGG GTACCGACAG GTCCCACCAG AGCTGGCTGC GCTGGCCGAA 420

GACGACGCCG ATCGTGCGGG CGTTGCGCTG CCGGTGCCGG TAGGGCTCCA GCCCGGCGAC 480

CGTGCAGCGG CCGGAGGTGG GGGTCATGAT GCCGGTCAGC ATCTTGATCG TGGTCGACTT 540

GCCGGCTCCG TTGGCGCCGA TGTAGGCGGT CTTCGTGCCG GCCGGTATCT CGAAGGAGAC 600

GTCGTCGACG GCGCGCACGA CGCGGTACCG GCGGGTCAGG AGGGTGGAGA GGCTGCCGAG 660

CAGGCCGGGC TCGCGTTCGG CCAGCCGGAA CTCCTTGACG AGGTGTTCGG CCACGATCAC 720

GCGATCACCC GCTCGACGGC CGTCTCCAGC AGGCGCAGGC CCTCGTCGAG CAGCGCCTCG 780

TCGAGGGTGA ACGGCGGTGC CAGCCGCAGG ATGTGGCCGC CCAGGGAGGT GCGCAGCCCC 840

AGGTCGAGGG CGGTGGTGTA GACGGCCCGG GCGGTCTCGG GGGCGGGTGC CCGGCCGACG 900

GCGTCGGTGA CGAACTCCAG GCCCCACAGC AGTCCGAGGC CGCGTACCTG GCCGAGCTGG 960

GGGAAGCGGG ACTCCAGGGC GCGCAGCCGC TCCTGGATGA GCTCGCCGAG GACGCGCACG 1020

CGGTCGATCA GCCGGTCGCG CTCGACGACC TCCAGCGTGG CGCGGGCGGC GGCGATCCCC 1080

AGTGGGTTGC TCGCGTACGT CGAGGCGTAC GCCCCGGGGT GGCCGCCTCC GGCCTGCGCA 1140

GCTTCCGCGC GTCCGGCCAG CACGGCGAAG GGGAATCCGC TCGCGGTGCC CTTGGACAGC 1200

ATCGCCAGGT CCGGCTCGAT GCCGAACAGT TCGCTGGCGA GGAAGGCGCC GGTGCGCCCG 1260

CCGCCGGTGA GGACCTCGTC GGCGACGAGC AGCACGCCGC CGTCCCGGCA GGCGCCGGCG 1320

ATCCGCTCCC AGTAGCCGGG GGGCGGCACG ATGACGCCTG CCGCGCCGAG GACGGGTTCG 1380

AAGACCAGGG CCGAGACGTT GGGCTTCTCC GCGATGTGCC GGCGCACGAG GGTCGCGCAC 1440

CGCACGTCGC ACGAGGGGTA CTCCAGGCCC AGGGGACAGC GGTAGCCAGT AGGGGCTGTA 1500

GCCAGCACGC TGTTGCCGCT GAAGGCCTGG TGGCCGATGT CCCAGTGGAC CAGCATCCGG 1560

GCGCCCATGG TCTTGCCGTG GAAGCCGTGG CGCAGGGCGC AGATCCGGTT GCGGCCCGGC 1620

GCGGCGGTCG CCTGGACGAC CCGCAGGGCG GCCTCGACCA CCTCCGCGCC GGTGGAGAAG 1680

AAGGCGTAGG TGTCGAGCTG TTCGGGCAGC AGCCTGGCGA GCAGTTCCAG CAGGCCGGCG 1740

CGGTCCGGCG TGGCGCTGTC GTGGACGTTC CACAGGCGGC GGGCCTGGGT GGTGAGTGCC 1800

TCGACGACCT CCGGGTGCCC GTGGCCCAGT GACTGGGTGA GGGTCCCGGC CGCGAAGTCG 1860

AGGTACTGGT TGCCGTCCAG GTCGGTCAGA ACGGGACCGC GTCCCTCGGC GAAGACCCGG 1920

CGTCCGTGGA CGGCTTCCTC GGAGGCGCCC GGCGCCAGGT GGCGGGCCTC CCGTGCCAGG 1980

TGCTGTGTCT GCCGTAAGCC TGTCATCGCT GCCTCTGCTC GTCGGACCGG CTGACGCGAT 2040

CGCCGGCGAA CTGCGTTGTG GCGCACCACG GTTGGGGCGG CTCGGCGCTG AGTCAAACAC 2100

TTGAACACAC ACCGCTGCAA GAGTTTGCGG GTTGTTTCAG AAAGTTGTTG CGAGCGGCCC 2160

CGGCACTCTG GTTGAGTCGA CGTGCTTACG GCGCCACCAC GCCTCACGTT CGAGGAGGGA 2220

CCTGTGAGAA CAAGCCCGCA GACCGACCCG CTCCCGCGGA GGCCGAGGTG AAGGCCCTGG 2280

TCCTGGCAGG TGGAACCGGC AGCAGACTGA GGCCGTTCAC CCACACCGCC GCCAAGCAGC 2340

TGCTCCCCAT CGCCAACAAG CCCGTGCTCT TCTACGCGCT GGAGTCCCTC GCCGCGGCGG 2400

GTGTCCGGGA GGCCGGCGTC GTCGTGGGCG CGTACGGCCG GGAGATCCGC GAACTCACCG 2460

GCGACGGCAC CGCGTTCGGG TTACGCATCA CCTACCTCCA CCAGCCCCGC CCGCTCGGTC 2520

TCGCGCACGC GGTGCGCATC GCCCGCGGCT TCCTGGGCGA CGACGACTTC CTGCTGTACC 2580

TGGGGGACAA CTACCTGCCC CAGGGCGTCA CCGACTTCGC CCGCCAATCG GCCGCCGATC 2640

CCGCGGCGGC CCGGCTGCTG CTCACCCCGG TCGCGGACCC GTCCGCCTTC GGCGTCGCGG 2700

AGGTCGACGC GGACGGGAAC GTGCTGCGCT TGGAGGAGAA ACCCGACGTC CCGCGCAGCT 2760

CGCTCGCGCT CATCGGCGTG TACGCCTTCA GCCCGGCCGT CCACGAGGCG GTACGGGCCA 2820

TCACCCCCTC CGCCCGCGGC GAGCTGGAGA TCACCCACGC CGTGCAGTGG ATGATCGACC 2880

GGGGCCTGCG CGTACGGGCC GAGACCACCA CCCGGCCCTG GCGCGACACC GGCAGCGCGG 2940

AGGACATGCT GGAGGTCAAC CGTCACGTCC TGGACGGACT GGAGGGCCGC ATCGAGGGGA 3000

AGGTCGACGC GCACAGCACG CTGGTCGGCC GGGTCCGGGT GGCCGAAGGC GCGATCGTGC 3060

GGGGGTCACA CGTGGTGGGC CCGGTGGTGA TCGGCGCGGG TGCCGTCGTC AGCAACTCCA 3120

GTGTCGGCCC GTACACCTCC ATCGGGGAGG ACTGCCGGGT CGAGGACAGC GCCATCGAGT 3180

ACTCCGTCCT GCTGCGCGGC GCCCAGGTCG AGGGGGCGTC CCGCATCGAG GCGTCCCTCA 3240

TCGGCCGCGG CGCCGTCGTC GGCCCGGCCC CCCGTCTCCC GCAGGCTCAC CGACTGGTGA 3300

TCGGCGACCA CAGCAAGGTG TATCTCACCC CATGACCACG ACCATCCTCG TCACCGGCGG 3360

AGCGGGCTTC ATTCGCTCCG CCTACGTCCG CCGGCTCCTG TCGCCCGGGG CCCCCGGCGG 3420

CGTCGCGGTG ACCGTCCTCG ACAAACTCAC CTACGCCGGC AGCCTCGCCC GCCTGCACGC 3480

GGTGCGTGAC CATCCCGGCC TCACCTTCGT CCAGGGCGAC GTGTGCGACA CCGCGCTCGT 3540

CGACACGCTG GCCGCGCGGC ACGACGACAT CGTGCACTTC GCGGCCGAGT CGCACGTCGA 3600

CCGCTCCATC ACCGACAGCG GTGCCTTCAC CCGCACCAAC GTGCTGGGCA CCCAGGTCCT 3660

GCTCGACGCC GCGCTCCGCC ACGGTGTGCG CACCTTCGTG CACGTCTCCA CCGACGAGGT 3720

GTACGGCTCC CTCCCGCACG GGGCCGCCGC GGAGAGCGAC CCCCTGCTTC CGACCTCGCC 3780

GTACGCGGCG TCGAAGGCGG CCTCGGACCT CATGGCGCTC GCCCACCACC GCACCCACGG 3840

CCTGGACGTC CGGGTGACCC GCTGTTCGAA CAACTTCGGC CCCCACCAGC ATCCCGAGAA 3900

GCTCATACCG CGCTTCCTGA CCAGCCTCCT GTCCGGCGGC ACCGTTCCCC TCTACGGCGA 3960

CGGGCGGCAC GTGCGCGACT GGCTGCACGT CGACGACCAC GTCAGGGCCG TCGAACTCGT 4020

CCGCGTGTCG GGCCGGCCGG GAGAGATCTA CAACATCGGG GGCGGCACCT CGCTGCCCAA 4080

CCTGGAGCTC ACGCACCGGT TGCTCGCACT GTGCGGCGCG GGCCCGGAGC GCATCGTCCA 4140

CGTCGAGAAC CGCAAGGGGC ACGACCGGCG CTACGCGGTC GACCACAGCA AGATCACCGC 4200

GGAACTCGGT TACCGGCCGC GCACCGACTT CGCGACCGCG CTGGCCGACA CCGCGAAGTG 4260

GTACGAGCGG CACGAGGACT GGTGGCGTCC CCTGCTCGCC GCGACATGAC GTCGGGCCGG 4320

ACCGCAACCA CCGGCCCCGG CCGGCACACC GCCGCCCGCG GCCGGTGGCC GGCCGGTCAG 4380

CGTCCGTGAG CCGGGCGCCG GCCGCCCCGC GGGCCGGCGG CGGTGGACCC CCGGACCACC 4440

AGTTCCGGCA TGAAGACGAA TTCGGTGCGC GGCGGCGGCG TTCCGCTCAT CTCCTCCAGC 4500

AGTGCGTCCA CGGCGACCTG CCCCATCGCC TTGACGGGCT GTCTGATGGT GGTCAGGGGA 4560

GGGTCGGTGA AGGCCATGAG CGGCGAGTCG TCGAAGCCGA CCACCGAGAT GTCACCGGGA 4620

ACCGTGAGAC CCCGCCGGCG CGCGGCCCGC ACGGCGCCGA GGGCCATCAT GTCGCTGGCG 4680

CACATGACGG CGGTGCAGCC CAGGTCGATC AGCGCGGACG CGGCGGCCTG GCCCCCCTCC 4740

AGGGAGAACA GCGAGTGCTG CACGAGCTCC TCGGACTCCC GCGCCGACAC TCCCAGGTGC 4800

TCCCGCACGC CGGCCCGGAA CCCCTCGATC TTCCGCTGCA CCGGCACGAA GCGGGCGGGC 4860

CCGACGGCGA GGCCGACGCG CTCGTGCCCC AGCTCCGCCA GGTGCGCCAC GGCCAGGCGC 4920

ATCGCGGCCC GGTCGTCCGG GGAGACGAAG GGTGCCTCGA TCCGGGGCGA GAACCCGTTC 4980

ACGAGGACGA AGGGCACCTG CCGCTCGTGC AGCCGGCCGT ACCGTCCGGT CTCGGCGGTG 5040

GTGTCCGCGT GCAGTCCGGA GACGAAGATG ATGCCGGACA CCCCGCGGTC CACGAGCATC 5100

TCCGTGAGTT CGTCCTCGGT CGAGCCGCCC GGGGTCTGCG TGGCGAGCAC GGGCGTGTAG 5160

CCCTGACGCG TGAGCGCCTG CCCCATCACC TGGGCCAGTG CGGGGAAGAA GGGGTTGTCC 5220

AGTTCGGGGG TGACCAGTCC GACCAGCTCG GCGCGGCGCT GTCGCGCCGG CTGCTCGTAG 5280

CCCAGCGCGT CCAGTGCGGT CAGCACCGAG TCGCGGGTGC CGGTGGCCAC ACCGCGCGCA 5340

CCGTTCAGCA CCCGGCTGAC CGTGGCCTTG CTGACGCCCG CCCGGGCTGC GATGTCGGCG 5400

AGCCGCATGG TCATGGCAAC GCACTCTACC TGTCGGGGCG TCAGGGCGTG CCCACCGCGC 5460

GCGGAACCGG CGGACTGCGG GGCACGGCCC GTCCGCCGCC CACGGACCAC GCGCCCGAAA 5520

CGATGGCTGA AAATGCTTGC AGCAAATTGC CGCAACGTCT TTCGGCGGCT TTTCGATCCT 5580

GTTACGTTCC TGGCAACCCC GGCGCCGCGC AGAAGCGGTT GGCGTGAGGC GTCCAGACCT 5640

CCGCCCGATT CCGGGATCAC TCAGGGGAGT TCACAATGCG GCGTGGCATT GCGGCCACCG 5700

CGCTGTTCGC GGCTGTGGCC ATGACGGCAT CGGCGTGTGG CGGGGGCGAC AACGGCGGAA 5760

GCGGTACCGA CGCGGGCGGC ACGGAGCTGT CGGGGACCGT CACCTTCTGG GACACGTCCA 5820

ACGAAGCCGA GAAGGCGACG TACCAGGCCC TCGCGGAGGG CTTCGAGAAG GAGCACCCGA 5880

AGGTCGACGT CAAGTACGTC AACGTCCCGT TCGGCGAGGC GAACGCCAAG TTCAAGAACG 5940

CCGCGGGCGG CAACTCCGGT GCCCCGGACG TGATGCGTAC GGAGGTCGCC TGGGTCGCGG 6000

ACTTCGCCAG CATCGGCTAC CTCGCCCCGC TCGACGGCAC GCCCGCCCTC GACGACGGGT 6060

CGGACCACCT TCCCCAGGGC GGCAGCACCA GGTACGAGGG GAAGACCTAC GCGGTCCCGC 6120

AGGTGATCGA CACCCTGGCG CTCTTCTACA ACAAGGAACT GCTGACGAAG GCCGGTGTCG 6180

AGGTGCCGGG CTCCCTCGCC GAGCTGAAGA CGGCCGCCGC CGAGATCACC GAGAAGACCG 6240

GCGCGAGCGG CCTCTACTGC GGGGCGACGA CCCGTACTTG GTTCCTGCCC TACCTCTACG 6300

GGGAGGGCGG CGACCTGGTC GACGAGAAGA ACAAGACCGT CACGGTCGAC GACGAAGCCG 6360

GTGTGCGCGC CTACCGCGTC ATCAAGGACC TCGTGGACAG CAAGGCGGCC ATCACCGACG 6420

CGTCCGACGG CTGGAACAAC ATGCAGAACG CCTTCAAGTC GGGCAAGGTC GCCATGATGG 6480

TCAACGGCCC CTGGGCCATC GAGGACGTCA AGGCGGGAGC CCGCTTCAAG GACGCCGGCA 6540

ACCTGGGGGT CGCCCCCGTC CCGGCCGGCA GTGCCGGACA GGGCTCTCCC CAGGGCGGGT 6600

GGAACCTCTC GGTGTACGCG GGCTCGAAGA ACCTCGACGC CTCCTACGCC TTCGTGAAGT 6660

ACATGAGCTC CGCCAAGGTG CAGCAGCAGA CCACCGAGAA GCTGAGCCTG CTGCCCACCC 6720

GCACGTCCGT CTACGAGGTC CCGTCCGTCG CGGACAACGA GATGGTGAAG TTCTTCAAGC 6780

CGGCCGTCGA CAAGGCCGTC GAACGGCCGT GGATCGCCGA GGGCAATGCC CTCTTCGAGC 6840

CGATCCGGCT GCAG 6854

240 amino acids

amino acid

<Unknown>

linear

protein

8

Val Ile Val Ala Glu His Leu Val Lys Glu Phe Arg Leu Ala Glu Arg

1 5 10 15

Glu Pro Gly Leu Leu Gly Ser Leu Ser Thr Leu Leu Thr Arg Arg Tyr

20 25 30

Arg Val Val Arg Ala Val Asp Asp Val Ser Phe Glu Ile Pro Ala Gly

35 40 45

Thr Lys Thr Ala Tyr Ile Gly Ala Asn Gly Ala Gly Lys Ser Thr Thr

50 55 60

Ile Lys Met Leu Thr Gly Ile Met Thr Pro Thr Ser Gly Arg Cys Thr

65 70 75 80

Val Ala Gly Leu Glu Pro Tyr Arg His Arg Gln Arg Asn Ala Arg Thr

85 90 95

Ile Gly Val Val Phe Gly Gln Arg Ser Gln Leu Trp Trp Asp Leu Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Met Arg Ala Glu Ile Ala Ala Ser Leu Leu His Asp Pro Ala Val Leu

165 170 175

Phe Trp Asp Glu Pro Thr Ile Gly Leu Asp Met Val Leu Lys Asp Ala

180 185 190

Val Arg Arg Leu Val Asn Arg Ala His Arg Glu Leu Gly Thr Thr Val

195 200 205

Val Leu Thr Ser His Asp Ile Ala Asp Ile Ala Ala Ile Cys Asp Ser

210 215 220

Ala Leu Val Val Asp Gln Gly Arg Val Val His Gln Gly Thr Leu Gln

225 230 235 240

429 amino acids

amino acid

<Unknown>

linear

protein

9

Met Thr Gly Leu Arg Gln Thr Gln His Leu Ala Arg Glu Ala Arg His

1 5 10 15

Leu Ala Pro Gly Ala Ser Glu Glu Ala Val His Gly Arg Arg Val Phe

20 25 30

Ala Glu Gly Arg Gly Pro Val Leu Thr Asp Leu Asp Gly Asn Gln Tyr

35 40 45

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

50 55 60

Pro Glu Val Val Glu Ala Leu Thr Thr Gln Ala Arg Arg Leu Trp Asn

65 70 75 80

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

85 90 95

Ala Arg Leu Leu Pro Glu Gln Leu Asp Thr Tyr Ala Phe Phe Ser Thr

100 105 110

Gly Ala Glu Val Val Glu Ala Ala Leu Arg Val Val Gln Ala Thr Ala

115 120 125

Ala Pro Gly Arg Asn Arg Ile Cys Ala Leu Arg His Gly Phe His Gly

130 135 140

Lys Thr Met Gly Ala Arg Met Leu Val His Trp Asp Ile Gly His Gln

145 150 155 160

Ala Phe Ser Gly Asn Ser Val Leu Ala Thr Ala Pro Thr Gly Tyr Arg

165 170 175

Cys Pro Leu Gly Leu Glu Tyr Pro Ser Cys Asp Val Arg Cys Ala Thr

180 185 190

Leu Val Arg Arg His Ile Ala Glu Lys Pro Asn Val Ser Ala Leu Val

195 200 205

Phe Glu Pro Val Leu Gly Ala Ala Gly Val Ile Val Pro Pro Pro Gly

210 215 220

Tyr Trp Glu Arg Ile Ala Gly Ala Cys Arg Asp Gly Gly Val Leu Leu

225 230 235 240

Val Ala Asp Glu Val Leu Thr Gly Gly Gly Arg Thr Gly Ala Phe Leu

245 250 255

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

260 265 270

Gly Thr Ala Ser Gly Phe Pro Phe Ala Val Leu Ala Gly Arg Ala Glu

275 280 285

Ala Ala Gln Ala Gly Gly Gly His Pro Gly Ala Tyr Ala Ser Thr Tyr

290 295 300

Ala Ser Asn Pro Leu Gly Ile Ala Ala Ala Arg Ala Thr Leu Glu Val

305 310 315 320

Val Glu Arg Asp Arg Leu Ile Asp Arg Val Arg Val Leu Gly Glu Leu

325 330 335

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

340 345 350

Gln Val Arg Gly Leu Gly Leu Leu Trp Gly Leu Glu Phe Val Thr Asp

355 360 365

Ala Val Gly Arg Ala Pro Ala Pro Glu Thr Ala Arg Ala Val Tyr Thr

370 375 380

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

385 390 395 400

Arg Leu Ala Pro Pro Phe Thr Leu Asp Glu Ala Leu Leu Asp Glu Gly

405 410 415

Leu Arg Leu Leu Glu Thr Ala Val Glu Arg Val Ile Ala

420 425

355 amino acids

amino acid

<Unknown>

linear

protein

10

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

1 5 10 15

Phe Thr His Thr Ala Ala Lys Gln Leu Leu Pro Ile Ala Asn Lys Pro

20 25 30

Val Leu Phe Tyr Ala Leu Glu Ser Leu Ala Ala Ala Gly Val Arg Glu

35 40 45

Ala Gly Val Val Val Gly Ala Tyr Gly Arg Glu Ile Arg Glu Leu Thr

50 55 60

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

65 70 75 80

Arg Pro Leu Gly Leu Ala His Ala Val Arg Ile Ala Arg Gly Phe Leu

85 90 95

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

100 105 110

Gly Val Thr Asp Phe Ala Arg Gln Ser Ala Ala Asp Pro Ala Ala Ala

115 120 125

Arg Leu Leu Leu Thr Pro Val Ala Asp Pro Ser Ala Phe Gly Val Ala

130 135 140

Glu Val Asp Ala Asp Gly Asn Val Leu Arg Leu Glu Glu Lys Pro Asp

145 150 155 160

Val Pro Arg Ser Ser Leu Ala Leu Ile Gly Val Tyr Ala Phe Ser Pro

165 170 175

Ala Val His Glu Ala Val Arg Ala Ile Thr Pro Ser Ala Arg Gly Glu

180 185 190

Leu Glu Ile Thr His Ala Val Gln Trp Met Ile Asp Arg Gly Leu Arg

195 200 205

Val Arg Ala Glu Thr Thr Thr Arg Pro Trp Arg Asp Thr Gly Ser Ala

210 215 220

Glu Asp Met Leu Glu Val Asn Arg His Val Leu Asp Gly Leu Glu Gly

225 230 235 240

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

245 250 255

Arg Val Ala Glu Gly Ala Ile Val Arg Gly Ser His Val Val Gly Pro

260 265 270

Val Val Ile Gly Ala Gly Ala Val Val Ser Asn Ser Ser Val Gly Pro

275 280 285

Tyr Thr Ser Ile Gly Glu Asp Cys Arg Val Glu Asp Ser Ala Ile Glu

290 295 300

Tyr Ser Val Leu Leu Arg Gly Ala Gln Val Glu Gly Ala Ser Arg Ile

305 310 315 320

Glu Ala Ser Leu Ile Gly Arg Gly Ala Val Val Gly Pro Ala Pro Arg

325 330 335

Leu Pro Gln Ala His Arg Leu Val Ile Gly Asp His Ser Lys Val Tyr

340 345 350

Leu Thr Pro

355

325 amino acids

amino acid

<Unknown>

linear

protein

11

Met Thr Thr Thr Ile Leu Val Thr Gly Gly Ala Gly Phe Ile Arg Ser

1 5 10 15

Ala Tyr Val Arg Arg Leu Leu Ser Pro Gly Ala Pro Gly Gly Val Ala

20 25 30

Val Thr Val Leu Asp Lys Leu Thr Tyr Ala Gly Ser Leu Ala Arg Leu

35 40 45

His Ala Val Arg Asp His Pro Gly Leu Thr Phe Val Gln Gly Asp Val

50 55 60

Cys Asp Thr Ala Leu Val Asp Thr Leu Ala Ala Arg His Asp Asp Ile

65 70 75 80

Val His Phe Ala Ala Glu Ser His Val Asp Arg Ser Ile Thr Asp Ser

85 90 95

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

100 105 110

Ala Ala Leu Arg His Gly Val Arg Thr Phe Val His Val Ser Thr Asp

115 120 125

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

130 135 140

Leu Leu Pro Thr Ser Pro Tyr Ala Ala Ser Lys Ala Ala Ser Asp Leu

145 150 155 160

Met Ala Leu Ala His His Arg Thr His Gly Leu Asp Val Arg Val Thr

165 170 175

Arg Cys Ser Asn Asn Phe Gly Pro His Gln His Pro Glu Lys Leu Ile

180 185 190

Pro Arg Phe Leu Thr Ser Leu Leu Ser Gly Gly Thr Val Pro Leu Tyr

195 200 205

Gly Asp Gly Arg His Val Arg Asp Trp Leu His Val Asp Asp His Val

210 215 220

Arg Ala Val Glu Leu Val Arg Val Ser Gly Arg Pro Gly Glu Ile Tyr

225 230 235 240

Asn Ile Gly Gly Gly Thr Ser Leu Pro Asn Leu Glu Leu Thr His Arg

245 250 255

Leu Leu Ala Leu Cys Gly Ala Gly Pro Glu Arg Ile Val His Val Glu

260 265 270

Asn Arg Lys Gly His Asp Arg Arg Tyr Ala Val Asp His Ser Lys Ile

275 280 285

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

290 295 300

Ala Asp Thr Ala Lys Trp Tyr Glu Arg His Glu Asp Trp Trp Arg Pro

305 310 315 320

Leu Leu Ala Ala Thr

325

345 amino acids

amino acid

<Unknown>

linear

protein

12

Met Thr Met Arg Leu Ala Asp Ile Ala Ala Arg Ala Gly Val Ser Lys

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Thr Arg Gln Gly Tyr Thr Pro Val Leu Ala Thr Gln Thr Pro Gly

85 90 95

Gly Ser Thr Glu Asp Glu Leu Thr Glu Met Leu Val Asp Arg Gly Val

100 105 110

Ser Gly Ile Ile Phe Val Ser Gly Leu His Ala Asp Thr Thr Ala Glu

115 120 125

Thr Gly Arg Tyr Gly Arg Leu His Glu Arg Gln Val Pro Phe Val Leu

130 135 140

Val Asn Gly Phe Ser Pro Arg Ile Glu Ala Pro Phe Val Ser Pro Asp

145 150 155 160

Asp Arg Ala Ala Met Arg Leu Ala Val Ala His Leu Ala Glu Leu Gly

165 170 175

His Glu Arg Val Gly Leu Ala Val Gly Pro Ala Arg Phe Val Pro Val

180 185 190

Gln Arg Lys Ile Glu Gly Phe Arg Ala Gly Val Arg Glu His Leu Gly

195 200 205

Val Ser Ala Arg Glu Ser Glu Glu Leu Val Gln His Ser Leu Phe Ser

210 215 220

Leu Glu Gly Gly Gln Ala Ala Ala Ser Ala Leu Ile Asp Leu Gly Cys

225 230 235 240

Thr Ala Val Met Cys Ala Ser Asp Met Met Ala Leu Gly Ala Val Arg

245 250 255

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

260 265 270

Gly Phe Asp Asp Ser Pro Leu Met Ala Phe Thr Asp Pro Pro Leu Thr

275 280 285

Thr Ile Arg Gln Pro Val Lys Ala Met Gly Gln Val Ala Val Asp Ala

290 295 300

Leu Leu Glu Glu Met Ser Gly Thr Pro Pro Pro Arg Thr Glu Phe Val

305 310 315 320

Phe Met Pro Glu Leu Val Val Arg Gly Ser Thr Ala Ala Gly Pro Arg

325 330 335

Gly Gly Arg Arg Pro Ala His Gly Arg

340 345

393 amino acids

amino acid

<Unknown>

linear

protein

13

Met Arg Arg Gly Ile Ala Ala Thr Ala Leu Phe Ala Ala Val Ala Met

1 5 10 15

Thr Ala Ser Ala Cys Gly Gly Gly Asp Asn Gly Gly Ser Gly Thr Asp

20 25 30

Ala Gly Gly Thr Glu Leu Ser Gly Thr Val Thr Phe Trp Asp Thr Ser

35 40 45

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

50 55 60

Lys Glu His Pro Lys Val Asp Val Lys Tyr Val Asn Val Pro Phe Gly

65 70 75 80

Glu Ala Asn Ala Lys Phe Lys Asn Ala Ala Gly Gly Asn Ser Gly Ala

85 90 95

Pro Asp Val Met Arg Thr Glu Val Ala Trp Val Ala Asp Phe Ala Ser

100 105 110

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

115 120 125

Ser Asp His Leu Pro Gln Gly Gly Ser Thr Arg Tyr Glu Gly Lys Thr

130 135 140

Tyr Ala Val Pro Gln Val Ile Asp Thr Leu Ala Leu Phe Tyr Asn Lys

145 150 155 160

Glu Leu Leu Thr Lys Ala Gly Val Glu Val Pro Gly Ser Leu Ala Glu

165 170 175

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

180 185 190

Leu Tyr Cys Gly Ala Thr Thr Arg Thr Trp Phe Leu Pro Tyr Leu Tyr

195 200 205

Gly Glu Gly Gly Asp Leu Val Asp Glu Lys Asn Lys Thr Val Thr Val

210 215 220

Asp Asp Glu Ala Gly Val Arg Ala Tyr Arg Val Ile Lys Asp Leu Val

225 230 235 240

Asp Ser Lys Ala Ala Ile Thr Asp Ala Ser Asp Gly Trp Asn Asn Met

245 250 255

Gln Asn Ala Phe Lys Ser Gly Lys Val Ala Met Met Val Asn Gly Pro

260 265 270

Trp Ala Ile Glu Asp Val Lys Ala Gly Ala Arg Phe Lys Asp Ala Gly

275 280 285

Asn Leu Gly Val Ala Pro Val Pro Ala Gly Ser Ala Gly Gln Gly Ser

290 295 300

Pro Gln Gly Gly Trp Asn Leu Ser Val Tyr Ala Gly Ser Lys Asn Leu

305 310 315 320

Asp Ala Ser Tyr Ala Phe Val Lys Tyr Met Ser Ser Ala Lys Val Gln

325 330 335

Gln Gln Thr Thr Glu Lys Leu Ser Leu Leu Pro Thr Arg Thr Ser Val

340 345 350

Tyr Glu Val Pro Ser Val Ala Asp Asn Glu Met Val Lys Phe Phe Lys

355 360 365

Pro Ala Val Asp Lys Ala Val Glu Arg Pro Trp Ile Ala Glu Gly Asn

370 375 380

Ala Leu Phe Glu Pro Ile Arg Leu Gln

385 390