A gene which contains the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 671 to nucleotide 6295 or a nucleotide sequence which can be obtained therefrom by substitution, insertion or deletion of up to 30%, preferably up to 10%, particularly preferably up to 20%, especially preferably up to 5%, of the nucleotides, and whose gene product has the enzymatic activity of an adenylate cyclase, and its use.

Description

13 parts

›The present invention relates to the change in…

The present invention relates to the change in activity of the cell cAMP signal transduction pathway, which leads to a change in the central cell metabolism and is thus exploited for increasing the production rates for fine chemicals in fermentative processes.

The enzyme adenylate cyclase catalyzes the production of cAMP from ATP and, with the aid of cAMP, governs, in eukaryotes, signal transduction by the enzyme protein kinase A. cAMP binds to the regulatory subunit of protein kinase A, which leads to protein kinase A being activated (Taylor et al., 1990, Annual Rev Biochem: 971-1005). This kinase alters the activity of target proteins by esterifying the hydroxyl groups of specific serines of these proteins with a phosphate group. Protein kinase A is involved in a large number of cellular regulatory processes. On the one hand, the kinase modulates the activity of some transcription factors such as, for example, CREB (Brindle and Montminy, 1992, Curr Opin Genet & Dev, 2, 199-204), which leads to the synthetic rates of metabolic enzymes being altered. Moreover, the product of adenylate cyclase (cAMP) takes part in regulating the central cell metabolism by governing both synthesis and activity of enzymes of glycolysis, gluconeogenesis and the glyoxylate pathway. Thus, cAMP inhibits the synthesis of key enzymes of gluconeogenesis and of the glyoxylate pathway, such as fructose bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase (Boy-Marcotte et al., 1996, Microbiology, 142:459-467). On the other hand, the activity of enzymes of glycolysis, such as phosphofructokinase, is increased by cAMP-mediated protein kinase A activity (Keβler and Eschrich, 1996, FEBS Lett., 395: 225-227). In addition, the cAMP-activated protein kinase A inactivates the formation of carbohydrate stores in yeasts and stimulates the degradation of carbohydrate stores in fungi (Pall and Robertson, 1988, Biochem Biophys Res Commun, 150: 365-370; Toda et al., 1985, Cell, 40: 27-36). The signal cascade, which is initiated by adenylate cyclase activity, thus plays a crucial role in regulating the substance fluxes in the cell. In particular, cAMP signal transduction alters the direction of the substance fluxes in the central metabolism so that the cAMP signal transduction chain plays a decisive role in providing substrates for synthesizing secondary metabolic products. Specific secondary metabolic products are commercially important fine chemicals. These include, for example, vitamins, carotinoids, amino acids, fragrances and antibiotics.

Fine chemicals have a wide range of industrial uses in animal and human nutrition, in the cosmetics industry and in medicine. In addition, carotinoids act as natural colorants. Fine chemicals are produced industrially by chemical synthesis or, increasingly, by fermentation.

Activation of adenylate cyclase in yeasts and fungi is governed by the RAS G proteins. RAS G proteins here act, for example, as sensors for the glucose concentration in the medium (Field et al., 1990, Science, 247: 464-467). Glucose in the medium leads to RAS being activated by exchanging the bound GDP for GTP. This process is made possible by an RAS-specific exchange factor. The RAS which is activated by GTP binding binds to, and activates, adenylate cyclase, which, in turn, forms cAMP. The signal pathway is switched off by the cAMP-specific phosphodiesterase, which converts cAMP into AMP (Ishikawa et al., 1988, Methods Enzymol., 159, 27-42). Adenylate cyclase is thus a central switchpoint for adaptation to changes in the nutritional environment of cells. In higher eukaryotes, adenylate cyclase is regulated by heterotrimeric G proteins (Federmann et al., 1992, Nature, 356: 159-161).

In prokaryotes, too, adenylate cyclase is involved in the regulation of metabolic processes. Here, cAMP regulates the expression of some catabolic operons by binding to a transcription factor (CRP) (Tagami et al., 1995 Mol Microbiol, 17, 251-258).

To optimize fermentative processes for the production of fine chemicals, the substance flux toward the desired product must be increased.

This can be effected in different ways:

Switching-off of negative regulatory mechanisms of metabolic pathways which lead to the product

Inactivation of enzymes which compete with the enzymes of the metabolic pathways leading to the desired product

Increase, in the cell, of the quantity or activity of enzymes of the metabolic pathways which lead to the product

Since cellular processes proceed in a coordinated fashion owing to higher regulatory mechanisms, such regulatory mechanisms are targets for altering the substance fluxes in the cell.

It is therefore an object to alter regulatory mechanisms in cells in such a way that they allow an increased substance flux toward the desired product.

We have found that this object is achieved according to the invention by altering the activity of the cAMP-dependent signal transduction pathway, which constitutes a central regulator of a large number of metabolic pathways in the cell, so that the substance fluxes can be directed toward the desired product. There is a multiplicity of possibilities of manipulating microorganisms in such a way that they carry an altered activity of the cAMP-dependent signal transduction pathway. However, an altered activity of the cAMP signal transduction pathway for increasing the production of fine chemicals in microorganisms has not been described as yet.

One possibility of increasing the production rates of fine chemicals in microorganisms by altering the activity of the cAMP signal transduction pathway consists in increasing or lowering, in the cell, the enzyme activity of adenylate cyclase, depending on the synthesis to be carried out.

An increase in the quantity of enzyme, and thus an increase in enzyme activity, can be achieved by introducing the gene which encodes adenylate cyclase into the microorganism to be altered at a higher repetition frequency, or by eliminating factors which repress enzyme synthesis. Alternatively, it is possible to exchange the sequences which govern adenylate cyclase expression for sequences which allow increased gene expression. In addition, an increase in enzyme activity can be achieved, for example, by mutating the enzyme to increase substrate conversion, or by disrupting the effect of enzyme inhibitors.

›Alternatively, cAMP or a chemical cAMP derivative may…

Alternatively, cAMP or a chemical cAMP derivative may be added to the medium, which also leads to protein kinase A being activated.

To reduce the enzyme activity of adenylate cyclase in the cell, the encoding gene can be disrupted, or the activity of adenylate cyclase synthesis activators can be reduced. Adenylate cyclase mutations, which have a reduced activity, can also be applied. Such mutations can be achieved either by traditional methods such as, for example, by UV irradiation or mutagenic chemicals, or directed by means of genetic engineering methods such as deletions, insertions or substitutions.

Not only can the gene activity of adenylate cyclase be altered, it is also possible in the same way to alter the enzyme activity of phosphodiesterase of protein kinase A (catalytic and regulatory domain) and of the RAS proteins (including proteins which have an effect on RAS activity) as described above.

It is preferred to alter the enzyme activity of adenylate cyclase. To reduce the enzyme activity, the adenylate cyclase gene is disrupted. To disrupt the adenylate cyclase gene, a selection marker is inserted into the DNA sequence which encodes adenylate cyclase. Then, this DNA construct is transformed into the cell, where it disrupts the adenylate cyclase gene locus by means of homologous recombination. Thus, the cell is incapable of synthesizing functional adenylate cyclase and thus cAMP.

The invention relates to a gene which contains the nucleotide sequence shown in SEQ ID NO:1 from nucleotide 671 to nucleotide 6295 or a nucleotide sequence which can be obtained therefrom by substitution, insertion or deletion of up to 30%, preferably up to 20%, particularly preferably up to 10%, especially preferably up to 5%, of the nucleotides and, whose gene product has the enzymatic activity of an adenylate cyclase.

The invention furthermore relates to the use of one or more of the abovementioned nucleic acid sequences for constructing, by genetic engineering, microorganisms which compared with the starting organism are capable of increased fine chemical production. A starting organism is to be understood as meaning, in the following text, those microorganisms which are capable of producing the desired fine chemicals, but which carry no genetic alteration with regard to the nucleic acid sequences according to the invention.

The invention is preferably applied to the production of riboflavin.

The preferred nucleic acid sequence used is the adenylate cyclase gene. The adenylate cyclase gene is preferably isolated from the microorganism which synthesizes the fine chemical in question. The preferred microorganisms which are employed in the process according to the invention are fungi and yeasts.

However, it is also possible to use other microorganisms, for example bacteria. Microorganisms which are especially suitable for the production of riboflavin are bacteria of the genus Bacillus and coryneform bacteria of the genus Corynebacteria or Brevibacteria, and yeasts of the genus Candida and Saccharomyces and fungi of the genus Ashbya and Eremothecium. Very particularly suitable for the production of riboflavin are Bacillus subtilis, Corynebacterium ammoniagenes, Candida flareri, Candida famata and Ashbya gossypii.

The adenylate cyclase gene can be isolated by sequencing a plasmid gene library, by homologous or heterologous complementation of a mutant which carries a defect in the adenylate cyclase gene, or else by heterologous probing or PCR with heterologous primers. For subcloning purposes, the insert of the complementing plasmid can subsequently be minimized in size by suitable cleavage with restriction enzymes. After sequencing and identification of the putative gene, it is fused to the DNA sequence of the selection marker by molecular biology techniques. The plasmid, which carries the sequence of the adenylate cyclase gene, is cleaved in the adenylate cyclase gene, and the selection marker is cloned into this cleavage site. The adenylate cyclase DNA, which frames the selection marker DNA, is subsequently introduced into the cell.

Isolation and sequencing of plasmids from a genomic plasmid library of Ashbya gossypii gives an adenylate cyclase gene which has nucleotide sequences shown in SEQ. ID NO:1 and which, from nucleotide 671 to nucleotide 6295, encode the amino acid sequence given in SEQ ID NO:2 or its allelic variations. Allelic variations encompass, in particular, derivatives which are obtainable from the sequence shown in SEQ.ID NO:1 by means of deletion, insertion or substitution of up to 30%, preferably up to 20%, particularly preferably up to 10%, especially preferably up to 5%, of the nucleotides.

Arranged upstream of the adenylate cyclase genes is, in particular, a promoter of the nucleotide sequence of nucleotide 1-670 as shown in SEQ.ID NO:1 or a sequence which acts essentially the same. Thus, for example, a promoter may be arranged upstream of the gene which differs from the promoter of the abovementioned nucleotide sequence by one or more nucleotide exchanges, by an insertion or insertions, and/or a deletion or deletions, but without the functionality or efficacy of the promoter being adversely affected. Moreover, the promoter may be altered with regard to its efficacy, due to its sequence being altered, or it may be exchanged completely by other promoters.

One or more DNA sequences can be arranged upstream and/or downstream of the adenylate cyclase gene with or without a promoter arranged upstream, or with or without a terminator sequence, so that the gene is retained in its gene structure.

Preference is given to increasing the production of the fine chemical riboflavin (vitamin B2) by altering the cAMP signal transduction pathway. However, other fermentative processes for the production of other fine chemicals are to be optimized by such an alteration. Riboflavin is produced in fungi, yeasts or bacteria, preferably in the fungus Ashbya gossypii.

If the adenylate cyclase gene in riboflavin-producing microorganism strains of Ashbya gossypii is disrupted, it emerges, surprisingly, that incubation on a glucose-containing medium leads to an increased riboflavin formation. This is seen, inter alia, on culture plates by the visual appearance of the cultures, which show a deep yellow coloration caused by riboflavin, which is weakened by adding cAMP to the medium.

›DESCRIPTION OF THE FIGURES

FIG. 1 describes the adenylate cyclase gene locus of Ashbya gossypii and identifies the subsequences covered by vectors pAG 1080, 1697 and 1612. Vector pAG 1080 and vector pAG1612 thus comprise the total sequence of the adenylate cyclase gene locus (encoding sequence nucleotide 671-6295).

FIG. 2 shows cleavage sites for important restriction enzymes in the adenylate cyclase gene fragment of plasmid pAG 1080. The Bgl II clevage site, which is also described in Example 3, is also shown.

FIG. 3 shows the NotI-EcoRI fragment of vector pAG-333b. This fragment can be employed for disrupting the adenylate cyclase gene locus.

EXAMPLES

›Examples9

›Example 1

Establishing a Genomic Gene Library of Ashbya gossypii

To establish a genomic DNA library of Ashbya gossypii , chromosomal DNA was isolated by the method of Wright and Philippsen (1991, Gene 109: 99-105) and Mohr (1995, PhD thesis, Center for Biological Studies, Basle University, Switzerland).

The genomic DNA was partially digested with Sau3A and fractioned in a sucrose density gradient. The largest fragments were ligated with vector pRS416 cleaved with Bam HI (Sikorski and Hieter, 1988, Genetics, 122; 19-27). The E.coli laboratory strain XL-1 blue was transformed with this ligation batch, and the resulting clones were employed for identifying the adenylate cyclase gene.

›Example 2

Selection of the Adenylate Cyclase Gene-encoding Fragment

Plasmids were purified from some clones described in Example 1, and the DNA of the inserts was sequenced. A computer-aided homology search based on the GCG program of Wisconsin University allowed DNA sequences to be identified which show homology to the Saccharomyces cerevisiae and Saccharomyces kluyveri adenylate cyclases. Plasmid pAG1080 (insert shown in FIG. 1 ), for example, contained a large portion of the adenylate cyclase gene and was sequenced. Screening of further plasmid libraries with a hybridizing probe complementary to plasmid pAG1080 led to the identification of plasmids pAG1687 and pAG1612, which, together with plasmid pAG1080, contain all of the DNA of the Ashbya gossypii adenylate cyclase gene (FIG. 1 ). The sequence is shown in SEQ. ID NO: 1. The portion which encodes adenylate cyclase starts at nucleotide 671 and ends at nucleotide 6295. At the nucleotide level, this encoding nucleic acid sequence from Ashbya gossypii shows an identity with Saccharomyces kluyveri adenylate cyclase of 66%. The amino acid sequence of the Ashbya gossypii adenylate cyclase, which was derived from the encoding region, shows an identity with the Klyveromyces lactis adenylate cyclase amino acid sequence of 63.5%.

›Example 3

Generation of the Construct for Disrupting the Adenylate Cyclase Gene in Ashbya gossypii

The resulting adenylate cyclase gene fragment in vector pAG 1080 was cleaved with Bgl II (FIG. 2 ). The resulting ends were then filled up with Klenow polymerase, treated with phosphatase and ligated to the 1.6 kb EcoRV-PvuII DNA fragment of pUG6 (Güldener et al., 1996, Nucleic Acids Research, 24: 2519-2524). The adenylate cyclase fragment of pAG 1080 now contains the DNA sequence of the Kanamycin resistance gene and the sequence of the promoter and terminator of the TEF gene (Wach et al., 1994, Yeast, 10: 1793-1808) flanked by loxP sequences from plasmid pUG6. From this vector construct, the NotI-EcoRI fragment, which, inter alia, contains the adenylate cyclase subsequence, was cloned into vector pBSII-SK which had been cleaved with NotI and EcoRI (termed vector pAG-333b hereinbelow, FIG. 3 ).

›Example 5

Transformation of the Adenylate Cyclase Gene Disruption Construct into Ashbya gossypii

The 6.2 kb NotI-EcoRI fragment was cleaved out of pAG-333b, purified and transformed into the fungus wild-type Ashbya gossypii strain by electroporation of freshly germinated spores. The wild-type strain is to be understood as meaning ATCC strain 10895. The transformants can be selected by adding the amino glycoside G418 (200 μg/ml) to the growth medium (1% casein peptone, 1% yeast extract, 2% glucose and 0.1% myo-inositol). The transformants were then clonally purified by micromanipulation (Steiner and Philipsen, 1995, Genetics, 140: 973-987) and examined for the disruption of the adenylate cyclase gene by Southern analysis. For example, a strain was found (AGΔ1-T333b) which showed the desired disruption in the adenylate cyclase gene.

›Example 6 · 1 of 5

Effect of the Adenylate Cyclase Gene Disruption on the Riboflavin Formation of Ashbya gossypii

A surprising indication of the effect of the cAMP quantities on riboflavin production was obtained in strain AGΔ1-T333b. In this adenylate cyclase disruptant of the fungus Ashbya gossypii , riboflavin concentrations of 36 mg/l are achieved. However, the addition of 1 mM cAMP to the medium (see Example 5) already leads to a drop in riboflavin concentration to 23 mg/l. To determine these values, a liquid culture of the selected transformants (Example 5) can be centrifuged and the OD 445 of the supernatant can be determined (ε(riboflavin)=12500 liter mol −1 cm 1 ).

When observing the visual appearance of strain AGΔ1-T333b when grown on solid medium (see medium of Example 5 with added agar), the addition of cAMP to the medium leads to a decrease in yellow coloration—and thus in riboflavin formation—of the Ashbya gossypii colonies. Also, when compared with the wild-type strain, strain AGΔ1-T333b, when grown on solid medium, shows an increased yellow coloration.

These experiments demonstrate the effect of cAMP on riboflavin production under the chosen experimental conditions.

6703 base pairs

nucleic acid

double

linear

GATCCGGCCA CGCATGTATT CCACTATGTA CGCTATATCG CGGCCTCGCC TCCGTGCGGG 60

CGCACCGCCT TACACCTCAA AGCCGTAGCC TTTCATGCAG TCCTTGTACT TTTGCACCAG 120

TTCCTGGCAC TTGACCGCAT CCACGCCGTT GAACAGCAGA CAGCTGTCTC TCGCTTCCTT 180

CTCGGGCTTG CACACACAGC ATGGCTTTGG CTTGTCGGTA CTACTACCTT GTGAAACACC 240

TGGCACAGAA GAGGATTCAG GCATGATTAA TGCTACAGTT CTTGGACGAT CTTCCACCAC 300

CGTCCGTTCC TTGAGCTTTT TTCACTTATA TAGCTCAACG CGCAAAAATG CTGACGAATA 360

CACATACAGC GCGCAGCAAC CGCTTAGTGG TTATTGGCTG CTGGTAGCAG CTGGACCCGA 420

GAGTCGGCAG TATCTTGGGC TTCGGCATTA GCGTAGTTCA GCGTAGGGCC ATGGGATTAT 480

TAATATCATA TCGTACGGCA AGCTTGTTTT AATAAGGAGC CTTCGGCATG GACACGGATT 540

GTCGTGAACC ACAGCCGCAA CGGGGCGAAG AGCTCTTCTA GCAGGTAAAA GAAGAGCACG 600

CGGGTCGTAC AGAACACGCT GAAGTCGTTC CAGAAGCTTA GCGGAGCGGA TTGCTGAGCA 660

GAATAATTCG ATG GAT AGA AAA GCG CAC AGA GCG GGC GAG CGC CGG GCT 709

Met Asp Arg Lys Ala His Arg Ala Gly Glu Arg Arg Ala

1 5 10

GCA GGC AGG ACA GAT GGG CGC GGA GGT TGG GAA CGC GGC GCG GAC GGG 757

Ala Gly Arg Thr Asp Gly Arg Gly Gly Trp Glu Arg Gly Ala Asp Gly

15 20 25

CTG GCG GCG GGG TCG CGG CGC GAC TCT GTC GAT GAC AGC TGG TCG AGC 805

Leu Ala Ala Gly Ser Arg Arg Asp Ser Val Asp Asp Ser Trp Ser Ser

30 35 40 45

CTA TCG AGC AAC TCG CAG GAG GCG CCA TTG GAG CAC GAG GTG CGG AGC 853

Leu Ser Ser Asn Ser Gln Glu Ala Pro Leu Glu His Glu Val Arg Ser

50 55 60

GGC GGA CGG ATT GTG GAG CCG GGC AGC GAC TTG CCG CCA CAT ATG AAG 901

Gly Gly Arg Ile Val Glu Pro Gly Ser Asp Leu Pro Pro His Met Lys

65 70 75

CAC AGT CGC GGG TTG CAT CAC GCG AGC TTT GCG CGC GTG AGC CAG CAC 949

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

80 85 90

CCG AGC TCG CCG CTG GCA AAG CAG CTG CGG CCT GTG CGT AAC GTG ACC 997

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

95 100 105

ATG CAG TCT GCC GCT GAG GAC GAC GAT GCG CGG GCC ACA GGC GCG CCT 1045

Met Gln Ser Ala Ala Glu Asp Asp Asp Ala Arg Ala Thr Gly Ala Pro

110 115 120 125

CCG CCA ATA CGG CGC ATA CCA TCG CGC GCA GGC TCG TTT TTT AAG CGC 1093

Pro Pro Ile Arg Arg Ile Pro Ser Arg Ala Gly Ser Phe Phe Lys Arg

130 135 140

CTG ACC GGC CGC AAG AGC TCC ATC AAT GGC TCG GAG ACT TCC GCG GCG 1141

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

145 150 155

GCT GAT GCG GAC GTC GCG GCG GCG CCG TCT TCG CTG CGG CGT AAG GTG 1189

Ala Asp Ala Asp Val Ala Ala Ala Pro Ser Ser Leu Arg Arg Lys Val

160 165 170

AAC ACA TTC ATC CAT GGA AAT GAA GCC AGG CGA GAC AGT AAG GGC ACA 1237

Asn Thr Phe Ile His Gly Asn Glu Ala Arg Arg Asp Ser Lys Gly Thr

175 180 185

ATA TCG ACG TCT TCA GCG GAC AGT AGG CGT GGG TCG ACG GCC AGC GGC 1285

Ile Ser Thr Ser Ser Ala Asp Ser Arg Arg Gly Ser Thr Ala Ser Gly

190 195 200 205

GGG AGT GCG TCG ATA ATT CAG CTT CCT GAC GGA CAG AGG CTC TCG TTG 1333

Gly Ser Ala Ser Ile Ile Gln Leu Pro Asp Gly Gln Arg Leu Ser Leu

210 215 220

CAG GAA TAC AAG CCT GTG TCG CCC ACT ACC ACT GCC CTT TCT AGC GCA 1381

Gln Glu Tyr Lys Pro Val Ser Pro Thr Thr Thr Ala Leu Ser Ser Ala

225 230 235

TCG AGC GCT GCT GCC CAG CAA GGC GGT GAC AGG AGC ACT GGC TCT CCT 1429

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

240 245 250

GTG CAT GTG CCG TGG GAT AAT GCG GAG GCT TCC AAC GAG CAC GAG TCG 1477

Val His Val Pro Trp Asp Asn Ala Glu Ala Ser Asn Glu His Glu Ser

255 260 265

GGC GTA AAC ACA ACA TTC TTT AAT TTA GAC ATG GAC CTG AAC AAC CTC 1525

Gly Val Asn Thr Thr Phe Phe Asn Leu Asp Met Asp Leu Asn Asn Leu

270 275 280 285

TCT GAC ATA ACG AGT GCC TAT CAG CAA CAG ACT GAG ACC ACT ACG AAT 1573

Ser Asp Ile Thr Ser Ala Tyr Gln Gln Gln Thr Glu Thr Thr Thr Asn

290 295 300

GCC AAT CTG GAG GTA AAT ATG GCA AAC GCC GGC AAG CCT GTT GTG CAC 1621

Ala Asn Leu Glu Val Asn Met Ala Asn Ala Gly Lys Pro Val Val His

305 310 315

ATG CCG CGG CCA CAG AAG CTT CCT GCG AGG GAA GTT CGC CAT ACC AAG 1669

Met Pro Arg Pro Gln Lys Leu Pro Ala Arg Glu Val Arg His Thr Lys

320 325 330

GGA GCT GCG CAG TGG ACG GCT CCG GAG TCA TGG GAT GTT GAT GAC CCC 1717

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

335 340 345

GTT ATC AAG CCA TCG AAA CAA AAA CAG AAG TGC CAC AAT CCA GCC CAT 1765

Val Ile Lys Pro Ser Lys Gln Lys Gln Lys Cys His Asn Pro Ala His

350 355 360 365

CGG CAC CAT CGG CAC CAC TAC AGT CCT CGT TCG GAT ATG ACC GGA ACG 1813

Arg His His Arg His His Tyr Ser Pro Arg Ser Asp Met Thr Gly Thr

370 375 380

TCT GGC GAT AAC AAT CGC TTG TCC GCA GAT CCA AAC CGG TAT TCA AAT 1861

Ser Gly Asp Asn Asn Arg Leu Ser Ala Asp Pro Asn Arg Tyr Ser Asn

385 390 395

CTG GAG GAC TCA CCC AAT CAT AGC CGT TTG ACA GCA GCC TTT TCT CGA 1909

Leu Glu Asp Ser Pro Asn His Ser Arg Leu Thr Ala Ala Phe Ser Arg

400 405 410

TCT CCA ATC CAC AGG GAT ATG TCT CCG CTT AGT ATC ATG AGT TCT GAT 1957

Ser Pro Ile His Arg Asp Met Ser Pro Leu Ser Ile Met Ser Ser Asp

415 420 425

AGC ATT GTA TCC AGT AAC TCA GAT TCC TGT AGC TTT TCC GTC GTG AGT 2005

Ser Ile Val Ser Ser Asn Ser Asp Ser Cys Ser Phe Ser Val Val Ser

430 435 440 445

AAC CCG GAA GAC ACG CCA AAA GAC ATC ACA CAT CCA GAC AAT GCT TCC 2053

›Example 6 · 2 of 5

Asn Pro Glu Asp Thr Pro Lys Asp Ile Thr His Pro Asp Asn Ala Ser

450 455 460

ACC TCT TCG ATC CAT GCA CAA GAA CTT GAA AAT AAT GAT ATT GAG GAT 2101

Thr Ser Ser Ile His Ala Gln Glu Leu Glu Asn Asn Asp Ile Glu Asp

465 470 475

GAT AAA CTG CAG CAT CAT TTA GAG AAG TAC TAC AAT CAC TTT AGT GAT 2149

Asp Lys Leu Gln His His Leu Glu Lys Tyr Tyr Asn His Phe Ser Asp

480 485 490

ATT GAC TAT CAC AAA AAA TAC ACG ATC CGT ATT TTT AAT ACG GAC GAT 2197

Ile Asp Tyr His Lys Lys Tyr Thr Ile Arg Ile Phe Asn Thr Asp Asp

495 500 505

ACT TTT ACA ACG CTA TCA TGT AGG CCG GAG ACT ACT GTA AAG GAA ATG 2245

Thr Phe Thr Thr Leu Ser Cys Arg Pro Glu Thr Thr Val Lys Glu Met

510 515 520 525

ATA CCC CAG ATA AAG CGG AAA TTT AAC GTG CCC CCA GGT AAC TAC CAG 2293

Ile Pro Gln Ile Lys Arg Lys Phe Asn Val Pro Pro Gly Asn Tyr Gln

530 535 540

GTT TCC CTT AAA GTC GGT AAG TTG TCA AAG GTA TTG AGG CCA ACA GCG 2341

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

545 550 555

AAG CCA ATC TTA ATT CAA ATA CGA CTC CTG TTA TTC AAT GGT TAT AAG 2389

Lys Pro Ile Leu Ile Gln Ile Arg Leu Leu Leu Phe Asn Gly Tyr Lys

560 565 570

AAG ACA GAT CGT TTA AAC ATT ATG GGC ATA GAA GAC TTG AGT TTT GTC 2437

Lys Thr Asp Arg Leu Asn Ile Met Gly Ile Glu Asp Leu Ser Phe Val

575 580 585

TTC AGT TTT GTC TTC CAC CCC GTT ATT ACA TCA CAG TTA ACA TAT GAA 2485

Phe Ser Phe Val Phe His Pro Val Ile Thr Ser Gln Leu Thr Tyr Glu

590 595 600 605

CAA GAG CAA AGG CTC AGT AAA GGT GAG TTC GTT CAT GTT GAC CTG CGT 2533

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

610 615 620

AAC ATG GAC CTT ACG ATT CCG CCG ATT ATT TTT TAT CAA CAT ACG TCT 2581

Asn Met Asp Leu Thr Ile Pro Pro Ile Ile Phe Tyr Gln His Thr Ser

625 630 635

GAC ATT GAG AGT CTC GAT GTA TCC AAT AAT GCC AAT ATA TTC CTG CCT 2629

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

640 645 650

TTG GAT TTC ATC GAG AGT GTG ATT AAG CTT TCC AGT TTA CGA ATG GTC 2677

Leu Asp Phe Ile Glu Ser Val Ile Lys Leu Ser Ser Leu Arg Met Val

655 660 665

AAT ATT AGA GCG TCC CGA TTT CCT TCA AAT ATC TGT GAG GCA ACA AAG 2725

Asn Ile Arg Ala Ser Arg Phe Pro Ser Asn Ile Cys Glu Ala Thr Lys

670 675 680 685

CTG ATC ACC CTT GAT TTG GAA AGG AAC TTT ATT AAA AGG GTA CCG GAC 2773

Leu Ile Thr Leu Asp Leu Glu Arg Asn Phe Ile Lys Arg Val Pro Asp

690 695 700

CAG ATG TCT AAG CTT ACA AAT TTA ACA ATT TTG AAT CTA AGG TGC AAT 2821

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

705 710 715

GAA TTG GAC AGG CTA CCA AGG GGG TTT AAA GAC TTA AAA AGT CTT CAA 2869

Glu Leu Asp Arg Leu Pro Arg Gly Phe Lys Asp Leu Lys Ser Leu Gln

720 725 730

CTA CTC GAT ATT TCT TCT AAT AAG TTT AAT ATC TAC CCC GAG GTT ATT 2917

Leu Leu Asp Ile Ser Ser Asn Lys Phe Asn Ile Tyr Pro Glu Val Ile

735 740 745

AAT TCA TGT ACA AAC TTG CTG CAG CTA GAC TTG TCC TAT AAC AAG ATT 2965

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

750 755 760 765

AGA TCC TTA CCG GAT AGT ATG AAT CAG CTG CAA AAA CTA GCC AAA ATT 3013

Arg Ser Leu Pro Asp Ser Met Asn Gln Leu Gln Lys Leu Ala Lys Ile

770 775 780

AAC CTA TCG AAC AAC CGC ATA ACA CAT GTC AAT GAT CTT TCG AAA ATG 3061

Asn Leu Ser Asn Asn Arg Ile Thr His Val Asn Asp Leu Ser Lys Met

785 790 795

ACT TCT CTG CGG ACC TTG GAC TTG AGA TAT AAT AGA ATT GAG TCT ATA 3109

Thr Ser Leu Arg Thr Leu Asp Leu Arg Tyr Asn Arg Ile Glu Ser Ile

800 805 810

AAA TGT CGG GTA CCA AAC CTC CAG AAT CTT TTC CTG ACC GAA AAT AGA 3157

Lys Cys Arg Val Pro Asn Leu Gln Asn Leu Phe Leu Thr Glu Asn Arg

815 820 825

CTC ACA ATG TTT GAT GAT GAC CAG CTG ATG CTT AGA ACA CTA GAA TTG 3205

Leu Thr Met Phe Asp Asp Asp Gln Leu Met Leu Arg Thr Leu Glu Leu

830 835 840 845

CAG AGA AAT CCA TTA TCG ATA CTC ACT TTG AAG AAC GAC TAC TTG GAA 3253

Gln Arg Asn Pro Leu Ser Ile Leu Thr Leu Lys Asn Asp Tyr Leu Glu

850 855 860

CAT TTG ACA AGT TTA TCT ATA AGC AAA GCA AAG TTA GCG GTT TTA CCT 3301

His Leu Thr Ser Leu Ser Ile Ser Lys Ala Lys Leu Ala Val Leu Pro

865 870 875

GAG AGC TTG CTA AGG CGG CTA CCA CGT TTA GAG AAA CTG GAA TTA AGT 3349

Glu Ser Leu Leu Arg Arg Leu Pro Arg Leu Glu Lys Leu Glu Leu Ser

880 885 890

GAA AAT AGC TTG ACT GTT CTG CCT CCA GAT ATC AAG CAC CTG AAG AAG 3397

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

895 900 905

TTA GTT CAT CTC TCA GTG GCC AAA AAT AAA CTG GAA TCA CTT CCA GAC 3445

Leu Val His Leu Ser Val Ala Lys Asn Lys Leu Glu Ser Leu Pro Asp

910 915 920 925

GAA ATC GCT TCT CTA AAA AAC CTT AAG ATG CTT GAC TTA CAT TGT AAT 3493

Glu Ile Ala Ser Leu Lys Asn Leu Lys Met Leu Asp Leu His Cys Asn

930 935 940

AAT TTG ATG ACA TTG CCT GCT GCT CTA TCA ACA CTC AGT TTG ACT TTT 3541

Asn Leu Met Thr Leu Pro Ala Ala Leu Ser Thr Leu Ser Leu Thr Phe

945 950 955

GTG AAC ATT TCT TCA AAT ATG TTA TCT GGG CAT CAT GAA TTG TAT CGC 3589

Val Asn Ile Ser Ser Asn Met Leu Ser Gly His His Glu Leu Tyr Arg

960 965 970

ACC TTC CAA GGA ACT TCA AAT ATT GCA AAA TCA TTG ATG TTT TTA AGC 3637

Thr Phe Gln Gly Thr Ser Asn Ile Ala Lys Ser Leu Met Phe Leu Ser

975 980 985

GCT GCA GAC AAC CAG ATG GGT GAT AAA TTC TGG GAG ATA TTC AAT ACT 3685

Ala Ala Asp Asn Gln Met Gly Asp Lys Phe Trp Glu Ile Phe Asn Thr

990 995 1000 1005

TTT AAG ACG TTG AAA GTT TTA AAC CTG TCA TAT AAC AAC TTT ATG GCC 3733

Phe Lys Thr Leu Lys Val Leu Asn Leu Ser Tyr Asn Asn Phe Met Ala

1010 1015 1020

CTG CCA GAA CTT GAG ATG GAG AAT TTA ACT GAA CTC TAC TTA TCG GGT 3781

Leu Pro Glu Leu Glu Met Glu Asn Leu Thr Glu Leu Tyr Leu Ser Gly

1025 1030 1035

AAT CAT TTA ACA ACG CTC TCT GGT GAA GCT TTC TTA AAG CTC AAA TCA 3829

Asn His Leu Thr Thr Leu Ser Gly Glu Ala Phe Leu Lys Leu Lys Ser

1040 1045 1050

CTT AGA GTT CTC ATG CTG AAT GCT AAT AAT TTG CAG TCC CTG CCA GCA 3877

Leu Arg Val Leu Met Leu Asn Ala Asn Asn Leu Gln Ser Leu Pro Ala

1055 1060 1065

GAG ATC TCG CAA CTC TCG CAA CTC TCC GTC ATT GAT GTC GGT TCG AAT 3925

Glu Ile Ser Gln Leu Ser Gln Leu Ser Val Ile Asp Val Gly Ser Asn

1070 1075 1080 1085

CAG CTC AAG TAC AAT ATA TCC AAC TAC CAT TAT GAC TGG AAT TGG AGA 3973

Gln Leu Lys Tyr Asn Ile Ser Asn Tyr His Tyr Asp Trp Asn Trp Arg

1090 1095 1100

CAG AAC ACC GAG CTA AAG TAC CTA AAC TTC TCC GGT AAC ACT AGA TTT 4021

Gln Asn Thr Glu Leu Lys Tyr Leu Asn Phe Ser Gly Asn Thr Arg Phe

1105 1110 1115

GAA ATC AAA AGT GCT ATG GAG TAT GGA ACC AAT ATG CAT TTA TCT GAC 4069

›Example 6 · 3 of 5

Glu Ile Lys Ser Ala Met Glu Tyr Gly Thr Asn Met His Leu Ser Asp

1120 1125 1130

CTT ACT GTC TTG AAA CAG CTC AGA GTT TTA GGC TTA ATG GAT GTG ACC 4117

Leu Thr Val Leu Lys Gln Leu Arg Val Leu Gly Leu Met Asp Val Thr

1135 1140 1145

CTA AAC ACT TCG CGA GTC CCT GAT GAT GGG GTC AAC TTC AGA TTA AGA 4165

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

1150 1155 1160 1165

ACA ATG GGT TCT ACA ATC AAT GGT ATG GAG TAT GGT GTA GCG GAC TCG 4213

Thr Met Gly Ser Thr Ile Asn Gly Met Glu Tyr Gly Val Ala Asp Ser

1170 1175 1180

TTG GGG CAG ACA GAT TCT GTT TCA ACC AGA GAC GTC ACT TTT GAA CGT 4261

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

1185 1190 1195

TTC AGG GGA AAT GAA GAT GAG TGT CTG ATT TGT TTG TAT GAT GGA ATT 4309

Phe Arg Gly Asn Glu Asp Glu Cys Leu Ile Cys Leu Tyr Asp Gly Ile

1200 1205 1210

AAC GAT AAT ACA AAC ACA GGC CAT AAA ATA TCA CAG GTC ATA AGA GAT 4357

Asn Asp Asn Thr Asn Thr Gly His Lys Ile Ser Gln Val Ile Arg Asp

1215 1220 1225

ATA TAT GAT CGC ATA TTA GTG CGG TCT GTG GAG AAA TAT GGT GAC TCA 4405

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

1230 1235 1240 1245

ACG CCA GCC GAA ATT CGC GAT GCC CTA CGC TTC AGT TTC CTG CAA CTA 4453

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

1250 1255 1260

AAC AAA GAA ATT AAT TCC TCG ATA CTT GCT GCT GGT AAT AAC AAG AAT 4501

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

1265 1270 1275

AGT AAC GGT AAC ACT AAT AAT AAT AAT AAC ACT AAT AAT AAT AAT AAT 4549

Ser Asn Gly Asn Thr Asn Asn Asn Asn Asn Thr Asn Asn Asn Asn Asn

1280 1285 1290

AAT GGA AAT AAT AAT ACT AAT TCA AAT GGT GAA TCT GTC GTC TTA ACT 4597

Asn Gly Asn Asn Asn Thr Asn Ser Asn Gly Glu Ser Val Val Leu Thr

1295 1300 1305

GCC GCC GAT TCA TTG AGT GGC GCG TCG GTC ACA GTT GTA TTC ATG AAG 4645

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

1310 1315 1320 1325

GGC AAA AAT ATC TAT ACT GCC AAC ATC GGA AAC GCA ACG GCT ATT CTC 4693

Gly Lys Asn Ile Tyr Thr Ala Asn Ile Gly Asn Ala Thr Ala Ile Leu

1330 1335 1340

TCT AAG GGC AAC GCT GAT TAT CGT ATT CTA ACA CGC AAA CAC GTT CCT 4741

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

1345 1350 1355

ACT GAT ACT GAA GAG TTT GAA AGA ATC CGA TTA TCC GGA GGA TAT GTC 4789

Thr Asp Thr Glu Glu Phe Glu Arg Ile Arg Leu Ser Gly Gly Tyr Val

1360 1365 1370

GAC AAT AAG AGG GTG AAT GGA GTA TCC GAA ATA TCC AGA GCA ATA GGA 4837

Asp Asn Lys Arg Val Asn Gly Val Ser Glu Ile Ser Arg Ala Ile Gly

1375 1380 1385

TTT TTT GAT CTC TTA CCA CAT ATT CAC GCC TCA CCT GAT ATT AGT GAG 4885

Phe Phe Asp Leu Leu Pro His Ile His Ala Ser Pro Asp Ile Ser Glu

1390 1395 1400 1405

ACC ACC TTA ACA TAT ACG GAC GAT ATG TTG GTA ATA GCG ACC CAG AGT 4933

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

1410 1415 1420

TTA CTA TCT TAT GTC AGT CTG GAA AAA ATA TGC GAT ATT GCT AGG GAA 4981

Leu Leu Ser Tyr Val Ser Leu Glu Lys Ile Cys Asp Ile Ala Arg Glu

1425 1430 1435

AAC AAG TCT CAA CCA ATG CTA GCA GCC GAA AGA ATG AAG GAT TAT GCG 5029

Asn Lys Ser Gln Pro Met Leu Ala Ala Glu Arg Met Lys Asp Tyr Ala

1440 1445 1450

ATT GCA TAT GGA TGT ACG GAT AAT ATA ACC ATA CTG TGC GTT TCA TTT 5077

Ile Ala Tyr Gly Cys Thr Asp Asn Ile Thr Ile Leu Cys Val Ser Phe

1455 1460 1465

AAT AAA AAT GTT GGC AAG CAG AAA CAA TTC TCT CTG AAC AAA AGC GAT 5125

Asn Lys Asn Val Gly Lys Gln Lys Gln Phe Ser Leu Asn Lys Ser Asp

1470 1475 1480 1485

CTA CTG GGT CGG CGT CTA AAC TTT GAA GAT GTT TCA CTG CGC AGA TTG 5173

Leu Leu Gly Arg Arg Leu Asn Phe Glu Asp Val Ser Leu Arg Arg Leu

1490 1495 1500

CGC CCA GAA ATT GCC CCA CCA ACT GGA CAT CTG GCA ATC GTA TTC ACA 5221

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

1505 1510 1515

GAT ATT AAA AAC TCA ACA TTC CTT TGG GAA CTA TTC CCC AAC GCA ATG 5269

Asp Ile Lys Asn Ser Thr Phe Leu Trp Glu Leu Phe Pro Asn Ala Met

1520 1525 1530

CGT GTG GCA ATA AAA GCT CAT AAC GAC ATT ATG CGT AGA ACA CTT CGA 5317

Arg Val Ala Ile Lys Ala His Asn Asp Ile Met Arg Arg Thr Leu Arg

1535 1540 1545

ATA TTT GGT GGA TAC GAA GTC AAG ACC GAA GGT GAT GCT TTT ATG GTT 5365

Ile Phe Gly Gly Tyr Glu Val Lys Thr Glu Gly Asp Ala Phe Met Val

1550 1555 1560 1565

GCT TTC CCA ACG CCT ACC AGT GCT CTG GTA TGG TGC TTA ACG ATT CAA 5413

Ala Phe Pro Thr Pro Thr Ser Ala Leu Val Trp Cys Leu Thr Ile Gln

1570 1575 1580

CTT AAA CTA CTG GAG CTT GAT TGG CCA GAG GAG ATC ACA TCC ATC AAA 5461

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

1585 1590 1595

TCC GGC TGT ATG ATT ACA GAT GAC AGC GGA AAT ACA ATA TAC CAG GGT 5509

Ser Gly Cys Met Ile Thr Asp Asp Ser Gly Asn Thr Ile Tyr Gln Gly

1600 1605 1610

CTC TCT GTT CGT ATG GGC ATC CAC TGG GGA TGT CCT GTG CCG GAG GTG 5557

Leu Ser Val Arg Met Gly Ile His Trp Gly Cys Pro Val Pro Glu Val

1615 1620 1625

GAT ATA GTG ACG CAG AGA ATG GAT TAC CTT GGT CCT ATG GTC AAC AAG 5605

Asp Ile Val Thr Gln Arg Met Asp Tyr Leu Gly Pro Met Val Asn Lys

1630 1635 1640 1645

GCA GCT AGA GTG TCA GCA ACC GCT GAT GGT GGC CAG ATT ACA CTG AGC 5653

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

1650 1655 1660

AGC GAC TTC CTA GCG GAA TTA AAC AAA ATA ATG AAA CTC CAC CAC ATG 5701

Ser Asp Phe Leu Ala Glu Leu Asn Lys Ile Met Lys Leu His His Met

1665 1670 1675

GTC GTA CAG GAT AAA AAG CCA CTG AAG GAG GTG TAT GGT GAA GAA TTC 5749

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

1680 1685 1690

GTG GGC GAA GTT TTA GAG CGT GAG ATC CAA ATG TTA GAT AAC GTT GGA 5797

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

1695 1700 1705

TTG GTT CTG GAA GAA CTG GGG GAG CAG AAG TTA AAG GGT CTG GAA ACT 5845

Leu Val Leu Glu Glu Leu Gly Glu Gln Lys Leu Lys Gly Leu Glu Thr

1710 1715 1720 1725

CGG GAA TTC ATA ACT ATT GCG TAT CCC AAG CAA TTG GCA GCT AGA CAT 5893

Arg Glu Phe Ile Thr Ile Ala Tyr Pro Lys Gln Leu Ala Ala Arg His

1730 1735 1740

ACC ATG TCC ACA GGG GAG AAA AGC ACC AAC ATT GTC AAC GAT AAA TAT 5941

Thr Met Ser Thr Gly Glu Lys Ser Thr Asn Ile Val Asn Asp Lys Tyr

1745 1750 1755

GTT CTC CAA TTG AAA GTA ATT TCC ACA ACT TTG GAG AAT CTT CTT TCA 5989

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

1760 1765 1770

ACT GTT AAC GGC GGT CTG ATC GAA ATC GAC AAT AGT GTC CTA CAA CTA 6037

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

›Example 6 · 4 of 5

1775 1780 1785

CCC ACA CAT ATG ACT CGT GAT AAA AAG GTT GAT AAA GCG CTG AGC CAG 6085

Pro Thr His Met Thr Arg Asp Lys Lys Val Asp Lys Ala Leu Ser Gln

1790 1795 1800 1805

AAC ACG GAA TAC GAT TGG ATA TCC TTG TTG GAT CAT CTT GTC ACT AGG 6133

Asn Thr Glu Tyr Asp Trp Ile Ser Leu Leu Asp His Leu Val Thr Arg

1810 1815 1820

CTG GAG TCT ACG GTA GCA ATG CTG CAG CTG CGG CAG CGA TTG GAG GGT 6181

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

1825 1830 1835

GGC TTG GAG ATC TGT CGA CCA GGT AAC AAT ACG AAA AAA TCA GTA TTC 6229

Gly Leu Glu Ile Cys Arg Pro Gly Asn Asn Thr Lys Lys Ser Val Phe

1840 1845 1850

GAG CTG CTG GAT CTG CTG TGC ACT GGA ACA GGT TTC AAG CGA GAT ACG 6277

Glu Leu Leu Asp Leu Leu Cys Thr Gly Thr Gly Phe Lys Arg Asp Thr

1855 1860 1865

CCG TCT GAA AAT AAA TAACTTAACA GTACATAATT GTCAGACTAT TTCGGAGTCC 6332

Pro Ser Glu Asn Lys

1870 187

TGCATCCGAA ATAACCAGCG TATGATATAT TACGTTATTA AAATGCTATT TACGAATGAT 6392

CAGTCTCACC GTGGCGCTAG AATGATCTCA AGCGTATTTA TAATGTAGGC TATTTCTGTG 6452

CCTCCAAGTC CTTCATCTCA AACTGCTGTC TGTTCTGAGA ACTGCCATGA TAGGGAGACT 6512

CAGCCCCACC CGTAATAGGT TCCTGATCTA TGAGACCCTC AAAAAACGTG TCGCGAAGCT 6572

CGCGCCACCT CGTGGTCCAC GTCCCCAAAA ACACCAAAAT CAAGCTCGAA CAGAGCTGCA 6632

GGAAAAACCA CGAGAACGAC GGAAGCTGCT TCGTGTGAAA CCACACCACT AGGAGATTTA 6692

TCGCATGGAT C 6703

1874 amino acids

amino acid

single

linear

Met Asp Arg Lys Ala His Arg Ala Gly Glu Arg Arg Ala Ala Gly Arg

1 5 10 15

Thr Asp Gly Arg Gly Gly Trp Glu Arg Gly Ala Asp Gly Leu Ala Ala

20 25 30

Gly Ser Arg Arg Asp Ser Val Asp Asp Ser Trp Ser Ser Leu Ser Ser

35 40 45

Asn Ser Gln Glu Ala Pro Leu Glu His Glu Val Arg Ser Gly Gly Arg

50 55 60

Ile Val Glu Pro Gly Ser Asp Leu Pro Pro His Met Lys His Ser Arg

65 70 75 80

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

85 90 95

Pro Leu Ala Lys Gln Leu Arg Pro Val Arg Asn Val Thr Met Gln Ser

100 105 110

Ala Ala Glu Asp Asp Asp Ala Arg Ala Thr Gly Ala Pro Pro Pro Ile

115 120 125

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

130 135 140

Arg Lys Ser Ser Ile Asn Gly Ser Glu Thr Ser Ala Ala Ala Asp Ala

145 150 155 160

Asp Val Ala Ala Ala Pro Ser Ser Leu Arg Arg Lys Val Asn Thr Phe

165 170 175

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

180 185 190

Ser Ser Ala Asp Ser Arg Arg Gly Ser Thr Ala Ser Gly Gly Ser Ala

195 200 205

Ser Ile Ile Gln Leu Pro Asp Gly Gln Arg Leu Ser Leu Gln Glu Tyr

210 215 220

Lys Pro Val Ser Pro Thr Thr Thr Ala Leu Ser Ser Ala Ser Ser Ala

225 230 235 240

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

245 250 255

Pro Trp Asp Asn Ala Glu Ala Ser Asn Glu His Glu Ser Gly Val Asn

260 265 270

Thr Thr Phe Phe Asn Leu Asp Met Asp Leu Asn Asn Leu Ser Asp Ile

275 280 285

Thr Ser Ala Tyr Gln Gln Gln Thr Glu Thr Thr Thr Asn Ala Asn Leu

290 295 300

Glu Val Asn Met Ala Asn Ala Gly Lys Pro Val Val His Met Pro Arg

305 310 315 320

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

325 330 335

Gln Trp Thr Ala Pro Glu Ser Trp Asp Val Asp Asp Pro Val Ile Lys

340 345 350

Pro Ser Lys Gln Lys Gln Lys Cys His Asn Pro Ala His Arg His His

355 360 365

Arg His His Tyr Ser Pro Arg Ser Asp Met Thr Gly Thr Ser Gly Asp

370 375 380

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

385 390 395 400

Ser Pro Asn His Ser Arg Leu Thr Ala Ala Phe Ser Arg Ser Pro Ile

405 410 415

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

420 425 430

Ser Ser Asn Ser Asp Ser Cys Ser Phe Ser Val Val Ser Asn Pro Glu

435 440 445

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

450 455 460

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

465 470 475 480

Gln His His Leu Glu Lys Tyr Tyr Asn His Phe Ser Asp Ile Asp Tyr

485 490 495

His Lys Lys Tyr Thr Ile Arg Ile Phe Asn Thr Asp Asp Thr Phe Thr

500 505 510

Thr Leu Ser Cys Arg Pro Glu Thr Thr Val Lys Glu Met Ile Pro Gln

515 520 525

Ile Lys Arg Lys Phe Asn Val Pro Pro Gly Asn Tyr Gln Val Ser Leu

530 535 540

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

545 550 555 560

Leu Ile Gln Ile Arg Leu Leu Leu Phe Asn Gly Tyr Lys Lys Thr Asp

565 570 575

Arg Leu Asn Ile Met Gly Ile Glu Asp Leu Ser Phe Val Phe Ser Phe

580 585 590

Val Phe His Pro Val Ile Thr Ser Gln Leu Thr Tyr Glu Gln Glu Gln

595 600 605

Arg Leu Ser Lys Gly Glu Phe Val His Val Asp Leu Arg Asn Met Asp

610 615 620

Leu Thr Ile Pro Pro Ile Ile Phe Tyr Gln His Thr Ser Asp Ile Glu

625 630 635 640

Ser Leu Asp Val Ser Asn Asn Ala Asn Ile Phe Leu Pro Leu Asp Phe

645 650 655

Ile Glu Ser Val Ile Lys Leu Ser Ser Leu Arg Met Val Asn Ile Arg

660 665 670

Ala Ser Arg Phe Pro Ser Asn Ile Cys Glu Ala Thr Lys Leu Ile Thr

675 680 685

Leu Asp Leu Glu Arg Asn Phe Ile Lys Arg Val Pro Asp Gln Met Ser

690 695 700

Lys Leu Thr Asn Leu Thr Ile Leu Asn Leu Arg Cys Asn Glu Leu Asp

705 710 715 720

Arg Leu Pro Arg Gly Phe Lys Asp Leu Lys Ser Leu Gln Leu Leu Asp

725 730 735

Ile Ser Ser Asn Lys Phe Asn Ile Tyr Pro Glu Val Ile Asn Ser Cys

740 745 750

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

755 760 765

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

770 775 780

Asn Asn Arg Ile Thr His Val Asn Asp Leu Ser Lys Met Thr Ser Leu

785 790 795 800

Arg Thr Leu Asp Leu Arg Tyr Asn Arg Ile Glu Ser Ile Lys Cys Arg

805 810 815

Val Pro Asn Leu Gln Asn Leu Phe Leu Thr Glu Asn Arg Leu Thr Met

820 825 830

Phe Asp Asp Asp Gln Leu Met Leu Arg Thr Leu Glu Leu Gln Arg Asn

835 840 845

Pro Leu Ser Ile Leu Thr Leu Lys Asn Asp Tyr Leu Glu His Leu Thr

850 855 860

Ser Leu Ser Ile Ser Lys Ala Lys Leu Ala Val Leu Pro Glu Ser Leu

865 870 875 880

Leu Arg Arg Leu Pro Arg Leu Glu Lys Leu Glu Leu Ser Glu Asn Ser

885 890 895

Leu Thr Val Leu Pro Pro Asp Ile Lys His Leu Lys Lys Leu Val His

900 905 910

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

915 920 925

Ser Leu Lys Asn Leu Lys Met Leu Asp Leu His Cys Asn Asn Leu Met

930 935 940

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

945 950 955 960

Ser Ser Asn Met Leu Ser Gly His His Glu Leu Tyr Arg Thr Phe Gln

965 970 975

Gly Thr Ser Asn Ile Ala Lys Ser Leu Met Phe Leu Ser Ala Ala Asp

980 985 990

Asn Gln Met Gly Asp Lys Phe Trp Glu Ile Phe Asn Thr Phe Lys Thr

995 1000 1005

›Example 6 · 5 of 5

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

1010 1015 1020

Leu Glu Met Glu Asn Leu Thr Glu Leu Tyr Leu Ser Gly Asn His Leu

1025 1030 1035 1040

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

1045 1050 1055

Leu Met Leu Asn Ala Asn Asn Leu Gln Ser Leu Pro Ala Glu Ile Ser

1060 1065 1070

Gln Leu Ser Gln Leu Ser Val Ile Asp Val Gly Ser Asn Gln Leu Lys

1075 1080 1085

Tyr Asn Ile Ser Asn Tyr His Tyr Asp Trp Asn Trp Arg Gln Asn Thr

1090 1095 1100

Glu Leu Lys Tyr Leu Asn Phe Ser Gly Asn Thr Arg Phe Glu Ile Lys

1105 1110 1115 1120

Ser Ala Met Glu Tyr Gly Thr Asn Met His Leu Ser Asp Leu Thr Val

1125 1130 1135

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

1140 1145 1150

Ser Arg Val Pro Asp Asp Gly Val Asn Phe Arg Leu Arg Thr Met Gly

1155 1160 1165

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

1170 1175 1180

Thr Asp Ser Val Ser Thr Arg Asp Val Thr Phe Glu Arg Phe Arg Gly

1185 1190 1195 1200

Asn Glu Asp Glu Cys Leu Ile Cys Leu Tyr Asp Gly Ile Asn Asp Asn

1205 1210 1215

Thr Asn Thr Gly His Lys Ile Ser Gln Val Ile Arg Asp Ile Tyr Asp

1220 1225 1230

Arg Ile Leu Val Arg Ser Val Glu Lys Tyr Gly Asp Ser Thr Pro Ala

1235 1240 1245

Glu Ile Arg Asp Ala Leu Arg Phe Ser Phe Leu Gln Leu Asn Lys Glu

1250 1255 1260

Ile Asn Ser Ser Ile Leu Ala Ala Gly Asn Asn Lys Asn Ser Asn Gly

1265 1270 1275 1280

Asn Thr Asn Asn Asn Asn Asn Thr Asn Asn Asn Asn Asn Asn Gly Asn

1285 1290 1295

Asn Asn Thr Asn Ser Asn Gly Glu Ser Val Val Leu Thr Ala Ala Asp

1300 1305 1310

Ser Leu Ser Gly Ala Ser Val Thr Val Val Phe Met Lys Gly Lys Asn

1315 1320 1325

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

1330 1335 1340

Asn Ala Asp Tyr Arg Ile Leu Thr Arg Lys His Val Pro Thr Asp Thr

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

Leu Leu Pro His Ile His Ala Ser Pro Asp Ile Ser Glu Thr Thr Leu

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

Gln Pro Met Leu Ala Ala Glu Arg Met Lys Asp Tyr Ala Ile Ala Tyr

1445 1450 1455

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

1460 1465 1470

Val Gly Lys Gln Lys Gln Phe Ser Leu Asn Lys Ser Asp Leu Leu Gly

1475 1480 1485

Arg Arg Leu Asn Phe Glu Asp Val Ser Leu Arg Arg Leu Arg Pro Glu

1490 1495 1500

Ile Ala Pro Pro Thr Gly His Leu Ala Ile Val Phe Thr Asp Ile Lys

1505 1510 1515 1520

Asn Ser Thr Phe Leu Trp Glu Leu Phe Pro Asn Ala Met Arg Val Ala

1525 1530 1535

Ile Lys Ala His Asn Asp Ile Met Arg Arg Thr Leu Arg Ile Phe Gly

1540 1545 1550

Gly Tyr Glu Val Lys Thr Glu Gly Asp Ala Phe Met Val Ala Phe Pro

1555 1560 1565

Thr Pro Thr Ser Ala Leu Val Trp Cys Leu Thr Ile Gln Leu Lys Leu

1570 1575 1580

Leu Glu Leu Asp Trp Pro Glu Glu Ile Thr Ser Ile Lys Ser Gly Cys

1585 1590 1595 1600

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

1605 1610 1615

Arg Met Gly Ile His Trp Gly Cys Pro Val Pro Glu Val Asp Ile Val

1620 1625 1630

Thr Gln Arg Met Asp Tyr Leu Gly Pro Met Val Asn Lys Ala Ala Arg

1635 1640 1645

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

1650 1655 1660

Leu Ala Glu Leu Asn Lys Ile Met Lys Leu His His Met Val Val Gln

1665 1670 1675 1680

Asp Lys Lys Pro Leu Lys Glu Val Tyr Gly Glu Glu Phe Val Gly Glu

1685 1690 1695

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

1700 1705 1710

Glu Glu Leu Gly Glu Gln Lys Leu Lys Gly Leu Glu Thr Arg Glu Phe

1715 1720 1725

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

1730 1735 1740

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

1745 1750 1755 1760

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

1765 1770 1775

Gly Gly Leu Ile Glu Ile Asp Asn Ser Val Leu Gln Leu Pro Thr His

1780 1785 1790

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

1795 1800 1805

Tyr Asp Trp Ile Ser Leu Leu Asp His Leu Val Thr Arg Leu Glu Ser

1810 1815 1820

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

1825 1830 1835 1840

Ile Cys Arg Pro Gly Asn Asn Thr Lys Lys Ser Val Phe Glu Leu Leu

1845 1850 1855

Asp Leu Leu Cys Thr Gly Thr Gly Phe Lys Arg Asp Thr Pro Ser Glu

1860 1865 1870

Asn Lys

2 of 13 part labels are ours — the grant heads the rest

Claims as granted

11 claims

Classifications

22 codes

IPC · International Patent Classification

Section C — Chemistry; metallurgy

C12N9/02
C12N9/88
C12N9/04
C12N9/10
C12P25/00
C12N15/09
C12N15/60
C12R1/645
C12Q1/68
C12N15/00
C12N15/31
C12R1/865
C12N1/15
C07K14/37
C12Q1/18

USPC · US Patent Classification

435/122435/252.3435/255.5435/255.4435/254.1435/232435/255.2

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Examiner

Charles L. Patterson, Jr.

art unit 1652 · TC 1600

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