Cellulase enzymes

Cellulases are enzymes that can hydrolyze the glycosidic linkages in polysaccharides such as cellulose. These enzymes are used in a number of industrial applications where breaking down biomass is beneficial. For example, cellulases can be used as a supplement in animal feed to decrease the production of fecal waste by increasing the digestibility of the feed. Cellulases can also be used to increase the efficiency of alcoholic fermentations (e.g., in beer brewing) by converting undigestible biomass into fermentable sugars. In addition, the “softening” of blue jeans to produce a “stone-washed” look can be facilitated by treating the jeans with cellulases.

The invention is based on the discovery of a new cellulase isolated from the fungus Piromyces rhizinflata . The gene encoding this cellulase is designated cbhA. A portion of an cbhA cDNA is described below.

Accordingly, the invention features a substantially pure polypeptide having an amino acid sequence at least 70% (e.g., at least 80, 90, or 95%) conserved with or identical to an amino acid sequence representing the catalytic domain of CBHA (SEQ ID NO:4; described below), the polypeptide encoded by cbhA. The polypeptide is capable of hydrolyzing a polysaccharide such as oat spelt xylan. Such a polysaccharide can also be cellulose (e.g., carboxymethyl cellulose), polysaccharides containing β-1,3′ or β-1,4′ glycosidic linkage (e.g., barley β-glycan), or lechinan.

The invention also includes an isolated nucleic acid encoding a polypeptide of the invention. For example, the invention includes an isolated nucleic acid having a sequence encoding a polypeptide that hydrolyzes a polysaccharide, provided that the nucleic acid hybridizes under stringent conditions to SEQ ID NO:1.

In addition, the invention features any vectors or transformed cells which contain a nucleic acid of the invention. Vectors include nucleic acid vectors, such as expression plasmids, or viral vectors. Transformed cells include eukaryotic and prokaryotic cells.

A “nucleic acid” encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized or modified) DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. An “isolated nucleic acid” refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the 5′ non-coding (e.g., promoter) sequences which are immediately contiguous to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. The term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.

By “hybridizes under stringent conditions” is meant specific and non-covalent binding to an immobilized reference nucleic acids in the presence of 0.2×SSC (1.75 g/l NaCl, 0.88 g/l Na 3 citrate.2H 2 O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68° C.

The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). The polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) by weight pure. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity or conservation to a reference polypeptide or nucleic acid, the percent identity or conservation is determined by the algorithm of Myers and Miller, CABIOS (1989), which is embodied in the ALIGN program (version 2.0), or its equivalent, using a gap length penalty of 12 and a gap penalty of 4 where such parameters are required. All other parameters are set to their default positions. Access to ALIGN is readily available. See, e.g., http://www2.igh.cnrs.fr\/bin/align-guess.cgi on the Internet.

Other features or advantages of the present invention will be apparent from the following detailed description, the drawings, and also from the claims.

The invention relates to a cellulase enzyme, nucleic acids encoding it, and vectors and cells containing such nucleic acids. Contemplated within the scope of this invention are recombinant nucleic acids or viruses which allow production of CBHA in a transformed cell or transgenic organism or allow ease of producing specific or non-specific mutations within the CBHA reading frame. These recombinant nucleic acids or viruses may further include any one of a variety of sequences flanking or within the CBHA coding sequences, such as strong constitutive promoters within the CBHA coding sequence, as introns containing cis-elements that allow high level expression, or efficient polyadenylation signals.

Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the isolation of CBHA polypeptides and nucleic acids described below, utilize the present invention to its fullest extent. The following examples are to be construed as merely illustrative of how one skilled in the art can isolate and use CBHA polypeptides and nucleic acids from biological sources, and are not limitative of the remainder of the disclosure in any way. For example, once the sequence of the cbhA cDNA is known, any cbhA sequence can be obtained by PCR amplification of mRNA or genomic DNA. Any publications cited in this disclosure are hereby incorporated by reference.

The anaerobic fungus Piromyces rhizinflata, strain 2301, was cultivated anaerobically at 39° C. in a modified semi-defined medium as described in Lowe et al., J. Gen. Microbiol. 131:2225-2229, 1985. The mycelia were harvested from the culture media, lyophilized, frozen in liquid nitrogen, and ground into a powder. The powder was homogenized in extraction buffer containing 100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl, 2% SDS, and 1% β-mercaptoethanol. An equal volume of a 1:1 mixture of phenol/chloroform was added, and the resulting mixture vortexed for 60 seconds and then centrifuged. The aqueous phase was extracted with the phenol/chloroform again. A one-third volume of 8 M LiCl was then added to the extracted mixture. The mixture was centrifuged sufficiently to pellet the RNA, which was washed with 2 M LiCl, followed by 80% ethanol. The washed RNA was then resuspended in diethyl pyrocarbonate (DEPC)-treated water.

Polyadenylated RNA was isolated from total RNA using a standard oligo-(dT)-cellulose chromatography column. The construction of a cDNA expression library was carried out using a Stratagene kit. The library was screened for cellulase activity by overlaying plaques with 0.7% (w/v) agarose containing 0.2% (w/v) carboxymethyl cellulose (CMC). The plates were incubated at 39° C. overnight, then stained with a 0.1% (w/v) aqueous solution of Congo red and destained with 1 M NaCl as described in Teather et al., App. Environ. Microbiol. 43:777-780, 1982. Cellulase-producing plaques were surrounded by a clear halo visible against a red background. The positive clones were excised and purified using standard procedures. One of the clones, designated pPr2301-16, was selected for further study. The mRNA and gene from which the cDNA residing in pPr2301-16 was designated cbhA.

The complete sequence of the cDNA insert in plasmid pPr2301-16 was determined using a commercial service (Bio S&T, Lachine, QC, Canada). Translation of one reading frame revealed a 1128 bp open reading frame, as shown below.

This partial cDNA sequence (SEQ ID NO:1) of an cbhA from Piromyces rhizinflata encodes the partial CBHA amino acid sequence (SEQ ID NO:2) shown immediately above. No translation initiation codon was found at the 5′ end, suggesting that the cDNA is incomplete.

Using previously known cellulase genes as a model, the cDNA of the pPr2301-16 clone appeared to be missing a N-terminal catalytic domain but includes a complete C-terminal catalytic domain. Based on this assumption, amino acids 44-376 of the above polypeptide sequence was considered to be a catalytic domain of CBHA and was further characterized.

The nucleic acid sequence encoding the putative CBHA catalytic domain was amplified by PCR using primers 16F (GCA GGATCC GGTAATGGTAGTAGTCAAA; SEQ ID NO:5) and 16R (GTAG CTCGAG TAGAATGGTGGATTAGC; SEQ ID NO:6). To facilitate cloning, 16F contains a BamHI site, while 16R contains a XhoI site; both restriction sites are underlined in the primer sequences immediately above. The PCR product was then digested with the appropriate enzymes and ligated into BamHI and XhoI digested pGEX-4T-3 (Pharmacia Biotech, Inc.) to produce the Glutathione S-transferase (GST)-fusion expression plasmid PGEX-CBHA. The CBHA amino acid sequence downstream of the GST is shown below. The underlined sequence at the 5′ and 3′ ends indicates the binding site for primers 16F and 16R.

The complete nucleic acid sequence immediately above is designated SEQ ID NO:3, and the complete amino acid sequence encoded by that nucleic acid sequence is designated SEQ ID NO:4. SEQ ID NO:3 correspond to nucleotides 131-1131 of SEQ ID NO:1. SEQ ID NO:4 corresponds to amino acids 44-377 of SEQ ID NO:2.

The CBHA catalytic domain expression plasmid was used to transformed E. coli to produce recombinant CBHA. GST-CBHA was purified on glutathione Sepharose 4B (Pharmacia Biotech, Inc.) following the manufacturer's protocols. Bound fusion protein was cleaved with thrombin to release only the CBHA catalytic domain.

The enzymatic activity of the CBHA fragment was determined as follows. The purified protein was suspended in 50 mM sodium phosphate buffer containing 1% CMC, 1% oat spelt xylan, 0.4% barley β-glucan, 1% lechinan, 5 mM pNP-β-D-glucoside, Avicel, or 5 mM pNP-β-D-cellobioside. The barley βglucan contains mixed β-1,3′-1,4′ glucan. Enzymatic activity was measured by detecting the amount of reducing sugar released from the substrate. After incubating the reaction at 37° C. for 15 minutes, the reaction was stopped by adding a half-volume each of 0.3% (w/v) 3,6-dinitrophthalic acid and stop solution (25% K 2 CO 3 and 5% Na 2 S 2 O 3 ). The stopped reaction was then boiled for 10 minutes before the absorbance at 450 nm was read. Protein concentrations were measured using a protein assay kit (BioRad). The results are summarized in Table 1 below.

No activity was detected using pNP-β-D-glucoside, Avicel, or pNP-β-D-cellobioside as a substrate under these conditions.

Using the assay described immediately above, the temperature or pH was varied to obtain conditions necessary for optimal activity. The optimal temperature for the CBHA catalytic domain was about 40° C., and the optimal pH for the catalytic domain was about 6.5.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of this invention.

1

gaa act ctt cca caa caa tct aac tcc gca aaa acg ctt cca caa caa 48

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

1 5 10 15

tct gac tct gca aaa aca att cca caa ccc act tca gca gaa tca caa 96

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

20 25 30

act tca aag aca ctt cca caa aca gga ggc agt ggt aat ggt agt agt 144

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

35 40 45

caa aac ttt ttc tta aat gaa att tat gct aat cca aaa ttc att gaa 192

Gln Asn Phe Phe Leu Asn Glu Ile Tyr Ala Asn Pro Lys Phe Ile Glu

50 55 60

gaa gtt gaa gat tcc att gaa aaa tta act cct gaa tta caa gct aag 240

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

65 70 75 80

gcc gaa aag gtc aag gat gtt cca act gct gtt tgg tta gct tgg gat 288

Ala Glu Lys Val Lys Asp Val Pro Thr Ala Val Trp Leu Ala Trp Asp

85 90 95

ggt tct cca ggt gaa gtt gaa ggt cat ctt gtt gct gcc ggt tct aag 336

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

100 105 110

act gtt gta ttc ctt ctt tac atg att cca act cgt gat tgt aac agt 384

Thr Val Val Phe Leu Leu Tyr Met Ile Pro Thr Arg Asp Cys Asn Ser

115 120 125

aat gct tct gct ggt ggt gct gct agt ctt gat aaa tat aag ggt tat 432

Asn Ala Ser Ala Gly Gly Ala Ala Ser Leu Asp Lys Tyr Lys Gly Tyr

130 135 140

atc gat gac att tca aac act atc aag agt cat cca gaa tca aag gtt 480

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

145 150 155 160

gtt atg gtt gtt gaa cca gat act ctc ggt aat ctc gtt act ggt aat 528

Val Met Val Val Glu Pro Asp Thr Leu Gly Asn Leu Val Thr Gly Asn

165 170 175

agt gaa gca tgt aaa aat gtt cac act tta cac aag aat gcc tta tct 576

Ser Glu Ala Cys Lys Asn Val His Thr Leu His Lys Asn Ala Leu Ser

180 185 190

tac gct gtt gat gtc ttt ggt gct atg agc aat gtt agt gtt tat ctt 624

Tyr Ala Val Asp Val Phe Gly Ala Met Ser Asn Val Ser Val Tyr Leu

195 200 205

gat gca gct cat ggt atg tgg tta ggt cct cac act gat aag gtt gct 672

Asp Ala Ala His Gly Met Trp Leu Gly Pro His Thr Asp Lys Val Ala

210 215 220

tct gtc att aaa gaa att tta aat aat gct cca aat ggt aag att cgt 720

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

225 230 235 240

ggt tta agt acc aat gtg tca aac tac caa cca gtc agt tct gaa tac 768

Gly Leu Ser Thr Asn Val Ser Asn Tyr Gln Pro Val Ser Ser Glu Tyr

245 250 255

caa tac cat caa aaa ctc gct gct tct ctt gcc gcc gtt ggt gtt aat 816

Gln Tyr His Gln Lys Leu Ala Ala Ser Leu Ala Ala Val Gly Val Asn

260 265 270

gac gtt cat ttc att gtc gat act ggt cgt agt ggt gtt gat gtt act 864

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

275 280 285

gaa act ttc agt aaa caa caa act tgg tgt aac ttt att ggt gct ggt 912

Glu Thr Phe Ser Lys Gln Gln Thr Trp Cys Asn Phe Ile Gly Ala Gly

290 295 300

tta ggt cca cgt cca caa ggt aac cca gat gct agt atg cca tta tta 960

Leu Gly Pro Arg Pro Gln Gly Asn Pro Asp Ala Ser Met Pro Leu Leu

305 310 315 320

gat gcc tac atg tgg ctc aag act cca ggg gaa gct gat gga tct gct 1008

Asp Ala Tyr Met Trp Leu Lys Thr Pro Gly Glu Ala Asp Gly Ser Ala

325 330 335

gtt ggt gac aga gct gat cca gtt tgt tct cat gaa gat tct ctt caa 1056

Val Gly Asp Arg Ala Asp Pro Val Cys Ser His Glu Asp Ser Leu Gln

340 345 350

gtt gca cca gat gca ggt caa tgg ttc cac gat tac ttc gtc ctc tta 1104

Val Ala Pro Asp Ala Gly Gln Trp Phe His Asp Tyr Phe Val Leu Leu

355 360 365

tta aaa aat gct aat cca cca ttc taaataaatt aaaaaaaaaa aaattatttt 1158

Leu Lys Asn Ala Asn Pro Pro Phe

370 375

tacatataat ataaaaatat aattattttt attttttttt tcatttacta ttaattaata 1218

ataattaata ataatacaaa aaatatttaa attatatttt tattaatgta ataatttata 1278

tttatttcta ttctttgttg tattattatt taatcatcaa tgaatgatta tcttatataa 1338

ataaaaatta taaacatata aattat 1364

2

376

Piromyces rhizinflata

2

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

1 5 10 15

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

20 25 30

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

35 40 45

Gln Asn Phe Phe Leu Asn Glu Ile Tyr Ala Asn Pro Lys Phe Ile Glu

50 55 60

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

65 70 75 80

Ala Glu Lys Val Lys Asp Val Pro Thr Ala Val Trp Leu Ala Trp Asp

85 90 95

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

100 105 110

Thr Val Val Phe Leu Leu Tyr Met Ile Pro Thr Arg Asp Cys Asn Ser

115 120 125

Asn Ala Ser Ala Gly Gly Ala Ala Ser Leu Asp Lys Tyr Lys Gly Tyr

130 135 140

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

145 150 155 160

Val Met Val Val Glu Pro Asp Thr Leu Gly Asn Leu Val Thr Gly Asn

165 170 175

Ser Glu Ala Cys Lys Asn Val His Thr Leu His Lys Asn Ala Leu Ser

180 185 190

Tyr Ala Val Asp Val Phe Gly Ala Met Ser Asn Val Ser Val Tyr Leu

195 200 205

Asp Ala Ala His Gly Met Trp Leu Gly Pro His Thr Asp Lys Val Ala

210 215 220

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

225 230 235 240

Gly Leu Ser Thr Asn Val Ser Asn Tyr Gln Pro Val Ser Ser Glu Tyr

245 250 255

Gln Tyr His Gln Lys Leu Ala Ala Ser Leu Ala Ala Val Gly Val Asn

260 265 270

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

275 280 285

Glu Thr Phe Ser Lys Gln Gln Thr Trp Cys Asn Phe Ile Gly Ala Gly

290 295 300

Leu Gly Pro Arg Pro Gln Gly Asn Pro Asp Ala Ser Met Pro Leu Leu

305 310 315 320

Asp Ala Tyr Met Trp Leu Lys Thr Pro Gly Glu Ala Asp Gly Ser Ala

325 330 335

Val Gly Asp Arg Ala Asp Pro Val Cys Ser His Glu Asp Ser Leu Gln

340 345 350

Val Ala Pro Asp Ala Gly Gln Trp Phe His Asp Tyr Phe Val Leu Leu

355 360 365

Leu Lys Asn Ala Asn Pro Pro Phe

370 375

3

ggt aat ggt agt agt caa aac ttt ttc tta aat gaa att tat gct aat 48

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

1 5 10 15

cca aaa ttc att gaa gaa gtt gaa gat tcc att gaa aaa tta act cct 96

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

20 25 30

gaa tta caa gct aag gcc gaa aag gtc aag gat gtt cca act gct gtt 144

Glu Leu Gln Ala Lys Ala Glu Lys Val Lys Asp Val Pro Thr Ala Val

35 40 45

tgg tta gct tgg gat ggt tct cca ggt gaa gtt gaa ggt cat ctt gtt 192

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

50 55 60

gct gcc ggt tct aag act gtt gta ttc ctt ctt tac atg att cca act 240

Ala Ala Gly Ser Lys Thr Val Val Phe Leu Leu Tyr Met Ile Pro Thr

65 70 75 80

cgt gat tgt aac agt aat gct tct gct ggt ggt gct gct agt ctt gat 288

Arg Asp Cys Asn Ser Asn Ala Ser Ala Gly Gly Ala Ala Ser Leu Asp

85 90 95

aaa tat aag ggt tat atc gat gac att tca aac act atc aag agt cat 336

Lys Tyr Lys Gly Tyr Ile Asp Asp Ile Ser Asn Thr Ile Lys Ser His

100 105 110

cca gaa tca aag gtt gtt atg gtt gtt gaa cca gat act ctc ggt aat 384

Pro Glu Ser Lys Val Val Met Val Val Glu Pro Asp Thr Leu Gly Asn

115 120 125

ctc gtt act ggt aat agt gaa gca tgt aaa aat gtt cac act tta cac 432

Leu Val Thr Gly Asn Ser Glu Ala Cys Lys Asn Val His Thr Leu His

130 135 140

aag aat gcc tta tct tac gct gtt gat gtc ttt ggt gct atg agc aat 480

Lys Asn Ala Leu Ser Tyr Ala Val Asp Val Phe Gly Ala Met Ser Asn

145 150 155 160

gtt agt gtt tat ctt gat gca gct cat ggt atg tgg tta ggt cct cac 528

Val Ser Val Tyr Leu Asp Ala Ala His Gly Met Trp Leu Gly Pro His

165 170 175

act gat aag gtt gct tct gtc att aaa gaa att tta aat aat gct cca 576

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

180 185 190

aat ggt aag att cgt ggt tta agt acc aat gtg tca aac tac caa cca 624

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

195 200 205

gtc agt tct gaa tac caa tac cat caa aaa ctc gct gct tct ctt gcc 672

Val Ser Ser Glu Tyr Gln Tyr His Gln Lys Leu Ala Ala Ser Leu Ala

210 215 220

gcc gtt ggt gtt aat gac gtt cat ttc att gtc gat act ggt cgt agt 720

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

225 230 235 240

ggt gtt gat gtt act gaa act ttc agt aaa caa caa act tgg tgt aac 768

Gly Val Asp Val Thr Glu Thr Phe Ser Lys Gln Gln Thr Trp Cys Asn

245 250 255

ttt att ggt gct ggt tta ggt cca cgt cca caa ggt aac cca gat gct 816

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

260 265 270

agt atg att att aga tgc tac atg tgg ctc aag act cca ggg gaa gct 864

Ser Met Ile Ile Arg Cys Tyr Met Trp Leu Lys Thr Pro Gly Glu Ala

275 280 285

gat gga tct gct gtt ggt gac aga gct gat cca gtt tgt tct cat gaa 912

Asp Gly Ser Ala Val Gly Asp Arg Ala Asp Pro Val Cys Ser His Glu

290 295 300

gat tct ctt caa gtt gca cca gat gca ggt caa tgg ttc cac gat tac 960

Asp Ser Leu Gln Val Ala Pro Asp Ala Gly Gln Trp Phe His Asp Tyr

305 310 315 320

ttc gtc ctc tta tta aaa aat gct aat cca cca ttc taa 999

Phe Val Leu Leu Leu Lys Asn Ala Asn Pro Pro Phe

325 330

4

332

Piromyces rhizinflata

4

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

1 5 10 15

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

20 25 30

Glu Leu Gln Ala Lys Ala Glu Lys Val Lys Asp Val Pro Thr Ala Val

35 40 45

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

50 55 60

Ala Ala Gly Ser Lys Thr Val Val Phe Leu Leu Tyr Met Ile Pro Thr

65 70 75 80

Arg Asp Cys Asn Ser Asn Ala Ser Ala Gly Gly Ala Ala Ser Leu Asp

85 90 95

Lys Tyr Lys Gly Tyr Ile Asp Asp Ile Ser Asn Thr Ile Lys Ser His

100 105 110

Pro Glu Ser Lys Val Val Met Val Val Glu Pro Asp Thr Leu Gly Asn

115 120 125

Leu Val Thr Gly Asn Ser Glu Ala Cys Lys Asn Val His Thr Leu His

130 135 140

Lys Asn Ala Leu Ser Tyr Ala Val Asp Val Phe Gly Ala Met Ser Asn

145 150 155 160

Val Ser Val Tyr Leu Asp Ala Ala His Gly Met Trp Leu Gly Pro His

165 170 175

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

180 185 190

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

195 200 205

Val Ser Ser Glu Tyr Gln Tyr His Gln Lys Leu Ala Ala Ser Leu Ala

210 215 220

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

225 230 235 240

Gly Val Asp Val Thr Glu Thr Phe Ser Lys Gln Gln Thr Trp Cys Asn

245 250 255

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

260 265 270

Ser Met Ile Ile Arg Cys Tyr Met Trp Leu Lys Thr Pro Gly Glu Ala

275 280 285

Asp Gly Ser Ala Val Gly Asp Arg Ala Asp Pro Val Cys Ser His Glu

290 295 300

Asp Ser Leu Gln Val Ala Pro Asp Ala Gly Gln Trp Phe His Asp Tyr

305 310 315 320

Phe Val Leu Leu Leu Lys Asn Ala Asn Pro Pro Phe

5

gcaggatccg gtaatggtag tagtcaaa 28

6

gtagctcgag tagaatggtg gattagc 27