Method for cloning and expression of BsaWI restriction endonuclease and BsaWI methylase in E. coli

The present invention relates to recombinant DNA that encodes the BsaWI restriction endonuclease (BsaWI endonuclease or BsaWI) as well as BsaWI methyltransferase (BsaWI methylase or M.BsaWI), expression of BsaWI endonuclease and methylase in E. coli cells containing the recombinant DNA.

BsaWI endonuclease is found in the strain of Bacillus stearothermophilus W1718 (New England Biolabs' strain collection). It recognizes the double-stranded DNA sequence 5′W/CCGGW3′ (W=A or T, / indicates the cleavage position) and cleaves between the W and C to generate 4-base cohesive ends. BsaWI methylase (M.BsaWI) is also found in the same strain, which recognizes the same DNA sequence and presumably modifies the cytosine at the C5 position on hemi-methylated or non-methylated BsaWI sites.

Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.

Restriction endonucleases recognize and bind particular sequences of nucleotides (the ‘recognition sequence’) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and twenty-eight restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res.29:268-269 (2001)).

Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′PuG/GNCCPy3′ and 5′CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.

A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.

With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by ‘shotgun’ procedures, when they occur at frequencies as low as 10 −3 to 10 −4 . Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.

A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78: 1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.

Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80: 402-406, (1983); Theriault and Roy, Gene 19: 355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164: 501-509, (1985); Bsa45I: Wayne et al. Gene 202:83-88, (1997)).

A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10: 219-225, (1980); BcnI: Janulaitis et al., Gene 20: 197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21: 111-119, (1983); and BsaI: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)).

A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.

There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer DNA sites resistant to restriction digestion. For example, Dcm methylase modification of 5′ CCWGG 3′ (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucleotide and make the NotI site (5′GCGGCCGC3′) refractory to NotI digestion (New England Biolabs'Catalog, 2000-01, page 220; Beverly, Mass.). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.

Type II methylase genes have been found in many sequenced bacterial genomes (GenBank, http://www.ncbi.nlm.nih.gov; and Rebase, http://rebase.neb.com/rebase). Direct cloning and over-expression of ORFs adjacent to the methylase genes yielded restriction enzymes with novel specificities (Kong et al. Nucl. Acids Res. 28:3216-3223 (2000)). Thus microbial genome mining emerged as a new way of screening/cloning new type II restriction enzymes and methylases and their isoschizomers.

Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes and methylases. Such over-expression strains should also simplify the task of enzyme purification.

The present invention relates to a method for cloning BsaWI restriction gene (bsaWIR) from Bacillus stearothermophilus W1718 into E. coli by inverse PCR and direct PCR from genomic DNA using primers that were based on the DNA sequences obtained via methylase selection.

The difficulty was to express bsaWIR gene in E. coli using high-copy-number expression vector such as pRRS or medium-copy-number vector such as pET21at. When a PCR fragment containing the bsaWIR gene was inserted in pRRS or pET21at and transformed pre-modified E. coli strain ER2502 [pLG339-BsaWIM], clones with inserts were found, but low BsaWI endonuclease activity was detected in IPTG-induced cell extracts. This negative result indicated there was selection pressure to isolate endonuclease mutants.

Since expression from a high/medium-copy-number vectors did not generate a stable high expression clone, efforts were made to express the bsaWIR gene in a low-copy-number plasmid pACYC-T7at. The tightly regulated T7 expression vector pACYC-T7at contains four transcription terminators inserted before the T7 promoter to reduce basal level expression. The successful expression strategy was to express bsaWIM gene in a high-copy-number plasmid pRRS and bsaWIM gene in a low-copy-number T7 vector within the same E. coli host.

FIG. 1 . Gene organization of BsaWI R-M system. bsaWIR, BsaWI restriction endonuclease gene; bsaWIM, BsaWI methylase gene.

FIG. 2 . BsaWI methylase gene sequence (bsaWIM, 1236 bp) (SEQ ID NO:1) and the encoded amino acid sequence (SEQ ID NO:2).

FIG. 3 . BsaWI endonuclease gene sequence (bsaWIR, 819 bp) (SEQ ID NO:3) and the encoded amino acid sequence (SEQ ID NO:4).

FIG. 4 . Recombinant BsaWI endonuclease activity in cell extract. λ DNA was used as the substrate. Lanes 1-4, 1×, ½, ⅕, {fraction (1/10)}, diluted cell extract added in the restriction digestions. Lanes 5 and 6, λ DNA and λ DNA digested with native BsaWI.

FIG. 5 . Recombinant BsaWI protein analyzed on SDS-PAGE. Lanes 1 and 2, recombinant BsaWI clone #3; lanes 3 and 4, heated cell extract; lane 5, protein size marker. Arrow indicates IPTG-induced BsaWI endonuclease. The predicted molecular mass of BsaWI endonuclease is 31.9 kDa. The apparent molecular wight of BsaWI endonuclease on SDS-PAGE gel was 30 kDa.

It proved very difficult to express bsaWIR gene in E. coli using high-copy-number expression vector such as pRRS or medium-copy-number vector such as pET21at. When a PCR fragment containing the bsaWIR gene was inserted in pRRS and transformed pre-modified E. coli strain ER2502 [pLG339-BsaWIM], many clones with inserts were found, but very low BsaWI endonuclease activity was detected in IPTG-induced cell extracts. This negative result suggested there was selection pressure to isolate endonuclease mutants with reduced activity. This was probably due to under-methylation by M.BsaWI on the host chromosome or lower specific activity of M.BsaWI at 37° C. than the physiological temperature of 60° C. The native strain has an optimal temperature of 60° C.

Since expression from a high-copy-number vector did not generate a stable high expression clone, efforts were made to express the bsaWIR gene in a medium-copy-number plasmid pET21at. The tightly regulated T7 expression vector pET21at contains four transcription terminators inserted before the T7 promoter to reduce basal level expression.

The bsaWIR gene was amplified in PCR from genomic DNA. Following purification and ethanol precipitation, the PCR DNA was digested with XbaI and XhoI and ligated to pET21at with compatible ends. The ligated DNA was transferred into pre-modified host ER2566 [pLG339-BsaWIM]. After screening 34 plasmids from Ap R Km R co-transformants, 7 clones were found to carry the PCR insert. These 7 clones were cultured and induced with IPTG. Cells were harvested and lysed by sonication. Clarified cell lysates were assayed for BsaWI activity on λ DNA. Three extracts generated BsaWI digestion pattern and one extract gave rise to partial digestion. The rest of three did not contain any activity. The recombinant BsaWI activity was relatively low (˜5,000 units/g wet cells), probably resulting from mutations introduced in PCR. Recombinant BsaWI activity was detected only in undiluted cell extract. Partial activity was observed after 10 and 20-fold dilution of the cell extracts.

The successful expression strategy was to express bsaWIM gene in a high-copy-number plasmid pRRS and bsaWIR gene in a low-copy-number T7 vector within the same E. coli host. The method described herein by which the bsaWIM and bsaWIR genes are preferably cloned and expressed in E. coli include the following steps:

1. Preparation of Genomic DNA, Restriction Digestion, and Construction of Genomic DNA Library

Genomic DNA was prepared from Bacillus stearothermophilus W1718 by the standard method.

ApoI and NlaIII were used to partially digest genomic DNA. The partially digested DNA was purified and ligated to EcoRI and SphI digested and CIP treated pRRS vector that contains multiple BsaWI sites. The ligated DNA was used to transform ER2502 by electroporation. Approximately 10,000 Ap R transformants were obtained for both partial libraries. All the colonies were pooled and amplified. Plasmid DNA was prepared, generating a mixed library.

2. Cloning of bsaWIM Gene by Methylase Selection

The primary plasmid library DNA was challenged with BsaWI. The digested DNA was transferred into ER2502 by transformation, resulting in six Ap R survivors. Plasmid DNA was prepared and following BsaWI digestion one resistant and one partial resistant clone were found.

3. Restriction Mapping and Subcloning of the Insert

The resistant clone DNA was digested with a number of restriction enzymes to estimate the insert size. The insert size was estimated to be 3.7 to 4.2 kb. The BamHI, EcoRV, HindIII, and PvuII fragments were gel-purified and sub-cloned into pUC19. The entire inserts were sequenced. One open reading frame (ORF) of 1236 bp was found that has extensive homology to other C5 methylases. This ORF was named bsaWIM gene.

4. Inverse PCR Amplification of DNA Downstream of BsaWI Methylase

After identification of the methylase gene, efforts were made to clone adjacent DNA. One truncated ORF was found downstream of the bsaWIM gene. DNA sequence at the C-terminus of bsaWIM gene was used as the template for inverse PCR.

The genomic DNA was digested with restriction enzymes, purified, and self-ligated. The circular DNA molecules were used as templates for inverse PCR. PCR products were found in the BspHI, DpnII, NlaIII, and TfiI templates. The PCR products were purified from a low-melting agarose gel, treated with β-agarase, precipitated with ethanol, and sequenced directly with primers. The BspHI, DpnII, NlaIII, and TfiI PCR fragments generated about 1700 bp, 250 bp, 275 bp, and 1340 bp new sequences, respectively. Therefore, the entire BspHI fragment was sequenced with four additional primers. After sequencing about 1.1 kb, an ORF with 819 bp long was found downstream of the bsaWIM gene. This gene was most likely the restriction gene (bsaWIR) coding for BsaWI endonuclease. Transcription of M and R genes is oriented in the same direction (see FIG. 1 for gene organization).

5. Expression of bsaWIR Gene in E. coli

The successful cloning/expression strategy was to express bsaWIM gene in a high-copy-number plasmid pRRS and the bsaWIR gene in a low-copy-number T7 vector.

The plasmid pRRS-BsaWIM was the original methylase positive clone isolated from methylase selection. Plasmid pRRS-BsaWIM was transferred into ER2566 to premodify E.coli host . The bsaWIR gene was amplified from genomic DNA by PCR with Vent® DNA polymerase. Following purification and digestion with BamHI, it was ligated to CIP-treated pACYC-T7at with compatible ends. The ligated DNA was transferred into pre-modified host ER2566 [pRRS-BsaWIM] by transformation. Ten ml of cell cultures were made from individual transformants and target protein production induced with IPTG. Cell extracts were prepared and assayed for BsaWI endonuclease activity on λ DNA substrate. Six highly active BsaWI-producing clones were found after screening 20 IPTG-induced cell extracts. The BsaWI activity of one active clone was shown in FIG. 4 . The cell extract of active clone #3 was heated at 60° C. for 30 min and heat-denatured E. coli proteins were removed by centrifugation. The remaining proteins were analyzed by SDS-PAGE. One induced protein of approximately 30 kDa apparent size was detected, in close agreement with the predicted size of 31.9 kDa.

In order to find out the exact start codon of the native protein, the native BsaWI endonuclease was purified from the native B. Stearothermophilus W1718 strain. The N-terminus of purified native protein was sequenced. The actual amino acid sequence is consistent with the predicted amino acid sequence from the coding DNA sequence. In addition, expression of a deletion mutant with 18 amino acid deletion generated an inactive protein. This deletion mutant started from the second ATG codon in the coding sequence.

The plasmid DNA pACYC-T7ter-BsaWIR clone #3 was prepared by Qiagen column and the entire insert was sequenced. It was found that the insert contained the wild type coding sequence.

<160> NUMBER OF SEQ ID NOS: 17

<210> SEQ ID NO 1

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<213> ORGANISM: Bacillus stearothermophilus W1718

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Met Thr Arg Arg Leu His Ile Ile Asp Cys Phe Ser Gly Pro Gly Gly

1 5 10 15

atc tgt aca gga ttt cga gca gca gga ttt gaa act tta tta gct atc 96

Ile Cys Thr Gly Phe Arg Ala Ala Gly Phe Glu Thr Leu Leu Ala Ile

20 25 30

gag ttg gta gaa tct tgt gta gaa aca tat acg gct aac cac aaa gat 144

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

35 40 45

gtt cct gtt atc aat aaa gat atc cgt gat gta act gaa gaa gag gtt 192

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

50 55 60

aag cgt atc gta ggg aat cgt gta gtg gat ata ctt aca gct gga atg 240

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

65 70 75 80

cct tgc gag acg ttt agt aca gcg gga gca act tct cgt agt ttc tat 288

Pro Cys Glu Thr Phe Ser Thr Ala Gly Ala Thr Ser Arg Ser Phe Tyr

85 90 95

gat cat cgt cag ttt tta tat cgt gaa gct att cgt ctt gct gat gct 336

Asp His Arg Gln Phe Leu Tyr Arg Glu Ala Ile Arg Leu Ala Asp Ala

100 105 110

gta aat gca cgg atg att tta ttt gaa aac gtt cct ggt atc cag acg 384

Val Asn Ala Arg Met Ile Leu Phe Glu Asn Val Pro Gly Ile Gln Thr

115 120 125

aag aaa gtg gca aaa gat agt gat cgt ttg att att gat gaa att tat 432

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

130 135 140

gat gat tta gcg aaa tac gga tac gtt cac cat ata tct acg ata tta 480

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

145 150 155 160

aat gca gag gat ttt ggt gtt cct caa tca aga gag cgt ttt ttt att 528

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

165 170 175

tta gcc agt cgt gaa aaa ttg gat tta cgt gtt cca gtt gct aaa aac 576

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

180 185 190

aga aaa att gtg acc gta gag gaa gca ttt ata gat tta cct gaa gaa 624

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

195 200 205

ccg aaa gaa acg att aat tct gca aag aag aag cta gag aat gga gag 672

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

210 215 220

gaa att gag gta gaa caa tat aaa gac att gat tct gct tac agc aag 720

Glu Ile Glu Val Glu Gln Tyr Lys Asp Ile Asp Ser Ala Tyr Ser Lys

225 230 235 240

ctt atg aag gat agg gat ttc tgg aag ttt gag aag aaa gaa tta gca 768

Leu Met Lys Asp Arg Asp Phe Trp Lys Phe Glu Lys Lys Glu Leu Ala

245 250 255

ttg aca tat cat ttc gct cct gtt cat cga aaa ggg act atc aaa cgt 816

Leu Thr Tyr His Phe Ala Pro Val His Arg Lys Gly Thr Ile Lys Arg

260 265 270

ttt gaa atg att aaa cag ggg gaa gga ttg aaa gat cta ttc gat aag 864

Phe Glu Met Ile Lys Gln Gly Glu Gly Leu Lys Asp Leu Phe Asp Lys

275 280 285

ttg atg gct gaa cat gga cct gaa gaa atc gag cgg ctt cag aag gag 912

Leu Met Ala Glu His Gly Pro Glu Glu Ile Glu Arg Leu Gln Lys Glu

290 295 300

aag ata att cca aat aag tgg tat atc caa aga aat cga aga tta tct 960

Lys Ile Ile Pro Asn Lys Trp Tyr Ile Gln Arg Asn Arg Arg Leu Ser

305 310 315 320

cct gat aag ccg agc gta act gta aca tca cat tgt tta agc gag ttg 1008

Pro Asp Lys Pro Ser Val Thr Val Thr Ser His Cys Leu Ser Glu Leu

325 330 335

gtt cat ccg atc aaa aat cga gca ttg acg att cgt gag gtt gca cgt 1056

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

340 345 350

ttg caa agt ttt cct gat ttc tat gat ttc aaa ggc gga aaa ttt gta 1104

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

355 360 365

gca cct cat aaa gac cca caa cag gat aaa tat gaa cag att ggg gat 1152

Ala Pro His Lys Asp Pro Gln Gln Asp Lys Tyr Glu Gln Ile Gly Asp

370 375 380

gcc gtg cct cct ttg ttg gct tat cat tgg ggg ctg gtt ata aaa gaa 1200

Ala Val Pro Pro Leu Leu Ala Tyr His Trp Gly Leu Val Ile Lys Glu

385 390 395 400

att ctt gaa gga gtt aaa gtt acg gag ggg gtc tag 1236

Ile Leu Glu Gly Val Lys Val Thr Glu Gly Val

405 410

<210> SEQ ID NO 2

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<213> ORGANISM: Bacillus stearothermophilus W1718

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Met Thr Arg Arg Leu His Ile Ile Asp Cys Phe Ser Gly Pro Gly Gly

1 5 10 15

Ile Cys Thr Gly Phe Arg Ala Ala Gly Phe Glu Thr Leu Leu Ala Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Cys Glu Thr Phe Ser Thr Ala Gly Ala Thr Ser Arg Ser Phe Tyr

85 90 95

Asp His Arg Gln Phe Leu Tyr Arg Glu Ala Ile Arg Leu Ala Asp Ala

100 105 110

Val Asn Ala Arg Met Ile Leu Phe Glu Asn Val Pro Gly Ile Gln Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Glu Ile Glu Val Glu Gln Tyr Lys Asp Ile Asp Ser Ala Tyr Ser Lys

225 230 235 240

Leu Met Lys Asp Arg Asp Phe Trp Lys Phe Glu Lys Lys Glu Leu Ala

245 250 255

Leu Thr Tyr His Phe Ala Pro Val His Arg Lys Gly Thr Ile Lys Arg

260 265 270

Phe Glu Met Ile Lys Gln Gly Glu Gly Leu Lys Asp Leu Phe Asp Lys

275 280 285

Leu Met Ala Glu His Gly Pro Glu Glu Ile Glu Arg Leu Gln Lys Glu

290 295 300

Lys Ile Ile Pro Asn Lys Trp Tyr Ile Gln Arg Asn Arg Arg Leu Ser

305 310 315 320

Pro Asp Lys Pro Ser Val Thr Val Thr Ser His Cys Leu Ser Glu Leu

325 330 335

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

340 345 350

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

355 360 365

Ala Pro His Lys Asp Pro Gln Gln Asp Lys Tyr Glu Gln Ile Gly Asp

370 375 380

Ala Val Pro Pro Leu Leu Ala Tyr His Trp Gly Leu Val Ile Lys Glu

385 390 395 400

Ile Leu Glu Gly Val Lys Val Thr Glu Gly Val

405 410

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Met Asn Phe Phe Glu Tyr Cys Ile Ser Thr Tyr Ala Lys Ile Phe Glu

1 5 10 15

gaa acg atg aat gca gtg ggt gat gaa cga gtt tcg caa aaa aag gca 96

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

20 25 30

atc cgt gac aca atg atc tca gct atg aga gag ttt cca aat gtc gaa 144

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

35 40 45

gcg gct gaa atc tgg aaa gct gtg tat tca gcc cat atg gat cgt aaa 192

Ala Ala Glu Ile Trp Lys Ala Val Tyr Ser Ala His Met Asp Arg Lys

50 55 60

tca ggt ata gca gac cca gat att att caa aag gtc ata tca gcg gag 240

Ser Gly Ile Ala Asp Pro Asp Ile Ile Gln Lys Val Ile Ser Ala Glu

65 70 75 80

aat agc tgg aaa aaa tcc agt gga cat gcc ttt gag gaa atg att aaa 288

Asn Ser Trp Lys Lys Ser Ser Gly His Ala Phe Glu Glu Met Ile Lys

85 90 95

cta cta ggg aat agc agt tta gaa gaa tat gga atg cgt att ttg ctc 336

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

100 105 110

caa aaa gac tta aac atg atg att gaa aac caa gag att gca aat gaa 384

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

115 120 125

cca cgc gac ata aat tgg tta aaa gag caa att tct tct aac gta ttt 432

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

130 135 140

gat ttg tac att aca gtg cgg aat aac gac aaa gag tat gtg ttt ggc 480

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

145 150 155 160

tgc att caa tcg aaa aca agc ata cgc gat aga gtc acc cgt gac cgt 528

Cys Ile Gln Ser Lys Thr Ser Ile Arg Asp Arg Val Thr Arg Asp Arg

165 170 175

gag cct tcg atg aag gcg atg gaa gct ttc ttt tgg tca gtt gct atc 576

Glu Pro Ser Met Lys Ala Met Glu Ala Phe Phe Trp Ser Val Ala Ile

180 185 190

tgt tta gat gga gac ttt tta aaa atg cca aaa ttt atc gca atg gta 624

Cys Leu Asp Gly Asp Phe Leu Lys Met Pro Lys Phe Ile Ala Met Val

195 200 205

aat gga gga aca agt aat tat aga ttg aat ggc tgg cat ggt atg tat 672

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

210 215 220

gta ttt tgg gac aag cca acg att gat cgt att tat ccg att gat atc 720

Val Phe Trp Asp Lys Pro Thr Ile Asp Arg Ile Tyr Pro Ile Asp Ile

225 230 235 240

aac ctt gaa ttg ttt gta caa cat gca cgt gag gca gca gag gat tgg 768

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

245 250 255

ctg cat aga cgg caa tgg ttt aat tat gag tgg aaa gca gga caa aaa 816

Leu His Arg Arg Gln Trp Phe Asn Tyr Glu Trp Lys Ala Gly Gln Lys

260 265 270

taa 819

<210> SEQ ID NO 4

<211> LENGTH: 272

<212> TYPE: PRT

<213> ORGANISM: Bacillus stearothermophilus W1718

<400> SEQUENCE: 4

Met Asn Phe Phe Glu Tyr Cys Ile Ser Thr Tyr Ala Lys Ile Phe Glu

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Glu Ile Trp Lys Ala Val Tyr Ser Ala His Met Asp Arg Lys

50 55 60

Ser Gly Ile Ala Asp Pro Asp Ile Ile Gln Lys Val Ile Ser Ala Glu

65 70 75 80

Asn Ser Trp Lys Lys Ser Ser Gly His Ala Phe Glu Glu Met Ile Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Cys Ile Gln Ser Lys Thr Ser Ile Arg Asp Arg Val Thr Arg Asp Arg

165 170 175

Glu Pro Ser Met Lys Ala Met Glu Ala Phe Phe Trp Ser Val Ala Ile

180 185 190

Cys Leu Asp Gly Asp Phe Leu Lys Met Pro Lys Phe Ile Ala Met Val

195 200 205

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

210 215 220

Val Phe Trp Asp Lys Pro Thr Ile Asp Arg Ile Tyr Pro Ile Asp Ile

225 230 235 240

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

245 250 255

Leu His Arg Arg Gln Trp Phe Asn Tyr Glu Trp Lys Ala Gly Gln Lys

260 265 270

<210> SEQ ID NO 5

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: primer

<400> SEQUENCE: 5

cggcatcccc aatctgttca tatt 24

<210> SEQ ID NO 6

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Primer

<400> SEQUENCE: 6

tgcctccttt gttggcttat catt 24

<210> SEQ ID NO 7

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Sequencing Primer

<400> SEQUENCE: 7

ctgtacagat tcctccaggc ccag 24

<210> SEQ ID NO 8

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Sequencing primer

<400> SEQUENCE: 8

taatgtgtaa tcgtctagtc atga 24

<210> SEQ ID NO 9

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Sequencing primer

<400> SEQUENCE: 9

tgtacattac agtgcggaat aacg 24

<210> SEQ ID NO 10

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Sequencing Primer

<400> SEQUENCE: 10

tatgtgtttg gctgcattca atcg 24

<210> SEQ ID NO 11

<211> LENGTH: 36

<212> TYPE: DNA

<213> ORGANISM: PCR Primer

<400> SEQUENCE: 11

gtgggatcca tgactagacg attacacatt atagat 36

<210> SEQ ID NO 12

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: PCR primer

<400> SEQUENCE: 12

tgatacgcat gcctagaccc cctccgtaac tttaactcc 39

<210> SEQ ID NO 13

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: PCR primer synthesized for PCR amplification

<400> SEQUENCE: 13

gaaggagcat gcggaggtaa ataaatgaat ttttttgagt attgtatcag c 51

<210> SEQ ID NO 14

<211> LENGTH: 36

<212> TYPE: DNA

<213> ORGANISM: PCR primer synthesized for PCR Amplification

<400> SEQUENCE: 14

gccggatcca gacataggtt gaacatctaa ttatta 36

<210> SEQ ID NO 15

<211> LENGTH: 54

<212> TYPE: DNA

<213> ORGANISM: PCR primer

<400> SEQUENCE: 15

gaaggatcta gaggatccgg aggtaaataa atgaattttt ttgagtattg tatc 54

<210> SEQ ID NO 16

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: PCR primer

<400> SEQUENCE: 16

caatttctcg agagacatag gttgaacatc taattatta 39

<210> SEQ ID NO 17

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: forward PCR primer which contains ribosome binding

site, 6 bp prior to start codon

<400> SEQUENCE: 17

ccgggatccg gaggtaaata aatgaatttt tttgagtatt gtatcagcac c 51