PATENT ABSTRACT
A hyperthermostable protease having the amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing or a sequence derived therefrom by deletion, substitution, insertion or addition of one to several amino acid residues, a gene encoding the hyperthermostable protease, and a process for preparing the protease, aiming at providing by genetic engineering techniques a hyperthermophile protease which is advantageous for industrial use.

PATENT DESCRIPTION
TECHNICAL FIELD  
         [0001]    The present invention relates to a hyperthermostable protease useful as an enzyme for industrial use, a gene encoding the same and a method of producing the enzyme by genetic engineering technique.  
         BACKGROUND ART  
         [0002]    A protease is an enzyme that cleaves peptide bonds in proteins. A number of such enzymes have been found in animals, plants and microorganisms. The protease is used as a reagent for laboratory use and as a pharmaceutical, as well as in industrial fields, for example, as an additive for a detergent, for processing foods and for chemical synthesis utilizing a reverse reaction. Therefore, it can be said that the protease is an extremely important enzyme for industries. Since high physical and chemical stability is required for a protease used in industrial fields, a thermostable enzyme is preferably used among others. Since proteases produced by bacteria of genus Bacillus exhibit relatively high thermostability, they are mainly used as proteases for industrial use. However, in search of a more superior enzyme, attempts have been made to obtain an enzyme from a microorganism growing at high temperature, for example, a thermophilic bacterium of genus Bacillus or a hyperthermophile.  
           [0003]    For example, a hyperthermophile  Pyrococcus furiosus  is known to produce a protease (Appl. Environ. Microbiol., 56:1992-1998 (1990); FEMS Microbiol. Letters, 71:17-20 (1990); J. Gen. Microbiol., 137:1193-1199 (1991)).  
           [0004]    In addition, a hyperthermophile, Pyrococcus sp. strain KOD1, is reported to produce a thiol protease (a cysteine protease) (Appl. Environ. Microbiol., 60:4559-4566 (1994)) Hyperthermophiles of genus Thermococcus, genus Staphylothermus and genus Thermobacteroides are also known to produce proteases (Appl. Microbiol. Biotechnol., 34:715-719 (1991)).  
           [0005]    The proteases from the hyperthermophiles as described above have high thermostability. Therefore, it is expected that they may be used in place of the thermostable proteases currently in use or in a field in which use of a protease has not been considered.  
           [0006]    However, most of the microorganisms producing these enzymes grow only at high temperature. For example,  Pyrococcus furiosus  needs to be cultured at 90-100° C. Culturing at such high temperature is disadvantageous in view of energy cost. Furthermore, the productivities of the proteases from the hyperthermophiles are lower than the productivities of the conventional microbial proteases. Thus, the methods for industrially producing the proteases from the hyperthermophiles have problems.  
           [0007]    By the way, production of an enzyme by genetic engineering technique by isolating the gene for the enzyme of interest and introducing it into a host microorganism that can readily be cultured is currently common in the art. However, the gene for the enzyme introduced into the host is not always expressed so efficiently as expected. It is believed that the main cause is that the GC content or the codon usage of the introduced gene is different from those of the genes of the host. Therefore, it is necessary to optimize the expression method for each gene to be introduced and/or each host in order to accomplish a suitable productivity of an enzyme for the intended use.  
         OBJECTS OF THE INVENTION  
         [0008]    The objects of the present invention are to provide a protease from a hyperthermophile which is advantageous for industrial use, to isolate a gene encoding the protease from the hyperthermophile, and to provide a method of producing the hyperthermostable protease using the gene by genetic engineering technique in order to solve the problems as described above.  
         SUMMARY OF THE INVENTION  
         [0009]    Among proteases produced by hyperthermophiles, some may be classified into the subtilisin-type of alkaline proteases based on the amino acid sequence homology. When a gene for such a protease is introduced into  Bacillus subtilis  which is generally used for production by genetic engineering technique, the productivity of this enzyme is much less than that of a protein inherently produced by  Bacillus subtilis.    
           [0010]    The present inventors have studied intensively and found that, by placing a gene encoding a signal peptide (signal sequence) derived from a subtilisin upstream a protease gene derived from a hyperthermophile to be expressed, and modifying the amino acid sequence around the cleavage site, the gene of interest is expressed in  Bacillus subtilis  with high efficiency. Furthermore, it has been found that the expression level of the enzyme can be increased by deleting a portion that is not essential for the enzymatic activity in the protease gene derived from the hyperthermophile of interest. Thus, the present invention has been completed.  
           [0011]    The present invention is outlined as follows. The first invention of the present invention is a thermostable protease having an amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing, and a protease having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing and having a thermostable protease activity.  
           [0012]    The second invention of the present invention is a gene encoding the thermostable protease of the first invention, and a thermostable protease gene that hybridizes with the gene.  
           [0013]    The third invention of the present invention is a gene to be used for producing a thermostable protease derived from a hyperthermophile by genetic engineering technique, characterized in that the gene encodes an amino acid sequence represented by formula I: 
           SIG-Ala-Gly-Gly-Asn-PRO  [I] 
           [0014]    wherein SIG represents an amino acid sequence of a signal peptide derived from a subtilisin, PRO represents an amino acid sequence of a protein to be expressed. Preferably, SIG is the amino acid sequence represented by the SEQ ID NO:3 of the Sequence Listing. Preferably, PRO is an amino acid sequence of a hyperthermostable protease derived from a hyperthermophile, more preferably, an amino acid sequence of a protease derived from  Pyrococcus furiosus.    
           [0015]    The fourth invention of the present invention relates to a method of producing a protein by genetic engineering technique, characterized in that the method comprises culturing a bacterium of genus Bacillus into which the gene of the third invention is introduced, and collecting the protein of interest from the culture.  
           [0016]    The fifth invention of the present invention is a plasmid used for producing a protein by genetic engineering technique, characterized in that the gene of the third invention is inserted into the plasmid.  
           [0017]    A mutation such as deletion, substitution, insertion or addition of one to several amino acid residues in an amino acid sequence may be generated in a naturally occurring protein including the protein disclosed by the present invention. Such mutation may be generated due to a polymorphism or a mutation of the gene encoding the protein, or due to a modification of the protein in vivo or during purification after synthesis may occur. Nevertheless, it is known that such a mutated protein may exhibit physiological and biological activities equivalent with those of a protein without a mutation. This is applicable to a protein in which such a mutation is introduced into its amino sequence artificially, in which case it is possible to generate a wide variety of mutations. For example, it is known that a polypeptide in which a cysteine residue in the amino acid sequence of human interleukin-2 (IL-2) is substituted with a serine residue retains an interleukin-2 activity (Science, 224:1431 (1984)). Thus, a protease having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence disclosed by the present invention and having a protease activity equivalent with that of the protease of the present invention is within the scope of the present invention.  
           [0018]    As used herein, “a gene which hybridizes (with a particular gene)” is a gene having a base sequence similar to that of the particular gene. It is likely that a gene having a base sequence similar to that of a particular gene encodes a protein having an amino acid sequence and a function similar to those of the protein encoded by the particular gene. Similarity of base sequences of genes can be examined by determining whether or not the genes or portions thereof form a hybrid (hybridize) each other under stringent conditions. By utilizing this procedure, a gene that encodes a protein having a similar function with that of the protein encoded by the particular gene can be obtained. That is, a gene having a similar base sequence with that of the gene of the present invention can be obtained by using the gene obtained by the present invention or a portion thereof as a probe to carry out hybridization according to a known method. Hybridization can be carried out according to the method, for example, as described in T. Maniatis et al. eds., Molecular Cloning: A Laboratory Manual 2nd ed., published by Cold Spring Harbor Laboratory, 1989. More specifically, hybridization can be carried out under the following conditions. Briefly, a membrane onto which DNAs are immobilized is incubated in 6×SSC (1×SSC represents 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrorridone, 0.1% Ficoll 400, 0.01% denatured salmon sperm DNA at 50° C. for 12-20 hours with a probe. After incubation, the membrane is washed until the signals for the immobilized DNAs can be distinguished from background, starting from washing in 2×SSC containing 0.5% SDS at 37° C. while decreasing the SSC concentration down to 0.1× and raising the temperature up to 50° C.  
           [0019]    Alternatively, instead of hybridization, a gene amplification method (e.g., PCR method) which employs portions of the base sequence of the gene obtained by the present invention as primers can be utilized. Whether or not the gene thus obtained encodes a protein having the function of interest can be determined by expressing the gene utilizing a suitable host and a suitable expression system and examining the activity of the resulting protein. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]    [0020]FIG. 1 is the restriction enzyme map of the plasmid pSTC3.  
         [0021]    [0021]FIG. 2 compares the amino acid sequences of Protease PFUS, Protease TCES and a subtilisin.  
         [0022]    [0022]FIG. 3 compares the amino acid sequences of Protease PFUS, Protease TCES and a subtilisin.  
         [0023]    [0023]FIG. 4 compares the amino acid sequences of Protease PFUS, Protease TCES and a subtilisin.  
         [0024]    [0024]FIG. 5 compares the amino acid sequences of Protease PFUS, Protease TCES and a subtilisin.  
         [0025]    [0025]FIG. 6 is the restriction enzyme map of the plasmid pSNP1.  
         [0026]    [0026]FIG. 7 is the restriction enzyme map of the plasmid pPS1.  
         [0027]    [0027]FIG. 8 is the restriction enzyme map of the plasmid pNAPS1. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0028]    The hyperthermostable protease according to the present invention includes proteases from various hyperthermophiles. For example, WO 95/34645 describes proteases from  Pyrococcus furiosus  and  Thermococcus celer.    
         [0029]    A protease gene from  Pyrococcus furiosus  DSM3638 was isolated from a genomic DNA library of the strain based on the expression of a thermostable protease activity. A plasmid containing this gene is designated as the plasmid pTPR12.  Escherichia coli  JM109 transformed with this plasmid is designated and indicated as  Escherichia coli  JM109/pTPR12, and deposited on May 24, 1994 (the date of the original deposit) under Budapest Treaty at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan under accession number FERM BP-5103.  
         [0030]    This protease is designated as Protease PFUL hereinafter. Protease PFUL is a protease having high thermostability and exhibits a protease activity even at 95° C.  
         [0031]    The base sequence of the DNA fragment derived from  Pyrococcus furiosus  inserted into the plasmid pTPR12 has been determined. The base sequence of the portion of approximately 4.8 kb bordered by two DraI sites in the DNA fragment inserted into the plasmid pTPR12 is shown in the SEQ ID NO:5 of the Sequence Listing. Furthermore, the amino acid sequence of the gene product deduced from the base sequence is shown in the SEQ ID NO:6 of the Sequence Listing. In other words, the amino acid sequence as shown in the SEQ ID NO:6 of the Sequence Listing is the amino acid sequence of Protease PFUL. As shown in the sequence, Protease PFUL consists of 1398 amino acid residues and is a protease with a high molecular weight of over 150,000.  
         [0032]    Comparison of the amino acid sequence of Protease PFUL as shown in SEQ ID NO:6 of the Sequence Listing with known amino acid sequences of proteases from microorganisms has revealed that the amino acid sequence of the first half portion of Protease PFUL is homologous to those of a series of alkaline serine proteases represented by a subtilisin (Protein Engineering, 4:719-737 (1991)), and that there is extremely high homology around the four amino acid residues which are believed to be important for the catalytic activity of the protease.  
         [0033]    As described above, it has been found that a region common among proteases derived from mesophiles is conserved in the amino acid sequence of Protease PFUL produced by a hyperthermophile  Pyrococcus furiosus.  Thus, it is expected that a homologous protease produced by a hyperthermophile other than  Pyrococcus furiosus  also has this region.  
         [0034]    For example, a gene for a hyperthermostable protease can be screened by performing PCR using a chromosomal DNA from various hyperthermophiles as a template and the oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4R in combination as primers. These oligonucleotides are synthesized based on the base sequence in the Protease PFUL gene which encodes a region exhibiting high homology with subtilisins or the like within the amino acid sequence of Protease PFUL. The base sequences of oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4R are shown in the SEQ ID NOS:7, 8, 9 and 10 of the Sequence Listing, respectively.  
         [0035]    As a hyperthermophile from which the protease according to the present invention is derived, a bacterium belonging to genus Pyrococcus, genus Thermococcus, genus Staphylothermus, genus Thermobacteroides and the like can be used. As a bacterium belonging to genus Thermococcus, for example,  Thermococcus celer  DSM2476 can be used. This strain is available from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. When performing PCR using a chromosomal DNA from  Thermococcus celer  DSM2476 as a template and a combination of the oligonucleotides PRO-1F and PRO-2R or the oligonucleotide PRO-2F and Pro-4R as primers, specific DNA fragments are amplified, indicating the presence of a protease gene. Furthermore, by creating recombinant plasmids in which the DNA fragments are inserted into an appropriate plasmid vector and determining the base sequences of the inserted DNA fragments by dideoxy method, the amino acid sequences encoded by the fragments can be deduced. As a result, it proved that such DNA fragments encode an amino acid sequence that is homologous to the amino acid sequences of Protease PFUL and alkaline serine proteases from various microorganisms and that the PCR-amplified DNA fragments were amplified from a protease gene as a template.  
         [0036]    Next, a gene for a hyperthermostable protease (for example, a gene for a hyperthermostable protease produced by  Thermococcus celer ) can be obtained by screening a gene library from a hyperthermophile using the PCR-amplified DNA fragment or the oligonucleotide as described above as a probe.  
         [0037]    For example, a phage clone containing the gene of interest can be obtained by performing plaque hybridization against a library using the PCR-amplified DNA fragment as a probe. Such library is generated by ligating lambda GEM-11 vector (Promega) and DNA fragments resulting from partial digestion of the chromosomal DNA from  Thermococcus celer  DSM2476 with a restriction enzyme Sau3AI, then packaging them into lambda phage particles by in vitro packaging method.  
         [0038]    It is found that a protease gene exists in a SacI fragment of approximately 1.9 kb by analyzing a DNA fragment contained in a phage clone thus obtained. Furthermore, it is found that this fragment lacks the 5′ region of the protease gene by determining its base sequence. The 5′ region can be obtained by PCR using a cassette and cassette primers (Takara Shuzo Gene Technology Product Guide, 1994-1995, pp.250-251). Thus, a DNA fragment which covers the 5′ region of the hyperthermostable protease gene which is absent in the plasmid pTCS6 can be obtained. Furthermore, the base sequence of the entire hyperthermostable protease gene derived from  Thermococcus celer  can be determined from the base sequences of the two DNA fragments.  
         [0039]    The base sequence of an open reading frame found in the determined base sequence is shown in the SEQ ID NO:11 of the Sequence Listing, and the amino acid sequence deduced from the base sequence is shown in the SEO ID NO:12 of the Sequence Listing. The base sequence of the gene encoding the hyperthermostable protease from  Thermococcus celer  and the amino acid sequence of the protease were thus determined. This protease is designated as Protease TCES.  
         [0040]    An expression vector in which the entire Protease TCES gene is reconstituted by combining the two DNA fragments can be constructed. However, when using  Escherichia coli  as a host, a transformant into which the expression plasmid of interest had been introduced was not obtained, probably because the generation of the product expressed from the gene in cells may be harmful or lethal to  Escherichia coli.  In such a case, for example, it is possible to use  Bacillus subtilis  as a host for extracellular secretion of the protease and to determine the activity.  
         [0041]    As a  Bacillus subtilis  strain,  Bacillus subtilis  DB104 can be used, which is a known strain as described in Gene, 83:215-233 (1989). As a cloning vector, the plasmid pUB18-P43 can be used, which is a generous gift from Dr. Sui-Lam Wong, University of Calgary. The plasmid contains a kanamycin-resistance gene as a selectable marker.  
         [0042]    A recombinant plasmid in which the Protease TCES gene is inserted downstream the P43 promoter in the plasmid vector pUB18-P43 is designated as the plasmid pSTC3.  Bacillus subtilis  DB104 transformed with this plasmid is designated and indicated as  Bacillus subtilis  DB104/pSTC3, and was deposited on Dec. 1, 1995 (the date of the original deposit) under Budapest Treaty at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan under accession number FERM BP-5635.  
         [0043]    The restriction enzyme map of the plasmid pSTC3 is shown in FIG. 1. In FIG. 1, the bold line indicates the DNA fragment inserted into the plasmid vector pUB18-P43.  
         [0044]    A thermostable protease activity is found in either of the culture supernatant and the cell extract of the culture of  Bacillus subtilis  DB104/pSTC3.  
         [0045]    Main properties of a crude enzyme preparation of the protease obtained from the culture of the transformant are as follows.  
         [0046]    (1) Action:  
         [0047]    Degrades casein and gelatin to generate short chain polypeptides.  
         [0048]    Hydrolyzes succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4-methylcoumarin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) to generate a fluorescent substance (7-amino-4-methylcoumarin).  
         [0049]    Hydrolyzes succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to generate a yellow substance (p-nitroaniline).  
         [0050]    (2) Optimal temperature:  
         [0051]    Exhibits an enzymatic activity at 37-95° C., with the optimal temperature being 70-80° C.  
         [0052]    (3) Optimal pH:  
         [0053]    Exhibits an enzymatic activity at pH 5.5-9, with the optimal pH being pH 7-8.  
         [0054]    (4) Thermostability:  
         [0055]    Retains 90% or more of its enzymatic activity after treatment at 80° C. for 3 hours.  
         [0056]    When aligning the amino acid sequences of Protease PFUL, Protease TCES and a subtilisin (subtilisin BNP′; Nucl. Acids Res., 11:7911-7925 (1983)) such that homologous regions match each other as shown in FIGS.  2 - 5 , it is found that, at the C-terminus and between the homologous regions of Protease PFUL, there are sequences which are not found in Protease TCES or the subtilisin. From these results, a protease having a molecular weight lower than that of Protease PFUL and similar to Protease TCES or subtilisins may exist in  Pyrococcus furiosus  in addition to Protease PFUL.  
         [0057]    Thereupon, Southern hybridization against a chromosomal DNA prepared from  Pyrococcus furiosus  was carried out using a DNA probe from the homologous region; and a signal other than that for the Protease PFUL gene was observed, indicating the existence of another protease gene.  
         [0058]    This novel protease gene can be isolated by the following procedure.  
         [0059]    For example, a DNA fragment containing a gene encoding the novel protease is obtained by digesting a chromosomal DNA from  Pyrococcus furiosus  with an appropriate restriction enzyme and performing Southern hybridization against the digested DNA as described above. The base sequence of the DNA fragment is determined to confirm that the base sequence encodes an amino acid sequence homologous to the above-mentioned protease. If the DNA fragment does not contain the entire gene of interest, the remaining portion is further obtained by inverse PCR method or the like.  
         [0060]    For example, when a chromosomal DNA from  Pyrococcus furiosus  is digested with restriction enzymes SacI and SpeI (Takara Shuzo) and is used for Southern hybridization, a signal of approximately 0.6 kb in size is observed. DNA fragments of this size are recovered, inserted between the SpeI-SacI sites in the plasmid vector pBluescript SK(−) (Stratagene), and  Escherichia coli  JM 109 is transformed with the resulting recombinant plasmids. A clone into which the fragment of interest is incorporated can be obtained from the transformants by colony hybridization using the same probe as that used for the Southern hybridization as described above. Whether or not the plasmid harbored by the obtained clone has the sequence that encodes the protease can be confirmed by determining the base sequence of the DNA fragment inserted into the plasmid. The presence of the protease gene in the plasmid was thus confirmed. This plasmid is designated as the plasmid pSS3.  
         [0061]    It is found that the amino acid sequence deduced from the base sequence of the DNA fragment inserted into the plasmid pSS3 has homology with sequences of subtilisins, Protease PFUL, Protease TCES and the like. The product of the protease gene distinct from the Protease PFUL gene, a portion of which was newly obtained from  Pyrococcus furiosus  as described above, is designated as Protease PFUS. The regions which encode the N-terminal and C-terminal regions of the protease can be obtained by inverse PCR method.  
         [0062]    Primers used for inverse PCR can be prepared based on the base sequence of the DNA fragment inserted into the plasmid pSS3. A chromosomal DNA from  Pyrococcus furiosus  is digested with an appropriate restriction enzyme, and the resulting DNA fragments are then subjected to an intramolecular ligation reaction. By performing PCR using the reaction mixture as a template and the above-mentioned primers, DNA fragments corresponding to the regions flanking the fragment for the protease gene contained in the plasmid pSS3 can be obtained. The amino acid sequence of the enzyme protein encoded by these regions can be deduced by analyzing the base sequences of the DNA fragments thus obtained. Furthermore, primers capable of amplifying the entire Protease PFUS gene using a chromosomal DNA from  Pyrococcus furiosus  as a template can be prepared. The primers NPF-4 and NPR-4 can be designed. The primer NPF-4 has the base sequence immediately upstream the initiation codon of the Protease PFUS gene and can introduce a BamHI site 5′ to the sequence. The primer NPR-4 has a sequence complementary to the 3′ portion of the Protease PFUS gene and can introduce a SphI site 5′ to the sequence.  
         [0063]    The base sequences of the primers NPF-4 and NPR-4 are shown in the SEQ ID NOS:13 and 14 of the Sequence Listing. These two primers can be used to amplify the entire Protease PFUS gene using a chromosomal DNA from  Pyrococcus furiosus  as a template.  
         [0064]    Like Protease TCES, Protease PFUS can be expressed in  Bacillus subtilis  as a host. A plasmid for expressing Protease PFUS can be constructed based on the expression plasmid for Protease TCES, pSTC3. Specifically, a plasmid for expressing Protease PFUS can be constructed by replacing the Protease TCES gene in the plasmid pSTC3 with the DNA fragment containing the entire Protease PFUS gene amplified by PCR with the primers as described above. The expression plasmid thus constructed is designated as the plasmid pSNP1.  Bacillus subtilis  DB104 transformed with this plasmid is designated and indicated as  Bacillus subtilis  DB104/pSNP1, and was deposited on Dec. 1, 1995 (the date of the original deposit) under Budapest Treaty at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan under accession number FERM BP-5634. The restriction enzyme map of the plasmid pSNP1 is shown in FIG. 6.  
         [0065]    The base sequence corresponding to an open reading frame in the gene encoding Protease PFUS and the amino acid sequence of Protease PFUS deduced from the base sequence are shown in the SEQ ID NOS: 15 and 16 of the Sequence Listing, respectively.  
         [0066]    A thermostable protease activity is found in either of the culture supernatant and the cell extract from the culture of  Bacillus subtilis  DB104/pSNP1. That is, a portion of the expressed Protease PFUS is secreted into the culture supernatant.  
         [0067]    Main properties of the protease obtained from the culture of the transformant are as follows.  
         [0068]    (1) Action:  
         [0069]    Degrades casein and gelatin to generate short chain polypeptides.  
         [0070]    Hydrolyzes succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4-methylcoumarin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) to generate a fluorescent substance (7-amino-4-methylcoumarin).  
         [0071]    Hydrolyzes succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to generate a yellow substance (p-nitroaniline).  
         [0072]    (2) Optimal temperature:  
         [0073]    Exhibits an enzymatic activity at 40-110° C., with the optimal temperature being 80-95° C.  
         [0074]    (3) Optimal pH:  
         [0075]    Exhibits an enzymatic activity at pH 5-10, with the optimal pH being pH 6-8.  
         [0076]    (4) Thermostability:  
         [0077]    Retains 90% or more of its enzymatic activity after treatment at 95° C. for 8 hours.  
         [0078]    (5) pH stability  
         [0079]    Retains 95% or more of its activity after treatment at pH 5-11 at 95° C. for 60 minutes.  
         [0080]    (6) Molecular weight  
         [0081]    Exhibits a molecular weight of approximately 45 kDa on SDS-PAGE.  
         [0082]    Protease genes homologous to the Protease TCES gene and the Protease PFUS gene can be obtained from hyperthermophiles other than  Pyrococcus furiosus  and  Thermococcus celer  using a method similar to that used to obtain the Protease TCES gene and the Protease PFUS gene.  
         [0083]    A DNA fragment of approximately 1 kb which encodes a sequence from the residue at position 323 to the residue at position 650 of the amino acid sequence of Protease PFUL as shown in the SEQ ID NO:6 of the Sequence Listing can be prepared and used as a probe for genomic Southern hybridization against chromosomal DNAs from  Staphylothermus marinus  DSM3639 and  Thermobacteroides proteoliticus  DSM 5265. As a result, signals are observed at the position of approximately 4.8 kb for the chromosomal DNA from  Staphylothermus marinus  digested with PstI (Takara Shuzo) and at the position of approximately 3.5 kb for the chromosomal DNA from  Thermobacteroides proteoliticus  digested with XbaI.  
         [0084]    From these results, it proved that there are sequences homologous to those of the genes for Protease PFUL, Protease PFUS and Protease TCES and the like on the chromosomal DNAs from  Staphylothermus marinus  and  Thermobacteroides proteoliticus.  The genes encoding the hyperthermostable proteases in  Staphylothermus marinus  and  Thermobacteroides proteoliticus  can be isolated and identified from the DNA fragments thus detected by using a method similar to that used to isolate and identify the genes encoding Protease TCES and Protease PFUS.  
         [0085]    In general, it is believed that use of a promoter that acts effectively in a host rather than a promoter that is inherently associated with the gene encoding the protein of interest would be advantageous in order to prepare a protein in a large quantity by genetic engineering technique. Although the P43 promoter used to construct the expression systems for Protease TCES and Protease PFUS is a promoter derived from  Bacillus subtilis,  it was not sufficiently effective to express the two proteases.  
         [0086]    Thereupon, a gene that is expressed at high level in  Bacillus subtilis,  particularly a gene for a secreted protein, may be utilized in order to increase the expression level. Genes for α-amylase or various extracellular proteases can be used. For example, it is expected that use of a promoter and a signal peptide-encoding region of a subtilisin gene may increase the expression level of Protease PFUS.  
         [0087]    Specifically, Protease PFUS can be expressed as a fused protein under control of the promoter of the subtilisin gene by placing the entire Protease PFUS gene downstream the region encoding the signal peptide of the subtilisin gene including the promoter region such that the translational frames of the two genes match each other.  
         [0088]    For example, the gene encoding subtilisin E can be used as the subtilisin gene used in the present invention. The promoter and the signal peptide-encoding region of the subtilisin E gene inserted in the plasmid pKWZ as described in J. Bacteriol., 171:2657-2665 (1989) can be used. The base sequence of the 5′ upstream region including the promoter sequence is described in the reference (supra) and the base sequence of the region encoding the subtilisin is described in J. Bacteriol., 158:411-418 (1984).  
         [0089]    Based on these sequences, the primer SUB4 for introducing an EcoRI site upstream the promoter sequence of the gene and the primer BmR1 for introducing a BamHI site downstream the region encoding the signal peptide of subtilisin E are synthesized. The base sequences of the primers SUB4 and BmR1 are shown in the SEQ ID NOS:17 and 18 of the Sequence Listing, respectively. The primers SUB4 and BmR1 can be used to amplify a DNA fragment of approximately 0.3 kb containing the promoter and the signal peptide-encoding region of the subtilisin E gene by PCR using the plasmid pKWZ as a template.  
         [0090]    The Protease PFUS gene to be placed downstream the DNA fragment can be obtained from a chromosomal DNA from  Pyrococcus furiosus  by PCR method. The primer NPF-4 can be used as a primer that hybridizes with the 5′ region of the gene. The primer NPM-1, which is designed based on the base sequence downstream from the termination codon of the gene and has a SphI site, can be used as a primer which hybridizes with the 3′ region of the gene. The sequence of the primer NPM-1 is shown in the SEQ ID NO:19 of the Sequence Listing.  
         [0091]    One BamHI site present in the gene would become a problem for a procedure in which a BamHI site is utilized for joining the Protease PFUS gene to the 0.3 kb DNA fragment. The primers mutRR and mutFR for eliminating the BamHI site by PCR-mutagenesis method can be prepared based on the base sequence of the Protease PFUS gene as shown in the SEQ ID NO:15 of the Sequence Listing. The base sequences of the primers mutRR and mutFR are shown in the SEQ ID NOS:20 and 21 of the Sequence Listing, respectively. When these primers are used to eliminate the BamHI site, the amino acid residue encoded by this site, i.e., glycine at position 560 in the amino acid sequence of Protease PFUS as shown in the SEQ ID NO:16 of the Sequence Listing, is substituted by valine due to the base substitution introduced into the site.  
         [0092]    The Protease PFUS gene to be joined to the promoter and the signal peptide-encoding region of the subtilisin E gene can be obtained by using these primers. Specifically, two PCRs are performed using a chromosomal DNA from  Pyrococcus furiosus  as a template and the pair of the primers mutRR and NPF-4 or the pair of the primers mutFR and NPM-1. In addition, a second round of PCR is performed using a heteroduplex formed by mixing the respective PCR-amplified DNA fragments as a template and the primers NPF-4 and NPM-1. Thus, the entire Protease PFUS gene of approximately 2.4 kb which does not contain an internal BamHI site can be amplified.  
         [0093]    A DNA fragment of approximately 2.4 kb obtained by digesting the PCR-amplified DNA fragment with BamHI and SphI is isolated and used to replace a BamHI-SphI fragment in the plasmid pSNP1 which contains the Protease PFUS gene. An expression vector thus constructed is designated as the plasmid pPS1.  Bacillus subtilis  DB104 transformed with this plasmid is designated as  Bacillus subtilis  DB104/pPS1. A similar protease activity is found in either of the culture supernatant and the cell extract of the culture of this transformant as observed for the transformant harboring the plasmid pSNP1, demonstrating that the amino acid substitution does not influence the enzymatic activity. The restriction enzyme map of the plasmid pPS1 is shown in FIG. 7.  
         [0094]    The DNA fragment of approximately 0.3 kb containing the promoter and the signal peptide-encoding region of the subtilisin E gene is digested with EcoRI and BamHI and is used to replace the EcoRI-BamHI fragment containing the P43 promoter and a ribosome binding site in the plasmid pPS1. An expression plasmid thus constructed is designated as pNAPS1.  Bacillus subtilis  DB/104 transformed with this plasmid is designated as  Bacillus subtilis  DB104/pNAPS1. A thermostable protease activity is found in either of the culture supernatant and the cell extract of the culture of the transformant, with the expression level being increased as compared with that of  Bacillus subtilis  DB104/pSNP1. The restriction enzyme map of the plasmid pNAPS1 is shown in FIG. 8.  
         [0095]    The protease expressed from the transformant exhibits enzymological properties equivalent to those of the protease expressed by  Bacillus subtilis  DB104/pSNP1 as described above. The protease expressed by the transformant was purified. The analysis of the N-terminal amino acid sequence of the purified protease provided the amino acid sequence as shown in the SEQ ID NO:22 of the Sequence Listing. This sequence is identical with the sequence from position 133 to position 144 of the amino acid sequence of Protease PFUS as shown in the SEQ ID NO:15 of the Sequence Listing, indicating that the mature Protease PFUS is an enzyme consisting of a polypeptide starting from this portion. The amino acid sequence of the mature Protease PFUS assumed from these results is shown in the SEQ ID NO:4 of the Sequence Listing.  
         [0096]    Although the amount of the protease produced by  Bacillus subtilis  DB104/pNAPS1 is increased as compared with the amount of the protease produced by  Bacillus subtilis  DB104/pSNP1 (FERM BP-5634), higher productivity is desired. It is expected that the expression level of the protease is increased by modifying the junction of the fused peptide encoded by pNAPS1 between the signal peptide of the subtilisin and Protease PFUS to make the removal of the signal peptide more efficient. In the plasmid pNAPS1, a peptide consisting of three amino acid residues Ala-Gly-Ser is inserted between the C-terminal amino acid residue of the signal peptide of subtilisin E as shown in the SEQ ID NO:3 of the Sequence Listing (Ala) and the N-terminal amino acid residue of Protease PFUS (Met). A transformant with increased expression level of the protease can be obtained by introducing a mutation into the DNA encoding this peptide in the plasmid pNAPS1 and examining the protease productivity of the transformant into which the mutant plasmid is introduced.  
         [0097]    First, a mutant plasmid is prepared in which the portion encoding Ser in the three amino acid peptide in the gene encoding the-fused protein: subtilisin E-Protease PFUS, in the plasmid pNAPS1 is modified such that the base sequence of the portion encodes random two amino acid residues. Such a mutant plasmid can be created by means of PCR. For example, the primers SPOF0 and SPOR0 having sequences in which the codon encoding Ser (TCC) is substituted by random six bases (the base sequences of the primers SPOF0 and SPOR0 are shown in the SEQ ID NOS:24 and 25 of the Sequence Listing, respectively) and the primers SUB3 and NPR-10 which are prepared based on the base sequence around this region (the base sequences of the primers SUB3 and NPR-10 are shown in the SEQ ID NOS:26 and 27 of the Sequence Listing, respectively) can be used to perform PCR to obtain a DNA fragment into which the intended mutation at the portion corresponding to the codon encoding Ser (TCC) is introduced. A mutant plasmid containing the protease gene with the introduced mutation can be obtained by replacing the resulting fragment for the corresponding region in the plasmid pNAPS1.  
         [0098]    A transformant with increased expression level can be then obtained by introducing the mutant plasmids thus obtained into an appropriate host, for example,  Bacillus subtilis  DB104, and determining the level of the protease expressed by the transformants. The expression level of the protease can be confirmed by determining the activity in the independent culture of the isolated transformant. Alternatively, a transformant with increased expression level can be readily selected by using an agar plate containing a substrate.  
         [0099]    Specifically, the transformants into which the mutant plasmids are introduced are grown on agar plates containing skim milk. Thereafter, the plates are incubated at temperature at which Protease PFUS exhibits its activity, for example, at 70° C. Skim milk around a colony of a transformant expressing a protease is degraded to become clear. The expression level of the protease can be estimated from the size of the clear zone.  
         [0100]    One of the transformants thus obtained which express high level of protease activity as compared with  Bacillus subtilis  DB104/pNAPS1 is designated as  Bacillus subtilis  DB104/pSPO124. The plasmid contained in this transformant was prepared (this plasmid is designated as pSPO124). Analysis of the base sequence of the plasmid revealed that the portion encoding Ser was changed into a base sequence GGGAAT, that is, that a protein in which Ser was changed into Gly-Asn was encoded by the plasmid.  
         [0101]    Thus, it proved that the expression level of the protein of interest can be increased in a bacterium of genus Bacillus as a host by placing a peptide consisting of four amino acid residues Ala-Gly-Gly-Asn downstream the signal peptide of a subtilisin, fusing it to the N-terminus of the protein of interest and expressing the fused protein. In addition to subtilisin E (from  Bacillus subtilis ) which is used in the present invention, subtilisin BPN′ from  Bacillus amyloliquefaciens  (Nucl. Acids Res., 11:7911-7925 (1983)), subtilisin Carlsberg from  Bacillus licheniformis  (Nucl. Acids Res., 13:8913-8926 (1985)) and the like are known as subtilisins produced by bacteria of genus Bacillus. The signal peptides from them can be preferably used for the present invention although their amino acid sequences slightly vary each other. Various promoters which function in a bacterium of genus Bacillus can be used in place of the promoter from the subtilisin E gene which is used in the present invention for controlling expression.  
         [0102]    There is no limitation regarding the protein to be expressed. It is possible to express a protein at high level by genetic engineering technique by applying the present invention as long as the gene for the protein is available. It is evident that the present invention can be utilized to express a protein derived from an organism other than the host from the fact that a protein derived from  Pyrococcus furiosus,  which is taxonomically different from bacteria of genus Bacillus, is expressed at high level. The present invention is preferably used to produce Protease PFUL, Protease TCES as well as proteases from  Staphylothermus marinus  and  Thermobacteroides proteoliticus  that are structurally similar to Protease PFUS by genetic engineering technique.  
         [0103]    Based on the homology with subtilisins, it is considered that Protease PFUS is expressed as a precursor protein having a signal peptide and a propeptide and then subjected to processing to generate a mature enzyme. Furthermore; based on the results of the N-terminal amino acid sequence analysis of the mature Protease PFUS enzyme, it may be assumed that the mature enzyme is an enzyme consisting of the amino acid sequence as shown in the SEQ ID NO:4 of the Sequence Listing. However, the molecular weight of the purified mature Protease PFUS is approximately 45 kDa which is smaller than that calculated from the amino acid sequence, suggesting that Protease PFUS expressed as a precursor is converted to a mature protease after being subjected to processing of its C-terminal peptide as well.  
         [0104]    If the C-terminal peptide removed by the processing is not essential to the enzymatic activity or the folding of the enzyme protein into proper structure, it is expected that the expression level of Protease PFUS can be also increased by deleting the region encoding this portion from the gene and expressing the protease.  
         [0105]    The molecular weight of the mature Protease PFUS obtained from  Bacillus subtilis  DB104/pNAPS1 can be precisely measured, for example, by using a mass spectrometer. It is found from the measured molecular weight and the N-terminal amino acid sequence of the mature Protease PFUS determined as described above that the protease is a polypeptide corresponding to Ala at position 133 to Thr at position 552 of the amino acid sequence as shown in the SEQ ID NO:15 of the Sequence Listing. Furthermore, a plasmid which expresses Protease PFUS lacking a polypeptide nonessential for its enzymatic activity can be constructed by introducing a termination codon in the vicinity of the portion encoding Thr at position 552 in the Protease PFUS gene contained in the plasmid pNAPS1. Specifically, a DNA fragment having a base sequence into which the intended termination codon is introduced can be obtained by PCR using the primer NPR544 which can introduce a termination codon (TGA) on the C-terminal side of the 544th amino acid residue encoding codon from the initiation codon in the Protease PFUS gene in the plasmid pNAPS1 (Ser) (the base sequence of the primer NPR544 is shown in the SEQ ID NO:28 of the Sequence Listing) and the primer NPFE81 which has the base sequence of the region upstream from the NspV site in the gene (the base sequence of the primer NPFE81 is shown in the SEQ ID NO:29 of the Sequence Listing). A mutant plasmid containing the protease gene into which the mutation of interest is introduced can be then obtained by replacing the fragment for the corresponding region in the plasmid pNAPS1. This plasmid is designated as the plasmid pNAPSΔC.  Bacillus subtilis  DB104 transformed with this plasmid is designated as  Bacillus subtilis  DB104/pNAPSΔC.  
         [0106]    This transformant expresses a protease activity having properties equivalent to those of Protease PFUS, with the expression level being higher than that of  Bacillus subtilis  DB104/pNAPS1.  
         [0107]    Thus, it was found that the Protease PFUS gene contained in the plasmid pNAPSΔC has a sufficient region to express the activity of the enzyme. The base sequence of the region encoding Protease PFUS present in the plasmid is shown in the SEQ ID NO:2 of the Sequence Listing. The amino acid sequence encoded by the base sequence is shown in the SEQ ID NO:1 of the Sequence Listing.  
         [0108]    Furthermore, Protease PFUS lacking its C-terminal peptide can be expressed by introducing a mutation similar to that in the plasmid pNAPSΔC into the Protease PFUS gene in the plasmid pSPO124.  
         [0109]    Specifically, the plasmid of interest can be constructed by mixing and ligating a DNA fragment of approximately 13 kb obtained by digesting the plasmid pNAPSΔC with NspV and SphI with the plasmid pSPO124 that has been digested with NsnV and SphI. This plasmid is designated as the plasmid pSO124ΔC.  Bacillus subtilis  DB104 transformed with this plasmid is designated and indicated as  Bacillus subtilis  DB104/pSO124ΔC, and deposited on May 16, 1997 (the date of the original deposit) under Budapest Treaty at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan under accession number FERM BP-6294. The expression level of protease of this transformant is increased as compared with that of  Bacillus subtilis  DB104/pNAPS1.  
         [0110]    The enzymological properties as well as the physical and chemical properties of the proteases produced by the transformants,  Bacillus subtilis  DB104/pNAPSΔC and  Bacillus subtilis  DB104/pSPO124ΔC appear to be identical with those of the protease produced by  Bacillus subtilis  DB104/pSNP1. The main properties of the proteases obtained from the cultures of the two transformants are as follows:  
         [0111]    (1) Action:  
         [0112]    Degrades casein and gelatin to generate short chain polypeptides.  
         [0113]    Hydrolyzes succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4-methylcoumarin-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) to generate a fluorescent substance (7-amino-4-methylcoumarin).  
         [0114]    Hydrolyzes succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-p-NA) to generate a yellow substance (p-nitroaniline).  
         [0115]    (2) Optimal temperature:  
         [0116]    Exhibits an enzymatic activity at 40-110° C., with the optimal temperature being 80-95° C.  
         [0117]    (3) Optimal pH:  
         [0118]    Exhibits an enzymatic activity at pH 5-10, with the optimal pH being pH 6-8.  
         [0119]    (4) Thermostability:  
         [0120]    Retains 90% or more of its enzymatic activity after treatment at 95° C. for 8 hours.  
         [0121]    (5) pH stability  
         [0122]    Retains 95% or more of its activity after treatment at pH 5-11 at 95° C. for 60 minutes.  
         [0123]    (6) Molecular weight  
         [0124]    Exhibits a molecular weight of approximately 45 kDa on SDS-PAGE.  
         [0125]    Thus, proteases having high thermostability and genes therefor are provided. Also, a novel system for expressing a protein, which enables the expression of the protease in large quantity is disclosed by the present invention. The expression system is useful in production of the protease of the present invention as well as various  
         [0126]    The following Examples illustrate the present invention in more detail, but are not to be construed to limit the scope thereof.  
       Example 1  
       [0127]    (1) Preparation of a chromosomal DNA from  Pyrococcus furiosus    
         [0128]    [0128] Pyrococcus furiosus  DSM3638 was cultured as follows.  
         [0129]    A medium containing 1% Tryptone, 0.5% yeast extract, 1% soluble starch, 3.5% Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (Jamarine Laboratory), 0.003% MgSO 4 , 0.001% NaCl, 0.0001% FeSO 4 .7H 2 O, 0.0001% CoSO 4 , 0.0001% CaCl 2 .7H 2 O, 0.0001% ZnSO 4 , 0.1 ppm CuSO 4 .5H 2 O, 0.1 ppm H 3 BO 3 , 0.1 ppm KAl(SO 4 ) 2 , 0.1 ppm Na 2 MoO 4 .2H 2 O, 0.25 ppm NiCl 2 .H 2 O was placed in a 2 L medium bottle, &#39;sterilized at 120° C. for 20 minutes, bubbled with nitrogen gas to remove dissolved oxygen, then the strain was inoculated into the medium and cultured at 95° C. for 16 hours without shaking. After cultivation, cells were collected by centrifugation.  
         [0130]    The resulting cells were then suspended in 4 mL of 50 mM Tris-HCl (pH 8.0) containing 25% sucrose. 2 mL of 0.2 M EDTA and 0.8 mL of lysozyme (5 mg/mL) were added to the suspension. The mixture was incubated at 20° C. for 1 hour. 24 mL of SET solution (150mM NaCl, 1mM EDTA, 20 mM Tris-HCl, pH 8.0), 4 mL of 5% SDS and 400 μL of proteinase K (10 mg/mL) were then added to the mixture. Incubation was further carried out at 37° C. for 1 hour. The reaction was terminated by extracting the mixture with phenol-chloroform. Then, ethanol precipitation was carried out to obtain approximately 3.2mg of chromosomal DNA.  
       EXAMPLE 2  
       [0131]    (1) Synthesis of primers for constructing the plasmid pNSP1  
         [0132]    In order to synthesize primers used to amplify the entire Protease PFUS gene, the plasmid pSNP1 that contains the entire gene was isolated from  Bacillus subtilis  DB104/pSNP1 (FERM BP-5634) and the base sequence of the required region was determined. Based on the base sequence, the primer NPF-4 for introducing a BamHI site immediately upstream the initiation codon of the Protease PFUS gene and the primer NPM-1 which hybridizes with the 3′ region of the gene and contains a recognition site for SphI were synthesized. The base sequences of the primers NPF-4 and NPM-1 are shown in the SEQ ID NOS:13 and 19 of the Sequence Listing, respectively.  
         [0133]    The primers mutRR and mutFR for removing the BamHI site present approximately 1.7 kb downstream from the initiation codon in the Protease PFUS gene were also synthesized. The base sequences of the primers mutRR and mutFR are shown in the SEQ ID NOS:20 and 21 of the Sequence Listing, respectively.  
         [0134]    (2) Preparation of the plasmid pPS1  
         [0135]    Two sets of LA-PCR reaction mixtures each of which containing a chromosomal DNA from  Pyrococcus furiosus  as a template and a combination of the primers NPF-4 and mutRR or a combination of the primers mutFR and NPM-1 were prepared and subjected to 30 cycles of reactions of 94° C. for 30 seconds-55° C. for 1 minute-68° C. for 3 minutes. LA PCR Kit Ver. 2 (Takara Shuzo) was used to prepare the LA-PCR reaction mixtures. Aliquots of the reaction mixtures were subjected to agarose gel electrophoresis, and amplification of a DNA fragment of approximately 1.8 kb with the primers NPF-4 and mutRR and a DNA fragment of approximately 0.6 kb with the primers mutFR and NPM-1 were observed, respectively.  
         [0136]    The primers were removed from the two PCR reaction mixtures using SUPREC-02 (Takara Shuzo) to prepare amplified DNA fragments. An LA-PCR reaction mixture which contained these two amplified DNA fragments and did not contain the primers or LA Taq was prepared, heat-denatured at 94° C. for 10 minutes, cooled to 30° C. within 30 minutes, then incubated at 30° C. for 15 minutes to form a to the reaction mixture to react at 72° C. for 30 minutes. The primers NPF-4 and NPM-1 were then added to the reaction mixture, which was then subjected to 25 cycles of reactions of 94° C. for 30 seconds-55° C. for 1 minute-68° C. for 3 minutes. Amplification of a DNA fragment of approximately 2.4 kb was observed in the reaction mixture.  
         [0137]    The DNA fragment of approximately 2.4 kb was digested with BamHI and SphI (both from Takara Shuzo). The fragment was mixed and ligated with the plasmid pSNP1 which had been digested with BamHI and SphI to remove the entire Protease PFUS gene, then introduced into  Bacillus subtilis  DB104. Plasmids were prepared from resulting kanamycin-resistant transformants, and a plasmid into which only one molecule of the fragment of approximately 2.4 kb was inserted was selected and designated as the plasmid pPS1.  Bacillus subtilis  DB104 transformed with this plasmid pPS1 was designated as  Bacillus subtilis  DB104/pPS1.  
         [0138]    The restriction enzyme map of the plasmid pPS1 is shown in FIG. 7.  
         [0139]    (3) Amplification of a DNA fragment for the promoter-signal peptide-encoding region of the subtilisin E gene.  
         [0140]    Primers for obtaining the promoter-signal peptide-encoding region of the subtilisin E gene were on the base sequence of the promoter region of the subtilisin E gene as described in J. Bacteriol., 171:2657-2665 (1989), which hybridizes with the sequence upstream this region and contains an EcoRI site (the base sequence of the primer SUB4 is shown in the SEQ ID NO:17 of the Sequence Listing). The primer BmR1 which is capable of introducing a BamHI site immediately downstream the signal peptide-encoding region was synthesized based on the base sequence of the subtilisin E gene as described in J. Bacteriol., 158:411-418 (1984) (the base sequence of the primer BmR1 is shown in the SEQ ID NO:18 of the Sequence Listing).  
         [0141]    A PCR reaction mixture containing the plasmid pKWZ, which contains the subtilisin E gene as described in J. Bacteriol., 171:2657-2665, as a template and the primers SUB4 and BmR1 was prepared and subjected to 30 cycles of reactions of 94° C. for 30 seconds-55° C. for 1 munute-68° C. for 2 minutes. An aliquot of the reaction mixture was subjected to agarose gel electrophoresis, and amplification of a DNA fragment of approximately 0.3 kb was observed.  
         [0142]    (4) Construction of the protease expression plasmid pNAPS1.  
         [0143]    The DNA fragment of approximately 0.3 kb as described above was digested with EcoRI (Takara Shuzo) and BamHI, mixed and ligeted with the plasmid pPS1 described in Example 3 which had been digested with EcoRI and BamHI, then introduced into  Bacillus subtilis  DB104. Plasmids were prepared from resulting kanamycin-resistant transformants, and a plasmid into which only one molecule of the fragment of approximately 0.3 kb was inserted was selected and designated as the plasmid pNAPS1.  Bacillus subtilis  DB104 transformed with the plasmid pNAPS1 was designated as  Bacillus subtilis  DB104/pNAPS1.  
         [0144]    The restriction enzyme map of the plasmid pNAPS1 is shown in FIG. 8.  
         [0145]    (5) Construction of the plasmid pSNP2  
         [0146]    The primer SUB17R for introducing a BamHI site upstream the signal peptide-encoding region of the subtilisin E gene in the above-mentioned plasmid pNAPS1 was synthesized (the base sequence of the primer SUB17R is shown in the SEQ ID NO:23 of the Sequence Listing). A PCR reaction mixture containing the plasmid pNAPS1 as a template and the primers SUB17R and SUB4 was prepared and subjected to 25 cycles of reactions of 94° C. for 30 seconds-55° C. for 1 munute-72° C. for 1 minute. The amplified DNA fragment of approximately 0.21 kb was digested with EcoRI and BamHI to obtain a DNA fragment of approximately 0.2 kb that contains the promoter and the SD sequence of the subtilisin E gene. This fragment was mixed and ligated with the plasmid pAPS1 that had been digested with EcoRI and BamHI. The reaction mixture was used to transform  Bacillus subtilis  DB104. Plasmids were prepared from resulting kanamycin-resistant transformants, and a plasmid into which the DNA fragment of approximately 0.2 kb was inserted was selected and designated as the plasmid pSNP2.  
         [0147]    (6) Generation of a mutant plasmid which expresses a protease at high level  
         [0148]    The primers SPOF0 and SPOR0 for substituting the sequence encoding the amino acid residue Ser (base sequence: TCC) at the junction between the signal peptide-encoding region of the subtilisin E gene in the plasmid pNAPS1 and the initiation codon of the Protease PFUS gene with a sequence for two random amino acid residues were synthesized (the base sequences of the primers SPOF0 and SPOR0 are shown in the SEQ ID NOS:24 and 25 of the Sequence Listing, respectively). The primer SUB3 for introducing a BamHI site immediately upstream the signal peptide-encoding region in the subtilisin E gene in the plasmid pNAPS1 and the primer NPR-10 which contains a SpeI site within the Protease PFUS encoding region were synthesized (the base sequences of the primers SUB3 and NPR-10 are shown in the SEQ ID NOS:26 and 27 of the Sequence Listing, respectively).  
         [0149]    PCR reaction mixtures each of which containing the plasmid pNAPS1 as a template and a combination of the SUB3 and SPOR0 were prepared and subjected to 20 cycles of reactions of 94° C. for 30 seconds-50° C. for 1 munute-72° C. for 1 minute. DNA fragments of approximately 0.13 kb and approximately 0.35 kb amplified in the two reaction mixtures were mixed together, denatured at 94° C. for 10 minutes, cooled gradually to 37° C. to form a heteroduplex. A double-stranded DNA was then generated from the heteroduplex by means of Taq polymerase (Takara Shuzo) . A PCR reaction mixture containing the double-stranded DNA thus obtained as a template and the primers SUB3 and NPR-10 was prepared and subjected to 25 cycles of reactions of 94° C. for 30 seconds-50° C. for 1 minute-72° C. for 1 minute. A DNA fragment obtained by digesting the amplified DNA fragment of approximately 0.43 kb with BamHI and SpeI (Takara Shuzo) was mixed and ligated with the plasmid pSNP2 that had been digested with BamHI and SpeI. The reaction mixture was used to transform  Bacillus subtilis  DB104.  
         [0150]    Resulting kanamycin-resistant transformants were inoculated on skim milk plates (LB-agar medium for high temperature cultivation containing 10 μg/mL of kanamycin and 1% skim milk) to form colonies. Subsequently, the plates were incubated at 70° C. and the protease activities expressed by the respective transformants were examined based on the degree of degradation of the skim milk around particularly high activity was isolated and a plasmid, which was designated as the plasmid pSPO124, was prepared from the clone.  Bacillus subtilis  DB104 transformed with this plasmid was designated as  Bacillus subtilis  DB104/pSPO124. The base sequence of the plasmid pSPO124 was analyzed, and it was found that the base sequence which encodes Ser in the plasmid pNAPS1 was substituted by a base sequence GGGAAT, that is, that a protein in which Ser was changed to two amino acid residues Gly-Asn was encoded. Additionally, it proved that the 25th codon from the initiation codon corresponding to Pro (CCA) of the Protease PFUS gene was changed to a codon encoding Leu (CTA) simultaneously with the mutation as described above.  
         [0151]    (7) Construction of the protease expression plasmid pNAPSΔC.  
         [0152]    A termination codon was introduced on the C-terminal side of the 544th amino acid residue from the initiation codon of the Protease PFUS gene in the plasmid pNAPS1 to construct a plasmid which expresses a protease lacking downstream from this site. The primer NPR544 which introduces a termination codon (base sequence: TGA) on the C-terminal side of the codon encoding the 544th amino acid residue in the gene and has an SphI site was synthesized (the base sequence of the primer NPR544 is shown in the SEQ ID NO:28 of the Sequence Listing). In addition, the primer NPFE81 was synthesized based on the base sequence of the portion upstream from the NspV site in the gene (the base sequence of the primer NPFE81 is shown in the SEQ ID NO:29 of the Sequence Listing).  
         [0153]    A PCR reaction mixture containing the plasmid pNAPS1 as a template and the primers NPFE81 and NPR544 was prepared and subjected to 20 cycles of reactions of 94° C. for 30 seconds-50° C. for 1 minute-72° C. for 1 minute. The amplified DNA fragment of approximately 0.61 kb was digested with NspV (Takara Shuzo) and SpeI to obtain a DNA fragment of approximately 0.13 kb containing the termination codon. This DNA fragment was mixed and ligated with the plasmid pNAPS1 that had been digested with restriction enzymes NspV and SphI. The reaction mixture was used to transform  Bacillus subtilis  DB104. Plasmids were prepared from the resulting kanamycin-resistant transformants, a plasmid into which the DNA fragment of approximately 0.13 kb was inserted was selected and designated as the plasmid pNAPSΔC.  Bacillus subtilis  DB104 transformed with the plasmid pNAPSΔC was designated as  Bacillus subtilis  DB104/pNAPSΔC.  
         [0154]    (8) Construction of the protease expression plasmid pSPO124ΔC.  
         [0155]    A DNA fragment of approximately 1.3 kb obtained by digesting the plasmid pNAPSΔC with NspV and SphI was isolated, then mixed and ligated with the plasmid pSPO124 that had been digested with NspV and SphI. The reaction mixture was used to transform  Bacillus subtilis  DB104. Plasmids were prepared from the resulting kanamycin-resistant transformants, a plasmid into which the DNA fragment of approximately 1.3 kb was inserted was selected and designated as the plasmid pSPO124ΔC.  Bacillus subtilis  DB104 transformed with the plasmid pSPO124ΔC was designated as  Bacillus subtilis  DB104/pSPO124ΔC.  
       EXAMPLE 3  
       [0156]    (1) Cultivation of  Bacillus subtilis  transformed with a plasmid containing the Protease PFUS gene and preparation of a crude enzyme solution  
         [0157]    [0157] Bacillus subtilis  DB104/pNAPS1, which is  Bacillus subtilis  DB104 into which the plasmid pNAPS1 containing the Protease PFUS gene was introduced as described in Example 2, was cultured in 2 mL of LB medium (Tryptone 10 g/L, yeast extract 5g/L, NaCl 5g/L, pH 7.2) containing 10 μg/mL of kanamycin at 37° C. for 24 hours. The culture was centrifuged to obtain a culture supernatant (the preparation 1-S) and cells.  
         [0158]    The cells were suspended in 100 μL of 50 mM Tris-HCl, pH 7.5 and digested at 37° C. for 45 minutes after an addition of 2 mg of lysozyme (Sigma). The digested sample was heat-treated at 95° C. for 10 minutes, and then a supernatant was collected by centrifugation to obtain a cell-free extract (the preparation 1-L).  
         [0159]    Similarly, culture supernatants and cell-free extracts were obtained from  Bacillus subtilis  DB104/pSPO124 containing the plasmid pSPO124,  Bacillus subtilis  DB104/pNAPSΔC containing the plasmid pNAPSΔC or  Bacillus subtilis  DB104/pSPO124ΔC containing the plasmid pSPO124ΔC. The culture supernatant and the cell-free extract from  Bacillus subtilis  DB104/pSPO124 were designated as 124-S and 124-L, respectively. The culture supernatant and the cell-free extract from  Bacillus subtilis  DB104/pNAPSΔC were designated as ΔC-S and ΔC-L, respectively. The culture supernatant and the cell-free extract from  Bacillus subtilis  DB104/pSPO124ΔC were designated as 124ΔC-S and 124ΔC-L, respectively. Protease activities were determined with these preparations and the concentration of the protease contained in each preparation was determined.  
         [0160]    (2) Comparison of protease productivities  
         [0161]    The activity of Protease PFUS was determined by spectroscopically measuring the amount of p-nitroaniline generated in an enzymatic hydrolysis reaction using Suc-Ala-Ala-Pro-Phe-p-NA (Sigma) as a substrate. Briefly, an enzyme preparation to be measured for its enzymatic Ala-Pro-Phe-p-NA solution in 100 mM phosphate buffer, pH 7.0 was added to 50 μL of the diluted sample solution. Then, the reaction was allowed to proceed at 95° C. for 30 minutes. After terminating the reaction by cooling on ice, absorbance at 405 nm was measured to calculate the amount of p-nitroaniline generated. One unit of the enzyme was defined as the amount of the enzyme which generated 1 μmole of p-nitroaniline per 1 minute at 95° C. The amount of enzyme protein expressed in the culture supernatant or the cells was calculated based on the measured enzymatic activity assuming the specific activity as 9.5 unit/mg protein of Protease PFUS.  
         [0162]    The protease activity of each enzyme preparation prepared in Example 3-(1) was measured. The productivity of Protease PFUS per 1 L of culture of each transformant calculated from the measurement is shown in Table 1.  
         [0163]    In  Bacillus subtilis  DB104/pSPO124, the productivity of Protease PFUS in the cells increased by 3.6 fold as compared with that of  Bacillus subtilis  DB104/pNAPS1. In  Bacillus subtilis  DB104/pNAPSΔC, the productivity of Protease PFUS increased in the culture supernatant by 2.4 fold and in the cells by 2.2 fold, respectively. Also, in  Bacillus subtilis  DB104/pSPO124ΔC, the productivity of Protease PFUS increased in the culture supernatant by 2 fold and in the cells by 2.4 fold, respectively. The productivity per cells also increased.  
         [0164]    The total amount of Protease PFUS produced in the culture supernatant and the cells increased by 2.1 fold for  Bacillus subtilis  DB104/pSPO124, by 2.1 fold for  Bacillus subtilis  DB104/pNAPSΔC and by 2.2 fold for  Bacillus subtilis  DB104/pSPO124ΔC, respectively, as compared with that of  Bacillus subtilis  DB104/pNAPS1.  
                                     TABLE 1                           The productivity of Protease PFUS (mg/L of culture)                            Culture           Transformant   Culture       Supernatant           (Plasmid)   Supernatant   Cells   + Cells                       pNAPS1   15.1   12.5   27.6           pSPO124   13.1   45.4   58.5           pNAPSΔC   35.5   28.1   63.6           pSPO124ΔC   30.5   30.1   60.6                      
 
       EXAMPLE 4  
       [0165]    (1) Preparation of purified enzyme preparation of the mature Protease PFUS.  
         [0166]    [0166] Bacillus subtilis  DB104/pNAPS1 and  Bacillus subtilis  DB104/pSPO124ΔC, both of which are  Bacillus subtilis  DB104 into which the gene for the hyperthermostable protease of the present invention was introduced as described in Example 2, were separately inoculated into 5 mL of LB medium containing 10 μg/mL kanamycin and cultured with shaking at 37° C. for 7 hours. The cultures of 5 mL were inoculated into 500 mL of TM medium (soybean powder 5 g/L, Polypeptone 10 g/L, meat extract 5 g/L, yeast extract 2 g/L, glucose 10 g/L, FeSO 4 .7H 2 O 10 mg/L, MnSO 4 .4H 2 O 10 mg/L, ZnSO 4 .7H 2 O 1 mg/L, pH 7.0) containing 10 μg/mL of kanamycin in 5 L Erlenmeyer flasks and cultured with shaking at 30° C. for 3 days. The resulting cultures were sonicated, heat-treated at 95° C. for 30 minutes, then centrifuged to collect supernatants. Ammonium sulfate was added to the supernatants to 25% saturation, then the supernatants obtained by subsequent centrifugation were applied to Micro-Prep Methyl HIC columns (Bio-Rad) equilibrated with 25 mM Tris-HCl buffer (pH 7.6) containing 25% saturated ammonium sulfate. After washing the gel with the same buffer, Protease PFUS adsorbed to the columns was eluted by stepwise elution using 25 mM Tris-HCl buffer (pH 7.6) containing 40% ethanol. The fractions containing Protease PFUS thus obtained were subjected to gel filtration using NAP-25 columns (Pharmacia) equilibrated with 0.05% trifluoroacetic acid containing 20% acetonitrile, desalted while denaturing Protease PFUS, then purified preparations of Protease PFUS were obtained. The preparations obtained from  Bacillus subtilis  DB104/pNAPS1 and  Bacillus subtilis  DB104/pSPO124ΔC were designated as NAPS-1 and SPO-124≢C, respectively.  
         [0167]    Electrophoresis of both of the purified enzyme preparations on 0.1% SDS-10% polyacrylamide gel followed by staining with Coomassie Brilliant Blue R-250 revealed single bands for both of the purified enzyme preparations NAPS-1 and SPO-124ΔC with an estimated molecular weight of approximately 45 kDa.  
         [0168]    (2) Analysis of the N-terminal amino acid sequence of the mature Protease PFUS.  
         [0169]    N-terminal amino acid sequences of the purified enzyme preparations NAPS-1 and SPO-124ΔC were analyzed by automated Edman method using G1000A protein sequencer (Hewlett-Packard). Both of the N-terminal amino acid sequences of the two purified enzyme preparations were as shown in the SEQ ID NO:22 of the Sequence Listing. This sequence coincides with the sequence from position 133 to position 144 of the amino acid sequence of Protease PFUS as shown in the SEQ ID NO:15 of the Sequence Listing, indicating that both of NAPS-1 and SPO-124ΔC are enzymes consisting of a polypeptide starting from this portion.  
         [0170]    (3) Mass spectrometric analysis of the mature Protease PFUS.  
         [0171]    Mass spectrometric analysis on the purified enzyme preparations NAPS-1 and SPO-124ΔC was carried out using API300 quadrupole triple mass spectrometer (Perkin-Elmer Sciex). Based on the estimated molecular weight of NAPS-1, 43,744 Da, it was demonstrated that the mature Protease PFUS produced by  Bacillus subtilis  DB104/pNAPS1 is an enzyme consisting of a polypeptide from Ala at position 133 to Thr at position 552 of the amino acid sequence of Protease PFUS as shown in the SEQ ID NO:15 of the Sequence Listing. Furthermore, based on the estimated molecular weight of SPO-124ΔC, 42,906 Da, it was demonstrated that the mature Protease PFUS produced by  Bacillus subtilis  DB104/pSPO124ΔC is an enzyme consisting of a polypeptide from Ala at position 133 to Ser at position 544 of the amino acid sequence of Protease PFUS as shown in the SEQ ID NO:15 of the Sequence Listing, i.e., the amino acid sequence as shown in the SEQ ID NO:2 of the Sequence Listing.  
     
       
       
         1 
         
           
             33  
           
           
             1  
             412  
             PRT  
             Pyrococcus furiosus  
           
            1 

Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln Val Met Ala Thr 
1               5                   10                  15 

Tyr Val Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile Thr Ile Gly Ile 
            20                  25                  30 

Ile Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu Gln Gly Lys Val 
        35                  40                  45 

Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr Pro Tyr Asp Asp 
    50                  55                  60 

His Gly His Gly Thr His Val Ala Ser Ile Ala Ala Gly Thr Gly Ala 
65                  70                  75                  80 

Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala Pro Gly Ala Lys Leu Ala 
                85                  90                  95 

Gly Ile Lys Val Leu Gly Ala Asp Gly Ser Gly Ser Ile Ser Thr Ile 
            100                 105                 110 

Ile Lys Gly Val Glu Trp Ala Val Asp Asn Lys Asp Lys Tyr Gly Ile 
        115                 120                 125 

Lys Val Ile Asn Leu Ser Leu Gly Ser Ser Gln Ser Ser Asp Gly Thr 
    130                 135                 140 

Asp Ala Leu Ser Gln Ala Val Asn Ala Ala Trp Asp Ala Gly Leu Val 
145                 150                 155                 160 

Val Val Val Ala Ala Gly Asn Ser Gly Pro Asn Lys Tyr Thr Ile Gly 
                165                 170                 175 

Ser Pro Ala Ala Ala Ser Lys Val Ile Thr Val Gly Ala Val Asp Lys 
            180                 185                 190 

Tyr Asp Val Ile Thr Ser Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly 
        195                 200                 205 

Arg Leu Lys Pro Glu Val Val Ala Pro Gly Asn Trp Ile Ile Ala Ala 
    210                 215                 220 

Arg Ala Ser Gly Thr Ser Met Gly Gln Pro Ile Asn Asp Tyr Tyr Thr 
225                 230                 235                 240 

Ala Ala Pro Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ile Ala 
                245                 250                 255 

Ala Leu Leu Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys Val Lys 
            260                 265                 270 

Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro Asp Glu Ile Ala 
        275                 280                 285 

Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr Lys Ala Ile Asn 
    290                 295                 300 

Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly Tyr Val Ala Asn Lys 
305                 310                 315                 320 

Gly Ser Gln Thr His Gln Phe Val Ile Ser Gly Ala Ser Phe Val Thr 
                325                 330                 335 

Ala Thr Leu Tyr Trp Asp Asn Ala Asn Ser Asp Leu Asp Leu Tyr Leu 
            340                 345                 350 

Tyr Asp Pro Asn Gly Asn Gln Val Asp Tyr Ser Tyr Thr Ala Tyr Tyr 
        355                 360                 365 

Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro Thr Asp Gly Thr Trp Thr 
    370                 375                 380 

Ile Lys Val Val Ser Tyr Ser Gly Ser Ala Asn Tyr Gln Val Asp Val 
385                 390                 395                 400 

Val Ser Asp Gly Ser Leu Ser Gln Pro Gly Ser Ser 
                405                 410 

 
           
             2  
             1236  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            2 

gcagaattag aaggactgga tgagtctgca gctcaagtta tggcaactta cgtttggaac     60 

ttgggatatg atggttctgg aatcacaata ggaataattg acactggaat tgacgcttct    120 

catccagatc tccaaggaaa agtaattggg tgggtagatt ttgtcaatgg taggagttat    180 

ccatacgatg accatggaca tggaactcat gtagcttcaa tagcagctgg tactggagca    240 

gcaagtaatg gcaagtacaa gggaatggct ccaggagcta agctggcggg aattaaggtt    300 

ctaggtgccg atggttctgg aagcatatct actataatta agggagttga gtgggccgtt    360 

gataacaaag ataagtacgg aattaaggtc attaatcttt ctcttggttc aagccagagc    420 

tcagatggta ctgacgctct aagtcaggct gttaatgcag cgtgggatgc tggattagtt    480 

gttgtggttg ccgctggaaa cagtggacct aacaagtata caatcggttc tccagcagct    540 

gcaagcaaag ttattacagt tggagccgtt gacaagtatg atgttataac aagcttctca    600 

agcagagggc caactgcaga cggcaggctt aagcctgagg ttgttgctcc aggaaactgg    660 

ataattgctg ccagagcaag tggaactagc atgggtcaac caattaatga ctattacaca    720 

gcagctcctg ggacatcaat ggcaactcct cacgtagctg gtattgcagc cctcttgctc    780 

caagcacacc cgagctggac tccagacaaa gtaaaaacag ccctcataga aactgctgat    840 

atcgtaaagc cagatgaaat agccgatata gcctacggtg caggtagggt taatgcatac    900 

aaggctataa actacgataa ctatgcaaag ctagtgttca ctggatatgt tgccaacaaa    960 

ggcagccaaa ctcaccagtt cgttattagc ggagcttcgt tcgtaactgc cacattatac   1020 

tgggacaatg ccaatagcga ccttgatctt tacctctacg atcccaatgg aaaccaggtt   1080 

gactactctt acaccgccta ctatggattc gaaaaggttg gttattacaa cccaactgat   1140 

ggaacatgga caattaaggt tgtaagctac agcggaagtg caaactatca agtagatgtg   1200 

gtaagtgatg gttccctttc acagcctgga agttca                             1236 

 
           
             3  
             29  
             PRT  
             Bacillus subtilis  
           
            3 

Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 
1               5                   10                  15 

Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala 
            20                  25 

 
           
             4  
             522  
             PRT  
             Pyrococcus furiosus  
             
               misc_feature  
               (428)..(428)  
               Xaa at position 428 is Gly or Val.  
             
           
            4 

Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln Val Met Ala Thr 
1               5                   10                  15 

Tyr Val Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile Thr Ile Gly Ile 
            20                  25                  30 

Ile Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu Gln Gly Lys Val 
        35                  40                  45 

Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr Pro Tyr Asp Asp 
    50                  55                  60 

His Gly His Gly Thr His Val Ala Ser Ile Ala Ala Gly Thr Gly Ala 
65                  70                  75                  80 

Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala Pro Gly Ala Lys Leu Ala 
                85                  90                  95 

Gly Ile Lys Val Leu Gly Ala Asp Gly Ser Gly Ser Ile Ser Thr Ile 
            100                 105                 110 

Ile Lys Gly Val Glu Trp Ala Val Asp Asn Lys Asp Lys Tyr Gly Ile 
        115                 120                 125 

Lys Val Ile Asn Leu Ser Leu Gly Ser Ser Gln Ser Ser Asp Gly Thr 
    130                 135                 140 

Asp Ala Leu Ser Gln Ala Val Asn Ala Ala Trp Asp Ala Gly Leu Val 
145                 150                 155                 160 

Val Val Val Ala Ala Gly Asn Ser Gly Pro Asn Lys Tyr Thr Ile Gly 
                165                 170                 175 

Ser Pro Ala Ala Ala Ser Lys Val Ile Thr Val Gly Ala Val Asp Lys 
            180                 185                 190 

Tyr Asp Val Ile Thr Ser Phe Ser Ser Arg Gly Pro Thr Ala Asp Gly 
        195                 200                 205 

Arg Leu Lys Pro Glu Val Val Ala Pro Gly Asn Trp Ile Ile Ala Ala 
    210                 215                 220 

Arg Ala Ser Gly Thr Ser Met Gly Gln Pro Ile Asn Asp Tyr Tyr Thr 
225                 230                 235                 240 

Ala Ala Pro Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ile Ala 
                245                 250                 255 

Ala Leu Leu Leu Gln Ala His Pro Ser Trp Thr Pro Asp Lys Val Lys 
            260                 265                 270 

Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro Asp Glu Ile Ala 
        275                 280                 285 

Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr Lys Ala Ile Asn 
    290                 295                 300 

Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly Tyr Val Ala Asn Lys 
305                 310                 315                 320 

Gly Ser Gln Thr His Gln Phe Val Ile Ser Gly Ala Ser Phe Val Thr 
                325                 330                 335 

Ala Thr Leu Tyr Trp Asp Asn Ala Asn Ser Asp Leu Asp Leu Tyr Leu 
            340                 345                 350 

Tyr Asp Pro Asn Gly Asn Gln Val Asp Tyr Ser Tyr Thr Ala Tyr Tyr 
        355                 360                 365 

Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro Thr Asp Gly Thr Trp Thr 
    370                 375                 380 

Ile Lys Val Val Ser Tyr Ser Gly Ser Ala Asn Tyr Gln Val Asp Val 
385                 390                 395                 400 

Val Ser Asp Gly Ser Leu Ser Gln Pro Gly Ser Ser Pro Ser Pro Gln 
                405                 410                 415 

Pro Glu Pro Thr Val Asp Ala Lys Thr Phe Gln Xaa Ser Asp His Tyr 
            420                 425                 430 

Tyr Tyr Asp Arg Ser Asp Thr Phe Thr Met Thr Val Asn Ser Gly Ala 
        435                 440                 445 

Thr Lys Ile Thr Gly Asp Leu Val Phe Asp Thr Ser Tyr His Asp Leu 
    450                 455                 460 

Asp Leu Tyr Leu Tyr Asp Pro Asn Gln Lys Leu Val Asp Arg Ser Glu 
465                 470                 475                 480 

Ser Pro Asn Ser Tyr Glu His Val Glu Tyr Leu Thr Pro Ala Pro Gly 
                485                 490                 495 

Thr Trp Tyr Phe Leu Val Tyr Ala Tyr Tyr Thr Tyr Gly Trp Ala Tyr 
            500                 505                 510 

Tyr Glu Leu Thr Ala Lys Val Tyr Tyr Gly 
        515                 520 

 
           
             5  
             4765  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            5 

tttaaattat aagatataat cactccgagt gatgagtaag atacatcatt acagtcccaa     60 

aatgtttata attggaacgc agtgaatata caaaatgaat ataacctcgg aggtgactgt    120 

agaatgaata agaagggact tactgtgcta tttatagcga taatgctcct ttcagtagtt    180 

ccagtgcact ttgtgtccgc agaaacacca ccggttagtt cagaaaattc aacaacttct    240 

atactcccta accaacaagt tgtgacaaaa gaagtttcac aagcggcgct taatgctata    300 

atgaaaggac aacccaacat ggttcttata atcaagacta aggaaggcaa acttgaagag    360 

gcaaaaaccg agcttgaaaa gctaggtgca gagattcttg acgaaaatag agttcttaac    420 

atgttgctag ttaagattaa gcctgagaaa gttaaagagc tcaactatat ctcatctctt    480 

gaaaaagcct ggcttaacag agaagttaag ctttcccctc caattgtcga aaaggacgtc    540 

aagactaagg agccctccct agaaccaaaa atgtataaca gcacctgggt aattaatgct    600 

ctccagttca tccaggaatt tggatatgat ggtagtggtg ttgttgttgc agtacttgac    660 

acgggagttg atccgaacca tcctttcttg agcataactc cagatggacg caggaaaatt    720 

atagaatgga aggattttac agacgaggga ttcgtggata catcattcag ctttagcaag    780 

gttgtaaatg ggactcttat aattaacaca acattccaag tggcctcagg tctcacgctg    840 

aatgaatcga caggacttat ggaatacgtt gttaagactg tttacgtgag caatgtgacc    900 

attggaaata tcacttctgc taatggcatc tatcacttcg gcctgctccc agaaagatac    960 

ttcgacttaa acttcgatgg tgatcaagag gacttctatc ctgtcttatt agttaactcc   1020 

actggcaatg gttatgacat tgcatatgtg gatactgacc ttgactacga cttcaccgac   1080 

gaagttccac ttggccagta caacgttact tatgatgttg ctgtttttag ctactactac   1140 

ggtcctctca actacgtgct tgcagaaata gatcctaacg gagaatatgc agtatttggg   1200 

tgggatggtc acggtcacgg aactcacgta gctggaactg ttgctggtta cgacagcaac   1260 

aatgatgctt gggattggct cagtatgtac tctggtgaat gggaagtgtt ctcaagactc   1320 

tatggttggg attatacgaa cgttaccaca gacaccgtgc agggtgttgc tccaggtgcc   1380 

caaataatgg caataagagt tcttaggagt gatggacggg gtagcatgtg ggatattata   1440 

gaaggtatga catacgcagc aacccatggt gcagacgtta taagcatgag tctcggtgga   1500 

aatgctccat acttagatgg tactgatcca gaaagcgttg ctgtggatga gcttaccgaa   1560 

aagtacggtg ttgtattcgt aatagctgca ggaaatgaag gtcctggcat taacatcgtt   1620 

ggaagtcctg gtgttgcaac aaaggcaata actgttggag ctgctgcagt gcccattaac   1680 

gttggagttt atgtttccca agcacttgga tatcctgatt actatggatt ctattacttc   1740 

cccgcctaca caaacgttag aatagcattc ttctcaagca gagggccgag aatagatggt   1800 

gaaataaaac ccaatgtagt ggctccaggt tacggaattt actcatccct gccgatgtgg   1860 

attggcggag ctgacttcat gtctggaact tcgatggcta ctccacatgt cagcggtgtc   1920 

gttgcactcc tcataagcgg ggcaaaggcc gagggaatat actacaatcc agatataatt   1980 

aagaaggttc ttgagagcgg tgcaacctgg cttgagggag atccatatac tgggcagaag   2040 

tacactgagc ttgaccaagg tcatggtctt gttaacgtta ccaagtcctg ggaaatcctt   2100 

aaggctataa acggcaccac tctcccaatt gttgatcact gggcagacaa gtcctacagc   2160 

gactttgcgg agtacttggg tgtggacgtt ataagaggtc tctacgcaag gaactctata   2220 

cctgacattg tcgagtggca cattaagtac gtaggggaca cggagtacag aacttttgag   2280 

atctatgcaa ctgagccatg gattaagcct tttgtcagtg gaagtgtaat tctagagaac   2340 

aataccgagt ttgtccttag ggtgaaatat gatgtagagg gtcttgagcc aggtctctat   2400 

gttggaagga taatcattga tgatccaaca acgccagtta ttgaagacga gatcttgaac   2460 

acaattgtta ttcccgagaa gttcactcct gagaacaatt acaccctcac ctggtatgat   2520 

attaatggtc cagaaatggt gactcaccac ttcttcactg tgcctgaggg agtggacgtt   2580 

ctctacgcga tgaccacata ctgggactac ggtctgtaca gaccagatgg aatgtttgtg   2640 

ttcccatacc agctagatta tcttcccgct gcagtctcaa atccaatgcc tggaaactgg   2700 

gagctagtat ggactggatt taactttgca cccctctatg agtcgggctt ccttgtaagg   2760 

atttacggag tagagataac tccaagcgtt tggtacatta acaggacata ccttgacact   2820 

aacactgaat tctcaattga attcaatatt actaacatct atgccccaat taatgcaact   2880 

ctaatcccca ttggccttgg aacctacaat gcgagcgttg aaagcgttgg tgatggagag   2940 

ttcttcataa agggcattga agttcctgaa ggcaccgcag agttgaagat taggataggc   3000 

aacccaagtg ttccgaattc agatctagac ttgtaccttt atgacagtaa aggcaattta   3060 

gtggccttag atggaaaccc aacagcagaa gaagaggttg tagttgagta tcctaagcct   3120 

ggagtttatt caatagtagt acatggttac agcgtcaggg acgaaaatgg taatccaacg   3180 

acaaccacct ttgacttagt tgttcaaatg acccttgata atggaaacat aaagcttgac   3240 

aaagactcga ttattcttgg aagcaatgaa agcgtagttg taactgcaaa cataacaatt   3300 

gatagagatc atcctacagg agtatactct ggtatcatag agattagaga taatgaggtc   3360 

taccaggata caaatacttc aattgcgaaa atacccataa ctttggtaat tgacaaggcg   3420 

gactttgccg ttggtctcac accagcagag ggagtacttg gagaggctag aaattacact   3480 

ctaattgtaa agcatgccct aacactagag cctgtgccaa atgctacagt gattatagga   3540 

aactacacct acctcacaga cgaaaacggt acagtgacat tcacgtatgc tccaactaag   3600 

ttaggcagtg atgaaatcac agtcatagtt aagaaagaga acttcaacac attagagaag   3660 

accttccaaa tcacagtatc agagcctgaa ataactgaag aggacataaa tgagcccaag   3720 

cttgcaatgt catcaccaga agcaaatgct accatagtat cagttgagat ggagagtgag   3780 

ggtggcgtta aaaagacagt gacagtggaa ataactataa acggaaccgc taatgagact   3840 

gcaacaatag tggttcctgt tcctaagaag gccgaaaaca tcgaggtaag tggagaccac   3900 

gtaatttcct atagtataga ggaaggagag tacgccaagt acgttataat tacagtgaag   3960 

tttgcatcac ctgtaacagt aactgttact tacactatct atgctggccc aagagtctca   4020 

atcttgacac ttaacttcct tggctactca tggtacagac tatattcaca gaagtttgac   4080 

gaattgtacc aaaaggccct tgaattggga gtggacaacg agacattagc tttagccctc   4140 

agctaccatg aaaaagccaa agagtactac gaaaaggccc ttgagcttag cgagggtaac   4200 

ataatccaat accttggaga cataagacta ttacctccat taagacaggc atacatcaat   4260 

gaaatgaagg cagttaagat actggaaaag gccatagaag aattagaggg tgaagagtaa   4320 

tctccaattt ttcccacttt ttcttttata acattccaag ccttttctta gcttcttcgc   4380 

tcattctatc aggagtccat ggaggatcaa aggtaagttc aacctccaca tctcttactc   4440 

ctgggatttc gagtactttc tcctctacag ctctaagaag ccagagagtt aaaggacacc   4500 

caggagttgt cattgtcatc tttatatata ccgttttgtc aggattaatc tttagctcat   4560 

aaattaatcc aaggtttaca acatccatcc caatttctgg gtcgataacc tcctttagct   4620 

tttccagaat catttcttca gtaatttcaa ggttctcatc tttggtttct ctcacaaacc   4680 

caatttcaac ctgcctgata ccttctaact ccctaagctt gttatatatc tccaaaagag   4740 

tggcatcatc aattttctct ttaaa                                         4765 

 
           
             6  
             1398  
             PRT  
             Pyrococcus furiosus  
           
            6 

Met Asn Lys Lys Gly Leu Thr Val Leu Phe Ile Ala Ile Met Leu Leu 
1               5                   10                  15 

Ser Val Val Pro Val His Phe Val Ser Ala Glu Thr Pro Pro Val Ser 
            20                  25                  30 

Ser Glu Asn Ser Thr Thr Ser Ile Leu Pro Asn Gln Gln Val Val Thr 
        35                  40                  45 

Lys Glu Val Ser Gln Ala Ala Leu Asn Ala Ile Met Lys Gly Gln Pro 
    50                  55                  60 

Asn Met Val Leu Ile Ile Lys Thr Lys Glu Gly Lys Leu Glu Glu Ala 
65                  70                  75                  80 

Lys Thr Glu Leu Glu Lys Leu Gly Ala Glu Ile Leu Asp Glu Asn Arg 
                85                  90                  95 

Val Leu Asn Met Leu Leu Val Lys Ile Lys Pro Glu Lys Val Lys Glu 
            100                 105                 110 

Leu Asn Tyr Ile Ser Ser Leu Glu Lys Ala Trp Leu Asn Arg Glu Val 
        115                 120                 125 

Lys Leu Ser Pro Pro Ile Val Glu Lys Asp Val Lys Thr Lys Glu Pro 
    130                 135                 140 

Ser Leu Glu Pro Lys Met Tyr Asn Ser Thr Trp Val Ile Asn Ala Leu 
145                 150                 155                 160 

Gln Phe Ile Gln Glu Phe Gly Tyr Asp Gly Ser Gly Val Val Val Ala 
                165                 170                 175 

Val Leu Asp Thr Gly Val Asp Pro Asn His Pro Phe Leu Ser Ile Thr 
            180                 185                 190 

Pro Asp Gly Arg Arg Lys Ile Ile Glu Trp Lys Asp Phe Thr Asp Glu 
        195                 200                 205 

Gly Phe Val Asp Thr Ser Phe Ser Phe Ser Lys Val Val Asn Gly Thr 
    210                 215                 220 

Leu Ile Ile Asn Thr Thr Phe Gln Val Ala Ser Gly Leu Thr Leu Asn 
225                 230                 235                 240 

Glu Ser Thr Gly Leu Met Glu Tyr Val Val Lys Thr Val Tyr Val Ser 
                245                 250                 255 

Asn Val Thr Ile Gly Asn Ile Thr Ser Ala Asn Gly Ile Tyr His Phe 
            260                 265                 270 

Gly Leu Leu Pro Glu Arg Tyr Phe Asp Leu Asn Phe Asp Gly Asp Gln 
        275                 280                 285 

Glu Asp Phe Tyr Pro Val Leu Leu Val Asn Ser Thr Gly Asn Gly Tyr 
    290                 295                 300 

Asp Ile Ala Tyr Val Asp Thr Asp Leu Asp Tyr Asp Phe Thr Asp Glu 
305                 310                 315                 320 

Val Pro Leu Gly Gln Tyr Asn Val Thr Tyr Asp Val Ala Val Phe Ser 
                325                 330                 335 

Tyr Tyr Tyr Gly Pro Leu Asn Tyr Val Leu Ala Glu Ile Asp Pro Asn 
            340                 345                 350 

Gly Glu Tyr Ala Val Phe Gly Trp Asp Gly His Gly His Gly Thr His 
        355                 360                 365 

Val Ala Gly Thr Val Ala Gly Tyr Asp Ser Asn Asn Asp Ala Trp Asp 
    370                 375                 380 

Trp Leu Ser Met Tyr Ser Gly Glu Trp Glu Val Phe Ser Arg Leu Tyr 
385                 390                 395                 400 

Gly Trp Asp Tyr Thr Asn Val Thr Thr Asp Thr Val Gln Gly Val Ala 
                405                 410                 415 

Pro Gly Ala Gln Ile Met Ala Ile Arg Val Leu Arg Ser Asp Gly Arg 
            420                 425                 430 

Gly Ser Met Trp Asp Ile Ile Glu Gly Met Thr Tyr Ala Ala Thr His 
        435                 440                 445 

Gly Ala Asp Val Ile Ser Met Ser Leu Gly Gly Asn Ala Pro Tyr Leu 
    450                 455                 460 

Asp Gly Thr Asp Pro Glu Ser Val Ala Val Asp Glu Leu Thr Glu Lys 
465                 470                 475                 480 

Tyr Gly Val Val Phe Val Ile Ala Ala Gly Asn Glu Gly Pro Gly Ile 
                485                 490                 495 

Asn Ile Val Gly Ser Pro Gly Val Ala Thr Lys Ala Ile Thr Val Gly 
            500                 505                 510 

Ala Ala Ala Val Pro Ile Asn Val Gly Val Tyr Val Ser Gln Ala Leu 
        515                 520                 525 

Gly Tyr Pro Asp Tyr Tyr Gly Phe Tyr Tyr Phe Pro Ala Tyr Thr Asn 
    530                 535                 540 

Val Arg Ile Ala Phe Phe Ser Ser Arg Gly Pro Arg Ile Asp Gly Glu 
545                 550                 555                 560 

Ile Lys Pro Asn Val Val Ala Pro Gly Tyr Gly Ile Tyr Ser Ser Leu 
                565                 570                 575 

Pro Met Trp Ile Gly Gly Ala Asp Phe Met Ser Gly Thr Ser Met Ala 
            580                 585                 590 

Thr Pro His Val Ser Gly Val Val Ala Leu Leu Ile Ser Gly Ala Lys 
        595                 600                 605 

Ala Glu Gly Ile Tyr Tyr Asn Pro Asp Ile Ile Lys Lys Val Leu Glu 
    610                 615                 620 

Ser Gly Ala Thr Trp Leu Glu Gly Asp Pro Tyr Thr Gly Gln Lys Tyr 
625                 630                 635                 640 

Thr Glu Leu Asp Gln Gly His Gly Leu Val Asn Val Thr Lys Ser Trp 
                645                 650                 655 

Glu Ile Leu Lys Ala Ile Asn Gly Thr Thr Leu Pro Ile Val Asp His 
            660                 665                 670 

Trp Ala Asp Lys Ser Tyr Ser Asp Phe Ala Glu Tyr Leu Gly Val Asp 
        675                 680                 685 

Val Ile Arg Gly Leu Tyr Ala Arg Asn Ser Ile Pro Asp Ile Val Glu 
    690                 695                 700 

Trp His Ile Lys Tyr Val Gly Asp Thr Glu Tyr Arg Thr Phe Glu Ile 
705                 710                 715                 720 

Tyr Ala Thr Glu Pro Trp Ile Lys Pro Phe Val Ser Gly Ser Val Ile 
                725                 730                 735 

Leu Glu Asn Asn Thr Glu Phe Val Leu Arg Val Lys Tyr Asp Val Glu 
            740                 745                 750 

Gly Leu Glu Pro Gly Leu Tyr Val Gly Arg Ile Ile Ile Asp Asp Pro 
        755                 760                 765 

Thr Thr Pro Val Ile Glu Asp Glu Ile Leu Asn Thr Ile Val Ile Pro 
    770                 775                 780 

Glu Lys Phe Thr Pro Glu Asn Asn Tyr Thr Leu Thr Trp Tyr Asp Ile 
785                 790                 795                 800 

Asn Gly Pro Glu Met Val Thr His His Phe Phe Thr Val Pro Glu Gly 
                805                 810                 815 

Val Asp Val Leu Tyr Ala Met Thr Thr Tyr Trp Asp Tyr Gly Leu Tyr 
            820                 825                 830 

Arg Pro Asp Gly Met Phe Val Phe Pro Tyr Gln Leu Asp Tyr Leu Pro 
        835                 840                 845 

Ala Ala Val Ser Asn Pro Met Pro Gly Asn Trp Glu Leu Val Trp Thr 
    850                 855                 860 

Gly Phe Asn Phe Ala Pro Leu Tyr Glu Ser Gly Phe Leu Val Arg Ile 
865                 870                 875                 880 

Tyr Gly Val Glu Ile Thr Pro Ser Val Trp Tyr Ile Asn Arg Thr Tyr 
                885                 890                 895 

Leu Asp Thr Asn Thr Glu Phe Ser Ile Glu Phe Asn Ile Thr Asn Ile 
            900                 905                 910 

Tyr Ala Pro Ile Asn Ala Thr Leu Ile Pro Ile Gly Leu Gly Thr Tyr 
        915                 920                 925 

Asn Ala Ser Val Glu Ser Val Gly Asp Gly Glu Phe Phe Ile Lys Gly 
    930                 935                 940 

Ile Glu Val Pro Glu Gly Thr Ala Glu Leu Lys Ile Arg Ile Gly Asn 
945                 950                 955                 960 

Pro Ser Val Pro Asn Ser Asp Leu Asp Leu Tyr Leu Tyr Asp Ser Lys 
                965                 970                 975 

Gly Asn Leu Val Ala Leu Asp Gly Asn Pro Thr Ala Glu Glu Glu Val 
            980                 985                 990 

Val Val Glu Tyr Pro Lys Pro Gly  Val Tyr Ser Ile Val  Val His Gly 
        995                 1000                 1005 

Tyr Ser  Val Arg Asp Glu Asn  Gly Asn Pro Thr Thr  Thr Thr Phe 
    1010                 1015                 1020 

Asp Leu  Val Val Gln Met Thr  Leu Asp Asn Gly Asn  Ile Lys Leu 
    1025                 1030                 1035 

Asp Lys  Asp Ser Ile Ile Leu  Gly Ser Asn Glu Ser  Val Val Val 
    1040                 1045                 1050 

Thr Ala  Asn Ile Thr Ile Asp  Arg Asp His Pro Thr  Gly Val Tyr 
    1055                 1060                 1065 

Ser Gly  Ile Ile Glu Ile Arg  Asp Asn Glu Val Tyr  Gln Asp Thr 
    1070                 1075                 1080 

Asn Thr  Ser Ile Ala Lys Ile  Pro Ile Thr Leu Val  Ile Asp Lys 
    1085                 1090                 1095 

Ala Asp  Phe Ala Val Gly Leu  Thr Pro Ala Glu Gly  Val Leu Gly 
    1100                 1105                 1110 

Glu Ala  Arg Asn Tyr Thr Leu  Ile Val Lys His Ala  Leu Thr Leu 
    1115                 1120                 1125 

Glu Pro  Val Pro Asn Ala Thr  Val Ile Ile Gly Asn  Tyr Thr Tyr 
    1130                 1135                 1140 

Leu Thr  Asp Glu Asn Gly Thr  Val Thr Phe Thr Tyr  Ala Pro Thr 
    1145                 1150                 1155 

Lys Leu  Gly Ser Asp Glu Ile  Thr Val Ile Val Lys  Lys Glu Asn 
    1160                 1165                 1170 

Phe Asn  Thr Leu Glu Lys Thr  Phe Gln Ile Thr Val  Ser Glu Pro 
    1175                 1180                 1185 

Glu Ile  Thr Glu Glu Asp Ile  Asn Glu Pro Lys Leu  Ala Met Ser 
    1190                 1195                 1200 

Ser Pro  Glu Ala Asn Ala Thr  Ile Val Ser Val Glu  Met Glu Ser 
    1205                 1210                 1215 

Glu Gly  Gly Val Lys Lys Thr  Val Thr Val Glu Ile  Thr Ile Asn 
    1220                 1225                 1230 

Gly Thr  Ala Asn Glu Thr Ala  Thr Ile Val Val Pro  Val Pro Lys 
    1235                 1240                 1245 

Lys Ala  Glu Asn Ile Glu Val  Ser Gly Asp His Val  Ile Ser Tyr 
    1250                 1255                 1260 

Ser Ile  Glu Glu Gly Glu Tyr  Ala Lys Tyr Val Ile  Ile Thr Val 
    1265                 1270                 1275 

Lys Phe  Ala Ser Pro Val Thr  Val Thr Val Thr Tyr  Thr Ile Tyr 
    1280                 1285                 1290 

Ala Gly  Pro Arg Val Ser Ile  Leu Thr Leu Asn Phe  Leu Gly Tyr 
    1295                 1300                 1305 

Ser Trp  Tyr Arg Leu Tyr Ser  Gln Lys Phe Asp Glu  Leu Tyr Gln 
    1310                 1315                 1320 

Lys Ala  Leu Glu Leu Gly Val  Asp Asn Glu Thr Leu  Ala Leu Ala 
    1325                 1330                 1335 

Leu Ser  Tyr His Glu Lys Ala  Lys Glu Tyr Tyr Glu  Lys Ala Leu 
    1340                 1345                 1350 

Glu Leu  Ser Glu Gly Asn Ile  Ile Gln Tyr Leu Gly  Asp Ile Arg 
    1355                 1360                 1365 

Leu Leu  Pro Pro Leu Arg Gln  Ala Tyr Ile Asn Glu  Met Lys Ala 
    1370                 1375                 1380 

Val Lys  Ile Leu Glu Lys Ala  Ile Glu Glu Leu Glu  Gly Glu Glu 
    1385                 1390                 1395 

 
           
             7  
             35  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            7 

ggwwsdrrtg ttrrhgthgc dgtdmtygac acbgg                                35 

 
           
             8  
             32  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            8 

kstcacggaa ctcacgtdgc bgghacdgtt gc                                   32 

 
           
             9  
             33  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            9 

ascmgcaach gtkccvgcha cgtgagttcc gtg                                  33 

 
           
             10  
             34  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            10 

chccgsyvac rtgbggagwd gccatbgavg tdcc                                 34 

 
           
             11  
             1977  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            11 

atgaagaggt taggtgctgt ggtgctggca ctggtgctcg tgggtcttct ggccggaacg     60 

gcccttgcgg cacccgtaaa accggttgtc aggaacaacg cggttcagca gaagaactac    120 

ggactgctga ccccgggact gttcaagaaa gtccagagga tgaactggaa ccaggaagtg    180 

gacaccgtca taatgttcgg gagctacgga gacagggaca gggcggttaa ggtactgagg    240 

ctcatgggcg cccaggtcaa gtactcctac aagataatcc ctgctgtcgc ggttaaaata    300 

aaggccaggg accttctgct gatcgcgggc atgatagaca cgggttactt cggtaacaca    360 

agggtctcgg gcataaagtt catacaggag gattacaagg ttcaggttga cgacgccact    420 

tccgtctccc agataggggc cgataccgtc tggaactccc tcggctacga cggaagcggt    480 

gtggtggttg ccatcgtcga tacgggtata gacgcgaacc accccgatct gaagggcaag    540 

gtcataggct ggtacgacgc cgtcaacggc aggtcgaccc cctacgatga ccagggacac    600 

ggaacccacg ttgcgggtat cgttgccgga accggcagcg ttaactccca gtacataggc    660 

gtcgcccccg gcgcgaagct cgtcggcgtc aaggttctcg gtgccgacgg ttcgggaagc    720 

gtctccacca tcatcgcggg tgttgactgg gtcgtccaga acaaggacaa gtacgggata    780 

agggtcatca acctctccct cggctcctcc cagagctccg acggaaccga ctccctcagt    840 

caggccgtca acaacgcctg ggacgccggt atagtagtct gcgtcgccgc cggcaacagc    900 

gggccgaaca cctacaccgt cggctcaccc gccgccgcga gcaaggtcat aaccgtcggt    960 

gcagttgaca gcaacgacaa catcgccagc ttctccagca ggggaccgac cgcggacgga   1020 

aggctcaagc cggaagtcgt cgcccccggc gttgacatca tagccccgcg cgccagcgga   1080 

accagcatgg gcaccccgat aaacgactac tacaccaagg cctctggaac cagcatggcc   1140 

accccgcacg tttcgggcgt tggcgcgctc atcctccagg cccacccgag ctggaccccg   1200 

gacaaggtga agaccgccct catcgagacc gccgacatag tcgcccccaa ggagatagcg   1260 

gacatcgcct acggtgcggg tagggtgaac gtctacaagg ccatcaagta cgacgactac   1320 

gccaagctca ccttcaccgg ctccgtcgcc gacaagggaa gcgccaccca caccttcgac   1380 

gtcagcggcg ccaccttcgt gaccgccacc ctctactggg acacgggctc gagcgacatc   1440 

gacctctacc tctacgaccc caacgggaac gaggttgact actcctacac cgcctactac   1500 

ggcttcgaga aggtcggcta ctacaacccg accgccggaa cctggacggt caaggtcgtc   1560 

agctacaagg gcgcggcgaa ctaccaggtc gacgtcgtca gcgacgggag cctcagccag   1620 

tccggcggcg gcaacccgaa tccaaacccc aacccgaacc caaccccgac caccgacacc   1680 

cagaccttca ccggttccgt taacgactac tgggacacca gcgacacctt caccatgaac   1740 

gtcaacagcg gtgccaccaa gataaccggt gacctgacct tcgatacttc ctacaacgac   1800 

ctcgacctct acctctacga ccccaacggc aacctcgttg acaggtccac gtcgagcaac   1860 

agctacgagc acgtcgagta cgccaacccc gccccgggaa cctggacgtt cctcgtctac   1920 

gcctacagca cctacggctg ggcggactac cagctcaagg ccgtcgtcta ctacggg      1977 

 
           
             12  
             659  
             PRT  
             Thermococcus celer  
           
            12 

Met Lys Arg Leu Gly Ala Val Val Leu Ala Leu Val Leu Val Gly Leu 
1               5                   10                  15 

Leu Ala Gly Thr Ala Leu Ala Ala Pro Val Lys Pro Val Val Arg Asn 
            20                  25                  30 

Asn Ala Val Gln Gln Lys Asn Tyr Gly Leu Leu Thr Pro Gly Leu Phe 
        35                  40                  45 

Lys Lys Val Gln Arg Met Asn Trp Asn Gln Glu Val Asp Thr Val Ile 
    50                  55                  60 

Met Phe Gly Ser Tyr Gly Asp Arg Asp Arg Ala Val Lys Val Leu Arg 
65                  70                  75                  80 

Leu Met Gly Ala Gln Val Lys Tyr Ser Tyr Lys Ile Ile Pro Ala Val 
                85                  90                  95 

Ala Val Lys Ile Lys Ala Arg Asp Leu Leu Leu Ile Ala Gly Met Ile 
            100                 105                 110 

Asp Thr Gly Tyr Phe Gly Asn Thr Arg Val Ser Gly Ile Lys Phe Ile 
        115                 120                 125 

Gln Glu Asp Tyr Lys Val Gln Val Asp Asp Ala Thr Ser Val Ser Gln 
    130                 135                 140 

Ile Gly Ala Asp Thr Val Trp Asn Ser Leu Gly Tyr Asp Gly Ser Gly 
145                 150                 155                 160 

Val Val Val Ala Ile Val Asp Thr Gly Ile Asp Ala Asn His Pro Asp 
                165                 170                 175 

Leu Lys Gly Lys Val Ile Gly Trp Tyr Asp Ala Val Asn Gly Arg Ser 
            180                 185                 190 

Thr Pro Tyr Asp Asp Gln Gly His Gly Thr His Val Ala Gly Ile Val 
        195                 200                 205 

Ala Gly Thr Gly Ser Val Asn Ser Gln Tyr Ile Gly Val Ala Pro Gly 
    210                 215                 220 

Ala Lys Leu Val Gly Val Lys Val Leu Gly Ala Asp Gly Ser Gly Ser 
225                 230                 235                 240 

Val Ser Thr Ile Ile Ala Gly Val Asp Trp Val Val Gln Asn Lys Asp 
                245                 250                 255 

Lys Tyr Gly Ile Arg Val Ile Asn Leu Ser Leu Gly Ser Ser Gln Ser 
            260                 265                 270 

Ser Asp Gly Thr Asp Ser Leu Ser Gln Ala Val Asn Asn Ala Trp Asp 
        275                 280                 285 

Ala Gly Ile Val Val Cys Val Ala Ala Gly Asn Ser Gly Pro Asn Thr 
    290                 295                 300 

Tyr Thr Val Gly Ser Pro Ala Ala Ala Ser Lys Val Ile Thr Val Gly 
305                 310                 315                 320 

Ala Val Asp Ser Asn Asp Asn Ile Ala Ser Phe Ser Ser Arg Gly Pro 
                325                 330                 335 

Thr Ala Asp Gly Arg Leu Lys Pro Glu Val Val Ala Pro Gly Val Asp 
            340                 345                 350 

Ile Ile Ala Pro Arg Ala Ser Gly Thr Ser Met Gly Thr Pro Ile Asn 
        355                 360                 365 

Asp Tyr Tyr Thr Lys Ala Ser Gly Thr Ser Met Ala Thr Pro His Val 
    370                 375                 380 

Ser Gly Val Gly Ala Leu Ile Leu Gln Ala His Pro Ser Trp Thr Pro 
385                 390                 395                 400 

Asp Lys Val Lys Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Ala Pro 
                405                 410                 415 

Lys Glu Ile Ala Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Val Tyr 
            420                 425                 430 

Lys Ala Ile Lys Tyr Asp Asp Tyr Ala Lys Leu Thr Phe Thr Gly Ser 
        435                 440                 445 

Val Ala Asp Lys Gly Ser Ala Thr His Thr Phe Asp Val Ser Gly Ala 
    450                 455                 460 

Thr Phe Val Thr Ala Thr Leu Tyr Trp Asp Thr Gly Ser Ser Asp Ile 
465                 470                 475                 480 

Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Glu Val Asp Tyr Ser Tyr 
                485                 490                 495 

Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro Thr Ala 
            500                 505                 510 

Gly Thr Trp Thr Val Lys Val Val Ser Tyr Lys Gly Ala Ala Asn Tyr 
        515                 520                 525 

Gln Val Asp Val Val Ser Asp Gly Ser Leu Ser Gln Ser Gly Gly Gly 
    530                 535                 540 

Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro Thr Pro Thr Thr Asp Thr 
545                 550                 555                 560 

Gln Thr Phe Thr Gly Ser Val Asn Asp Tyr Trp Asp Thr Ser Asp Thr 
                565                 570                 575 

Phe Thr Met Asn Val Asn Ser Gly Ala Thr Lys Ile Thr Gly Asp Leu 
            580                 585                 590 

Thr Phe Asp Thr Ser Tyr Asn Asp Leu Asp Leu Tyr Leu Tyr Asp Pro 
        595                 600                 605 

Asn Gly Asn Leu Val Asp Arg Ser Thr Ser Ser Asn Ser Tyr Glu His 
    610                 615                 620 

Val Glu Tyr Ala Asn Pro Ala Pro Gly Thr Trp Thr Phe Leu Val Tyr 
625                 630                 635                 640 

Ala Tyr Ser Thr Tyr Gly Trp Ala Asp Tyr Gln Leu Lys Ala Val Val 
                645                 650                 655 

Tyr Tyr Gly 

 
           
             13  
             28  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            13 

agagggatcc atgaaggggc tgaaagct                                        28 

 
           
             14  
             30  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            14 

agaggcatgc gctctagact ctgggagagt                                      30 

 
           
             15  
             1962  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            15 

atgaaggggc tgaaagctct catattagtg attttagttc taggtttggt agtagggagc     60 

gtagcggcag ctccagagaa gaaagttgaa caagtaagaa atgttgagaa gaactatggt    120 

ctgctaacgc caggactgtt cagaaaaatt caaaaattga atcctaacga ggaaatcagc    180 

acagtaattg tatttgaaaa ccatagggaa aaagaaattg cagtaagagt tcttgagtta    240 

atgggtgcaa aagttaggta tgtgtaccat attatacccg caatagctgc cgatcttaag    300 

gttagagact tactagtcat ctcaggttta acagggggta aagctaagct ttcaggtgtt    360 

aggtttatcc aggaagacta caaagttaca gtttcagcag aattagaagg actggatgag    420 

tctgcagctc aagttatggc aacttacgtt tggaacttgg gatatgatgg ttctggaatc    480 

acaataggaa taattgacac tggaattgac gcttctcatc cagatctcca aggaaaagta    540 

attgggtggg tagattttgt caatggtagg agttatccat acgatgacca tggacatgga    600 

actcatgtag cttcaatagc agctggtact ggagcagcaa gtaatggcaa gtacaaggga    660 

atggctccag gagctaagct ggcgggaatt aaggttctag gtgccgatgg ttctggaagc    720 

atatctacta taattaaggg agttgagtgg gccgttgata acaaagataa gtacggaatt    780 

aaggtcatta atctttctct tggttcaagc cagagctcag atggtactga cgctctaagt    840 

caggctgtta atgcagcgtg ggatgctgga ttagttgttg tggttgccgc tggaaacagt    900 

ggacctaaca agtatacaat cggttctcca gcagctgcaa gcaaagttat tacagttgga    960 

gccgttgaca agtatgatgt tataacaagc ttctcaagca gagggccaac tgcagacggc   1020 

aggcttaagc ctgaggttgt tgctccagga aactggataa ttgctgccag agcaagtgga   1080 

actagcatgg gtcaaccaat taatgactat tacacagcag ctcctgggac atcaatggca   1140 

actcctcacg tagctggtat tgcagccctc ttgctccaag cacacccgag ctggactcca   1200 

gacaaagtaa aaacagccct catagaaact gctgatatcg taaagccaga tgaaatagcc   1260 

gatatagcct acggtgcagg tagggttaat gcatacaagg ctataaacta cgataactat   1320 

gcaaagctag tgttcactgg atatgttgcc aacaaaggca gccaaactca ccagttcgtt   1380 

attagcggag cttcgttcgt aactgccaca ttatactggg acaatgccaa tagcgacctt   1440 

gatctttacc tctacgatcc caatggaaac caggttgact actcttacac cgcctactat   1500 

ggattcgaaa aggttggtta ttacaaccca actgatggaa catggacaat taaggttgta   1560 

agctacagcg gaagtgcaaa ctatcaagta gatgtggtaa gtgatggttc cctttcacag   1620 

cctggaagtt caccatctcc acaaccagaa ccaacagtag acgcaaagac gttccaagga   1680 

tccgatcact actactatga caggagcgac acctttacaa tgaccgttaa ctctggggct   1740 

acaaagatta ctggagacct agtgtttgac acaagctacc atgatcttga cctttacctc   1800 

tacgatccta accagaagct tgtagataga tcggagagtc ccaacagcta cgaacacgta   1860 

gaatacttaa cccccgcccc aggaacctgg tacttcctag tatatgccta ctacacttac   1920 

ggttgggctt actacgagct gacggctaaa gtttattatg gc                      1962 

 
           
             16  
             654  
             PRT  
             Pyrococcus furiosus  
           
            16 

Met Lys Gly Leu Lys Ala Leu Ile Leu Val Ile Leu Val Leu Gly Leu 
1               5                   10                  15 

Val Val Gly Ser Val Ala Ala Ala Pro Glu Lys Lys Val Glu Gln Val 
            20                  25                  30 

Arg Asn Val Glu Lys Asn Tyr Gly Leu Leu Thr Pro Gly Leu Phe Arg 
        35                  40                  45 

Lys Ile Gln Lys Leu Asn Pro Asn Glu Glu Ile Ser Thr Val Ile Val 
    50                  55                  60 

Phe Glu Asn His Arg Glu Lys Glu Ile Ala Val Arg Val Leu Glu Leu 
65                  70                  75                  80 

Met Gly Ala Lys Val Arg Tyr Val Tyr His Ile Ile Pro Ala Ile Ala 
                85                  90                  95 

Ala Asp Leu Lys Val Arg Asp Leu Leu Val Ile Ser Gly Leu Thr Gly 
            100                 105                 110 

Gly Lys Ala Lys Leu Ser Gly Val Arg Phe Ile Gln Glu Asp Tyr Lys 
        115                 120                 125 

Val Thr Val Ser Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln 
    130                 135                 140 

Val Met Ala Thr Tyr Val Trp Asn Leu Gly Tyr Asp Gly Ser Gly Ile 
145                 150                 155                 160 

Thr Ile Gly Ile Ile Asp Thr Gly Ile Asp Ala Ser His Pro Asp Leu 
                165                 170                 175 

Gln Gly Lys Val Ile Gly Trp Val Asp Phe Val Asn Gly Arg Ser Tyr 
            180                 185                 190 

Pro Tyr Asp Asp His Gly His Gly Thr His Val Ala Ser Ile Ala Ala 
        195                 200                 205 

Gly Thr Gly Ala Ala Ser Asn Gly Lys Tyr Lys Gly Met Ala Pro Gly 
    210                 215                 220 

Ala Lys Leu Ala Gly Ile Lys Val Leu Gly Ala Asp Gly Ser Gly Ser 
225                 230                 235                 240 

Ile Ser Thr Ile Ile Lys Gly Val Glu Trp Ala Val Asp Asn Lys Asp 
                245                 250                 255 

Lys Tyr Gly Ile Lys Val Ile Asn Leu Ser Leu Gly Ser Ser Gln Ser 
            260                 265                 270 

Ser Asp Gly Thr Asp Ala Leu Ser Gln Ala Val Asn Ala Ala Trp Asp 
        275                 280                 285 

Ala Gly Leu Val Val Val Val Ala Ala Gly Asn Ser Gly Pro Asn Lys 
    290                 295                 300 

Tyr Thr Ile Gly Ser Pro Ala Ala Ala Ser Lys Val Ile Thr Val Gly 
305                 310                 315                 320 

Ala Val Asp Lys Tyr Asp Val Ile Thr Ser Phe Ser Ser Arg Gly Pro 
                325                 330                 335 

Thr Ala Asp Gly Arg Leu Lys Pro Glu Val Val Ala Pro Gly Asn Trp 
            340                 345                 350 

Ile Ile Ala Ala Arg Ala Ser Gly Thr Ser Met Gly Gln Pro Ile Asn 
        355                 360                 365 

Asp Tyr Tyr Thr Ala Ala Pro Gly Thr Ser Met Ala Thr Pro His Val 
    370                 375                 380 

Ala Gly Ile Ala Ala Leu Leu Leu Gln Ala His Pro Ser Trp Thr Pro 
385                 390                 395                 400 

Asp Lys Val Lys Thr Ala Leu Ile Glu Thr Ala Asp Ile Val Lys Pro 
                405                 410                 415 

Asp Glu Ile Ala Asp Ile Ala Tyr Gly Ala Gly Arg Val Asn Ala Tyr 
            420                 425                 430 

Lys Ala Ile Asn Tyr Asp Asn Tyr Ala Lys Leu Val Phe Thr Gly Tyr 
        435                 440                 445 

Val Ala Asn Lys Gly Ser Gln Thr His Gln Phe Val Ile Ser Gly Ala 
    450                 455                 460 

Ser Phe Val Thr Ala Thr Leu Tyr Trp Asp Asn Ala Asn Ser Asp Leu 
465                 470                 475                 480 

Asp Leu Tyr Leu Tyr Asp Pro Asn Gly Asn Gln Val Asp Tyr Ser Tyr 
                485                 490                 495 

Thr Ala Tyr Tyr Gly Phe Glu Lys Val Gly Tyr Tyr Asn Pro Thr Asp 
            500                 505                 510 

Gly Thr Trp Thr Ile Lys Val Val Ser Tyr Ser Gly Ser Ala Asn Tyr 
        515                 520                 525 

Gln Val Asp Val Val Ser Asp Gly Ser Leu Ser Gln Pro Gly Ser Ser 
    530                 535                 540 

Pro Ser Pro Gln Pro Glu Pro Thr Val Asp Ala Lys Thr Phe Gln Gly 
545                 550                 555                 560 

Ser Asp His Tyr Tyr Tyr Asp Arg Ser Asp Thr Phe Thr Met Thr Val 
                565                 570                 575 

Asn Ser Gly Ala Thr Lys Ile Thr Gly Asp Leu Val Phe Asp Thr Ser 
            580                 585                 590 

Tyr His Asp Leu Asp Leu Tyr Leu Tyr Asp Pro Asn Gln Lys Leu Val 
        595                 600                 605 

Asp Arg Ser Glu Ser Pro Asn Ser Tyr Glu His Val Glu Tyr Leu Thr 
    610                 615                 620 

Pro Ala Pro Gly Thr Trp Tyr Phe Leu Val Tyr Ala Tyr Tyr Thr Tyr 
625                 630                 635                 640 

Gly Trp Ala Tyr Tyr Glu Leu Thr Ala Lys Val Tyr Tyr Gly 
                645                 650 

 
           
             17  
             25  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            17 

tctgaattcg ttcttttctg tatgg                                           25 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            18 

tgtactgctg gatccggcag                                                 20 

 
           
             19  
             30  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            19 

agaggcatgc gtatccatca gatttttgag                                      30 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            20 

agtgaacgga tacttggaac                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            21 

gttccaagta tccgttcact                                                 20 

 
           
             22  
             12  
             PRT  
             Pyrococcus furiosus  
           
            22 

Ala Glu Leu Glu Gly Leu Asp Glu Ser Ala Ala Gln 
1               5                   10 

 
           
             23  
             24  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            23 

tcatggatcc accctctcct ttta                                            24 

 
           
             24  
             46  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            24 

gtctgcgcag gctgccggan nnnnnatgaa ggggctgaaa gctctc                    46 

 
           
             25  
             49  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            25 

gagagctttc agccccttca tnnnnnntcc ggcagcctgc gcagacatg                 49 

 
           
             26  
             27  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            26 

agagggggat ccgtgagaag caaaaaa                                         27 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            27 

gatgactagt aagtctctaa                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            28 

aagcctgagg ttgttgctcc                                                 20 

 
           
             29  
             29  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            29 

gggcatgctc atgaacttcc aggctgtga                                       29 

 
           
             30  
             4  
             PRT  
             Artificial Sequence  
             
               Synthetic  
             
           
            30 

Ala Gly Gly Asn 
1 

 
           
             31  
             382  
             PRT  
             Bacillus subtilis  
           
            31 

Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu 
1               5                   10                  15 

Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly 
            20                  25                  30 

Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met 
        35                  40                  45 

Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly 
    50                  55                  60 

Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr 
65                  70                  75                  80 

Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala 
                85                  90                  95 

Tyr Val Glu Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro 
            100                 105                 110 

Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr 
        115                 120                 125 

Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser 
    130                 135                 140 

Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser 
145                 150                 155                 160 

Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala 
                165                 170                 175 

Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala 
            180                 185                 190 

Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 
        195                 200                 205 

Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 
    210                 215                 220 

Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala 
225                 230                 235                 240 

Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val 
                245                 250                 255 

Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val 
            260                 265                 270 

Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp 
        275                 280                 285 

Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Pro Glu Leu Asp 
    290                 295                 300 

Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys 
305                 310                 315                 320 

Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly 
                325                 330                 335 

Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln 
            340                 345                 350 

Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe 
        355                 360                 365 

Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln 
    370                 375                 380 

 
           
             32  
             4  
             PRT  
             Artificial Sequence  
             
               Synthetic  
             
           
            32 

Leu Leu Val Tyr 
1 

 
           
             33  
             4  
             PRT  
             Artificial Sequence  
             
               Synthetic  
             
           
            33 

Ala Ala Pro Phe 
1