There is disclosed a hyperthermostable protease gene originating in Pyrococcus furiosus, in particular, a hyperthermostable protease gene encoding the amino acid sequence represented by the SEQ ID NO 1 in the Sequence Listing or a part thereof which retains the activity of the hyperthermostable protease. There is also disclosed a process for producing the protease by culturing a transformant transformed with a plasmid into which the above gene has been inserted.

FIELD OF THE INVENTION 
The present invention relates to a gene encoding a hyperthermostable 
protease which is useful as an enzyme for industrial application and a 
process for producing the enzyme by genetic engineering. 
BACKGROUND OF THE INVENTION 
Proteases are enzymes which cleave peptide bonds in proteins and various 
proteases have been found in animals, plants and microorganisms. They are 
used not only as reagents for research works and medical supplies, but 
also in industrial fields such as additives for detergents, food 
processing and chemical syntheses utilizing their reverse reactions and it 
can be said that they are very important enzymes from an industrial 
viewpoint. For proteases to be used in industrial fields, since very high 
physical and chemical stabilities are required, in particular, enzymes 
having high thermostability are preferred to use. At present, proteases 
predominantly used in industrial fields are those produced by bacteria of 
the genus Bacillus because they have relatively high thermostabilities. 
However, enzymes having further superior properties are desired and 
activities have been attempted to obtain enzymes from microorganisms which 
can grow at high temperatures, for example, thermophiles of the genus 
Bacillus. 
On the other hand, a group of microorganisms, named as hyperthermophiles, 
are well adapted themselves to high temperature environment and therefore 
they are expected to be supply sources for various thermostable enzymes. 
It has been known that one of these hyperthermophiles, Pyrococcus 
furiosus, produces proteases Appl. Environ. Microbiol., 56, 1992-1998 
(1990); FEMS Microbiol. Letters, 71, 17-20 (1990); J. Gen. Microbiol., 
137, 1193-1199 (1991)!. 
In addition, as for hyperthermophiles of the genera Thermococcus, 
Staphylothermus and Thermobacteroides, the production of proteases have 
also been known Applied Microbiology and Biotechnology, 34, 715-719 
(1991)!. 
OBJECTS OF THE INVENTION 
Since proteases produced by these hyperthermophiles have high 
thermostabilities, they are expected to be applicable to new applications 
to which any known enzyme has not been utilized. However, the above 
publications merely teach that thermostable protease activities are 
present in cell-free extracts or crude enzyme solutions obtained from 
culture supernatants and there is no disclosure about properties of 
isolated and purified enzymes and the like. Moreover, since a cultivation 
of microorganisms at high temperature is required to obtain enzymes from 
these hyperthermophiles, there is a problem in industrial production of 
the enzymes. 
In order to solve the above problems, an object of the present invention is 
to isolate a gene encoding a protease of a hyperthermophile. Another 
object of the present invention is to provide a process for producing the 
protease by genetic engineering using the gene.

DISCLOSURE OF THE INVENTION 
In order to obtain a hyperthermostable protease gene, the present inventors 
attempted to purify a protease from microbial cells and a culture 
supernatant of Pyrococcus furiosus DSM 3638 so as to determine a partial 
amino acid sequence of the enzyme, independently. However, purification of 
the protease was very difficult in either case of using the microbial 
cells or the culture supernatant and the present inventors failed to 
obtain an enzyme sample having sufficient purity for determination of its 
partial amino acid sequence. 
As a method for cloning a gene for an objective enzyme without any 
information about a primary structure of the enzyme, there is an 
expression cloning method and, for example, a pullulanase gene originating 
in Pyrococcus woesei has been obtained according to this method (WO 
92/02614). However, in an expression cloning method, a plasmid vector is 
generally used and, in such case, it is necessary to use restriction 
enzymes which can cleave an objective gene into relatively small DNA 
fragments so that the fragments can be inserted into the plasmid vector 
without cleavage of any internal portion of the objective gene. Then, the 
method is not always applicable to cloning of all kinds of enzyme genes. 
Furthermore, it is necessary to test for an enzyme activity of a large 
number of clones and this operation is complicated. 
The present inventors have attempted to isolate a protease gene by using a 
cosmid vector which can maintain a larger DNA fragment (35-50 kb) instead 
of a plasmid vector to prepare a cosmid library of Pyrococcus furiosus 
genome and investigating cosmid clones in the library to find out a clone 
expressing a protease activity. By using a cosmid vector, the number of 
transformants to be screened can be reduced in addition to lowering of 
possibilities of cleavage of an internal portion of the enzyme gene. On 
the other hand, since the copy number of a cosmid vector in a host is not 
higher than that of a plasmid vector, it may be that an amount of the 
enzyme expressed is too small to detect its enzyme activity. 
In view of high thermostability of the objective enzyme, firstly, the 
present inventors have cultured respective transformants in a cosmid 
library, separately, and have combined this step with a step for preparing 
lysates containing ing only thermostable proteins from the microbial cells 
thus obtained. This group of lysates have been named as a cosmid protein 
library. By using the cosmid protein library in detection of the enzyme 
activity, detection sensitivity can be increased higher than that of a 
method using transformant colonies. 
In addition, the present inventors have made possible to detect a trace 
amount of the enzyme activity by performing SDS-polyacrylamide 
gel-electrophoresis with a gel containing gelatin. According to this 
method, a trace amount of a protease activity contained in a sample can be 
detected with high sensitivity as a band concentrated in the gel. 
In this manner, the present inventors have screened a cosmid protein 
library originating in Pyrococcus furiosus and have obtained several 
cosmid clones which express the protease activity. 
Furthermore, the present inventors have succeeded in isolation of 
hyperthermostable protease genes from inserted DNA fragments contained in 
the clones by utilizing various gene engineering techniques and also have 
found that products expressed from the genes are resistant to surfactants. 
By comparing an amino acid sequence of the hyperthermostable protease 
deduced from the nucleotide sequence of the gene with amino acid sequences 
of known proteases originating in microorganisms, homology of the amino 
acid sequence of the front half portion of the protease encoded by the 
gene with those of a group of alkaline proteases, whose representative 
example is subtilisin, has been shown and, in particular, very high 
homology has been found at each region around the four amino acid residues 
which are known to be of importance for a catalytic activity of the 
enzymes. Thus, since the protease produced by Pyrococcus furiosus, which 
is active at such high temperatures that proteases originating in 
mesophiles are readily inactivated, has been shown to retain a structure 
similar to those of enzymes from mesophiles, it has been suggested that 
similar proteases would also be produced by hyperthermophiles other than 
Pyrococcus furiosus. 
Then, the present inventors have noted possibilities that, in the 
nucleotide sequence of the hyperthermostable protease gene obtained, the 
nucleotide sequences encoding regions showing high homology with 
subtilisin and the like can be used as probes for investigating 
hyperthermostable protease genes and have attempted to detect protease 
genes originating in hyperthermophiles by PCR using synthetic DNA designed 
based on the above nucleotide sequences as primers so as to clone DNA 
fragments containing the protease genes. As a result, the present 
inventors have found a protease gene in a hyperthermophile, Thermococcus 
celer DSM2476, and have obtained a DNA fragment containing the gene. 
Furthermore, the present inventors have confirmed that an amino acid 
sequence encoded by the DNA fragment contains amino acids sequences having 
high homology with the amino acid sequences of the hyperthermostable 
protease represented by SEQ NO 1 of the Sequence Listing. Thus, the 
present inventors have completed the present invention. 
That is, the present invention provides an isolated hyperthermostable 
protease genes originating in Pyrococcus furiosus, in particular, a 
hyperthermostable protease gene which comprises the amino acid sequence 
represented by SEQ ID NO 1 in the Sequence Listing or a part thereof 
encoding the active portion of the hyperthermostable protease, especially, 
the hyperthermostable protease gene having the DNA sequence represented by 
SEQ ID NO 2 in the Sequence Listing. 
In addition, the present invention provides hyperthermostable protease 
genes hybridizable with the above hyperthermostable protease genes. For 
example, there is provided a hyperthermostable protease gene containing 
the nucleotide sequence represented by SEQ ID NO 7 in the Sequence 
Listing. 
Moreover, the present invention provides a process for producing the 
hyperthermostable protease which comprising culturing a transformant 
transformed with a recombinant plasmid into which the hyperthermostable 
protease gene of the present invention has been inserted, and collecting 
the hyperthermostable protease from the culture. 
The hyperthermostable protease genes of the present invention can be 
obtained by screening of gene libraries of hyperthermophiles. As the 
hyperthermophiles, bacteria belonging to the genus Pyrococcus can be used 
and the desired genes can be obtained by screening a cosmid library of 
Pyrococcus furiosus genome. 
For example, Pyrococcus furiosus DSM3638 can be used as Pyrococcus furiosus 
and this strain is available from Deutsch Sammlung von Microorganismen und 
Zellkulturen GmbH. 
One example of cosmid libraries of Pyrococcus furiosus can be obtained by 
partially digesting the genomic DNA of Pyrococcus furiosus DSM3638 with a 
restriction enzyme, Sau3AI (manufactured by Takara Shuzo, Co., Ltd.), to 
obtain DNA fragments, ligating the DNA fragments with a triple helix 
cosmid vector (manufactured by Stratagene) and packaging into lambda phage 
particles by in vitro packaging method. Then, the library is transduced 
into a suitable E. coli, for example, E. coli DH5.alpha.MCR (manufactured 
by BRL) to obtain transformants, followed by culturing them, collecting 
the microbial cells, subjecting them to heat treatment (100.degree. C. for 
10 minutes), sonicating and subjecting heat treatment (100.degree. C. for 
10 minutes) again. The lysates thus obtained can be subjected to screening 
for the protease activity by performing SDS-polyamide gel-electrophoresis 
with a gel containing gelatin. 
In this manner, a cosmid clone containing a hyperthermostable protease gene 
capable of expressing a protease which is resistant to the above heat 
treatment can be obtained. 
Furthermore, a cosmid DNA prepared from the above-obtained cosmid clone can 
be digested with suitable restriction enzymes to form fragments to prepare 
recombinant plasmids into which the respective fragments thus obtained are 
inserted. A recombinant plasmid containing the desired hyperthermostable 
protease gene can be obtained by transforming a suitable microorganism 
with the above-obtained plasmids and testing for the protease activity 
expressed by the resultant transformants. 
That is, a cosmid DNA prepared from one of the above-obtained cosmid clones 
can be digested with SphI (manufactured by Takara Shuzo, Co., Ltd.), 
followed by inserting the resultant DNA fragment into SphI site of a 
plasmid vector, pUC119 (manufactured by Takara Shuzo, Co., Ltd.) to obtain 
a recombinant plasmid. Then, the recombinant plasmid is introduced into E. 
coli JM109 (manufactured by Takara Shuzo, Co., Ltd.) and the protease 
activity of the resultant transformant is tested by the same method as 
that used for screening of the cosmid protein library. The transformant 
having the activity is used for preparation of a plasmid. 
As is seen from Examples hereinafter, one of the recombinant plasmids has 
been named as pTPR1 and E. coli JM109 transformed with the plasmid has 
been named as Escherichia coli JM109/pTPR1. FIG. 1 illustrates a 
restriction map of the plasmid pTPR1. In FIG. 1, the thick solid line 
represents the DNA fragment inserted into the plasmid vector pUC119. The 
recombinant plasmid contains SphI fragment of about 7.0 kb. 
In addition, a DNA fragment of about 2.5 kb which does not contain the 
hyperthermostable protease gene can be removed from the recombinant 
plasmid. That is, among three fragments of about 2.5 kb, about 3.3 kb and 
about 4.3 kb obtained by digesting the above plasmid pTPR1 with XbaI 
(manufactured by Takara Shuzo, Co., Ltd.), only the DNA fragment of about 
2.5 kb is removed and the remaining fragments are ligated and introduced 
into E. coli JM109. The protease activity of the resultant transformant is 
tested by the same method as that used for screening of the cosmid protein 
library. The resultant transformant having the protease activity is used 
for preparation of a plasmid. The plasmid has been named as pTPR9 and E. 
coli JM109 transformed with the plasmid has been named as Escherichia coli 
JM109/pTPR9. FIG. 2 illustrates a restriction map of the plasmid pTPR9. In 
FIG. 2, the thick solid line represents the DNA fragment inserted into the 
plasmid vector pUC119. 
The protease activities expressed by both plasmids pTPR1 and pTPR9 show 
high thermostability. However, since the activities are observed at 
positions different from that for the protease activity expressed by above 
cosmid clone on a SDS-polyacrylamide gel containing gelatin, these 
plasmids are estimated to be defect in a part of the protease gene on the 
cosmid DNA. A DNA fragment containing the whole length of the protease 
gene can be obtained from the cosmid DNA by, for example, using a part of 
the inserted DNA fragment of the above plasmid pTPR9 as a probe. That is, 
the cosmid DNA used for preparation of the plasmid pTPR1 is digested with 
NotI (manufactured by Takara Shuzo, Co., Ltd.) and several restriction 
enzymes which do not cleave any internal portion of the DNA fragment 
inserted into the plasmid pTPR1. After agarose gel-electrophoresis, the 
DNA fragments in the gel are blotted on a nylon membrane. Regarding the 
membrane thus obtained, hybridization is carried out by using a PstI-XbaI 
fragment of about 0.7 kb obtained from the DNA fragment inserted into the 
plasmid pTPR9 as a probe to detect a DNA fragment containing the same 
sequence as that of the PstI-XbaI fragment. 
In the cosmid DNA digested with two enzymes, NotI and PvuII (manufactured 
by Takara Shuzo Co., Ltd.), a DNA fragment of about 7.5 kb is hybridized 
with the PstI-XbaI fragment. This fragment of about 7.5 kb can be isolated 
to insert into a plasmid vector, pUC19 (manufactured by Takara Shuzo, Co., 
Ltd.) into which NotI linker (manufactured by Takara Shuzo Co., Ltd.) has 
been introduced at a HincII site, at a site between NotI and SmaI. The 
plasmid has been named as pTPR12 and E. coli JM109 transformed by the 
plasmid has been named and indicated as Escherichia coli JM109/pTPR12. 
This strain has been deposited with National Institute of Bioscience and 
Human-Technology (NIBH), Agency of Industrial Science & Technology, 
Ministry of International Trade & Industry under the accession number of 
FERM BP-5103 under Budapest Treaty since May 24, 1994 (the date of the 
original deposit). 
A lysate of Escherichia coli JM109/pTPR12 shows the protease activity 
similar to that of the cosmid clone on a SDS-polyacrylamide gel containing 
gelatin. FIG. 3 illustrates a restriction map of the plasmid pTPR12. In 
FIG. 3, the thick solid line is the DNA fragment inserted into the plasmid 
vector pUC19. 
FIG. 4 illustrates restriction maps of the DNA fragments originating in 
Pyrococcus furiosus which are inserted into the plasmids pTPR1, pTPR9 and 
pTPR12, respectively. According to FIG. 4, a fragment of about 1 kb which 
does not contain a hyperthermostable protease gene can be removed from the 
DNA fragment inserted into the plasmid pTPR12. That is, the plasmid pTPR12 
is digested with XbaI and KpnI (manufactured by Takara Shuzo, Co., Ltd.) 
and thus-obtained XbaI-XbaI fragment of about 3.3 kb and XbaI-KpnI 
fragment of about 3.2 kb are isolated, respectively. Then, firstly, the 
XbaI-KpnI fragment of about 3.2 kb is inserted into the plasmid vector 
pUC19 at a site between XbaI and KpnI to prepare a recombinant plasmid. 
This plasmid has been named as pTPR14 and FIG. 5 illustrates its 
restriction map. In FIG. 5, the thick solid line represents the DNA 
fragment inserted into the plasmid vector pUC19. 
Then, the above XbaI-XbaI fragment of about 3.3 kb is inserted into the 
plasmid pTPR14 at XbaI site and introduced into E. coli JM109. The 
protease activity of the transformant is tested by using the method used 
for screening the cosmid protein library. A plasmid is prepared by the 
transformant having the activity. The plasmid has been named as pTPR15 and 
E. coli JM109 transformed with the plasmid has been named as Escherichia 
coli JM109/pTPR15. FIG. 6 illustrates a restriction map of pTPR15. In FIG. 
6, the thick solid line represents the DNA fragment inserted into the 
plasmid vector pUC19. 
Further, in the nucleotide sequences of the DNA fragment originating in 
Pyrococcus furiosus and inserted into the plasmid pTPR15, the nucleotide 
sequence of the DNA fragment of about 4.8 kb between two DraI sites are 
shown as SEQ ID NO 8 in the Sequence Listing. That is, SEQ ID NO 8 of the 
Sequence Listing is an example of the nucleotide sequence of the 
hyperthermostable protease gene of the present invention. And, an amino 
acid sequence of a product of the gene deduced from the nucleotide 
sequence of SEQ ID NO 8 is shown as SEQ ID NO 9 in the Sequence Listing. 
That is, SEQ ID NO 9 in the Sequence Listing is an example of the amino 
acid sequence of an enzyme protein produced by using the hyperthermostable 
protease gene obtained according to the present invention. 
Because it has been found that the hyperthermostable protease gene of the 
present invention is contained in DraI fragment of about 4.8 kb in the DNA 
fragment inserted into the above plasmid pTPR15, a recombinant plasmid 
containing only this DraI fragment can be prepared. 
That is, the above plasmid pTPR15 is digested with DraI (manufactured by 
Takara Shuzo Co., Ltd.) to isolate the resultant DNA fragment of about 4.8 
kb. Then, it can be inserted into the plasmid vector pUC19 at SmaI site to 
prepare a recombinant plasmid. The recombinant plasmid has been named as 
pTPR13 and E. coli JM109 transformed with the plasmid has been named as 
Escherichia coli JM109/pTPR13. 
A lysate of Escherichia coli JM109/pTPR13 shows the same protease activity 
as that of the cosmid clone on a SDS-polyacrylamide gel containing 
gelatin. FIG. 7 illustrates a restriction map of the plasmid pTPR13. In 
FIG. 7, the thick solid line represents the DNA fragment inserted into the 
plasmid vector pUC19. 
In addition, the hyperthermostable protease gene of the present invention 
can be expressed in Bacillus subtilis. As the Bacillus subtilis, Bacillus 
subtilis DB104 can be used and this strain is a known strain described in 
Gene, Vol. 83, pp. 215-233 (1989). As a cloning vector, a plasmid 
pUB18-P43 can be used and this plasmid has been given by Dr. Sui-Lam Wong 
of Calgary University. This plasmid contains a kanamycin resistant gene as 
a selection marker. 
The above-described plasmid pTPR13 can be digested with KpnI (manufactured 
by Takara Shuzo Co., Ltd.) and BamHI (manufactured by Takara Shuzo Co., 
Ltd.) to obtain a DNA fragment of about 4.8 kb, followed by isolating and 
ligating the fragment between KpnI site and BamHI site of the plasmid 
pUB18-P43 to prepare a recombinant plasmid. The plasmid has been named as 
pUBP13 and Bacillus subtilis DB104 transformed with the plasmid has been 
named as Bacillus subtilis DB104/pUBP13. A lysate of Bacillus subtilis 
DB104/pUBP13 shows the same protease activity as that of the cosmid clone 
on a SDS-polyacrylamide gel containing gelatin. FIG. 8 illustrates a 
restriction map of the plasmid pUBP13. In Fig. 8, the thick solid line 
represents the DNA fragment inserted into the plasmid vector pUB18-P43. 
By comparing the amino acid sequence shown by SEQ ID NO 9 of the Sequence 
Listing with amino acid sequences of proteases originating in known 
microorganisms, it is shown that there is homology between the front half 
portion of the sequence of the hyperthermostable protease of the present 
invention and those of a group of alkaline serine proteases whose 
representative example is subtilisin Protein Engineering, Vol. 4, pp. 
719-737 (1991)!, in particular, there is high homology between each region 
around the four amino acid residues which are known to be of importance 
for protease activity. On the other hand, such homology cannot be observed 
between the back half portions of the amino acid sequences and it is 
considered that this portion may not be essential to a protease activity. 
Therefore, a mutant protease wherein an appropriate peptide chain is 
removed from its back half portion is expected to show the enzymatic 
activity. Examples of such mutant protease include a protease having an 
amino acid sequence corresponding to SEQ ID. NO 9 of the Sequence Listing 
from which the 904th amino acid, Ser, and the subsequent sequence has been 
removed. This can be prepared by the following process. 
Firstly, a KpnI-EcoRI fragment of about 2.8 kb wherein the EcoRI site is 
blunted is prepared from the above plasmid pTPR13 and the fragment is 
ligated between the KpnI site and the blunted XbaI site of the plasmid 
vector pUC119. A protease gene contained in the recombinant plasmid thus 
obtained encodes an amino acid sequence corresponding to the SEQ ID NO 9 
of the Sequence Listing except that the nucleotide sequence TCA encoding 
the 904th amino acid, Ser, has been replaced with the termination codon 
TAG and the subsequent nucleotide sequence has been deleted. The plasmid 
has been named as pTPR36 and E. coli JM109 transformed with the plasmid 
has been named Escherichia coli JM109/pTPR36. A lysate of Escherichia coli 
JM109/pTPR36 shows an protease activity on a SDS-polyacrylamide gel 
containing gelatin. FIG. 9 illustrates a restriction map of the plasmid 
pTPR36. In FIG. 9, the thick solid line represents the DNA fragment 
inserted into the plasmid vector pUC119. SEQ ID NO 2 in the Sequence 
Listing is a nucleotide sequence of the open reading frame contained in 
the DNA fragment inserted in the plasmid pTPR36. That is, SEQ ID NO 2 of 
the Sequence Listing is an example of nucleotide sequences of the 
hyperthermostable protease genes obtained in the present invention. In 
addition, SEQ ID NO 1 of the Sequence Listing is an amino acid sequence of 
the gene product deduced from the nucleotide sequence of SEQ ID NO 2. That 
is, SEQ ID NO 1 of the Sequence Listing is an example of amino acid 
sequences of enzyme proteins produced by using hyperthermostable protease 
genes obtained by the present invention. 
As described above, it has been found that the regions commonly present in 
alkaline serine proteases originating in mesophiles are conserved in the 
amino acid sequence of the hyperthermostable protease produced by the 
hyperthermophile Pyrococcus furiosus. Therefore, the presence of the 
regions is expected in the same kind of proteases produced by 
hyperthermophiles other than Pyrococcus furiosus. That is, it is possible 
to obtain genes for hyperthermostable proteases similar to the 
above-described hyperthermostable protease by preparing suitable synthetic 
DNA fragments based on parts of the nucleotide sequence of SEQ ID NO 2 of 
the Sequence Listing which encode amino acid sequences having high 
homology with those of subtilisin and the like, and using them as probes 
or primers. 
FIGS. 10, 11 and 12 illustrate the relation among the amino acid sequences 
of regions in the amino acid sequence of the hyperthermostable protease of 
the present invention which have high homology with those of subtilisin 
and the like, the nucleotide sequences of the hyperthermostable protease 
gene of the present invention which encode the regions, and the nucleotide 
sequences of oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4R 
synthesized based on the above sequences, respectively. In addition, SEQ 
ID NO 3, 4, 5 and 6 of the Sequence Listing illustrate the nucleotide 
sequences of the oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4R. That 
is, SEQ ID NO 3, 4, 5 and 6 of the Sequence Listing are examples of 
oligonucleotides which can be used for detection of the hyperthermostable 
protease genes of the present invention by hybridization. 
A combination of above oligonucleotides can be used as primers to carry out 
PCR using genomic DNA of various hyperthermophiles as templates to detect 
protease genes present in hyperthermophiles. As the hyperthermophiles, 
bacteria belonging to the genera Pyrococcus, Thermococcus, 
Staphylothermus, Thermobacteroides and the like can be used. As bacteria 
belonging to the genus Thermococcus, Thermococcus celer DSM2476 can be 
used and the strain is available from Deutsch Sammlung von Microorganismen 
und Zellkulturen GmbH. When PCR is carried out by using genomic DNA of 
Thermococcus celer DSM2476 as a template and a combination of the above 
oligonucleotides PRO-1F and PRO-2R or a combination of PRO-2F and PRO-4R 
as primers, specific amplification of DNA fragments is observed and the 
presence of a protease gene can be indicated. In addition, an amino acid 
sequence encoded by the fragment can be estimated by ligating the fragment 
to suitable plasmid vector to prepare a recombinant plasmid and 
determining the nucleotide sequence of the inserted DNA fragment by 
dideoxy method. 
A DNA fragment of about 150 bp amplified by using the oligonucleotides 
PRO-1F and PRO-2R and a DNA fragment of about 550 bp amplified by using 
the oligonucleotides PRO-2F and PRO-4R are ligated to HincII site of the 
plasmid vector pUC18 to obtain recombinant plasmids, respectively. The 
recombinant plasmids have been named as p1F-2R(2) and p2F-4R, 
respectively. SEQ ID NO 10 of the Sequence Listing illustrates the 
nucleotide sequence of the DNA fragment inserted into the plasmid 
p1F-2R(2) and an amino acid sequence deduced therefrom. SEQ ID NO 11 of 
the Sequence Listing illustrates the nucleotide sequence of the DNA 
fragment inserted into the plasmid p2F-4R and an amino acid sequence 
deduced therefrom. In the nucleotide sequence shown by SEQ ID NO 10 of the 
Sequence Listing, the sequence from the 1st to the 21st nucleotides and 
the sequence from the 113th to the 145th nucleotides and, in the 
nucleotide sequence shown by SEQ ID NO 11 of the Sequence Listing, the 
sequence from the 1st to the 32nd nucleotides and the sequence from the 
532nd to the 564th nucleotides are the nucleotide sequences derived from 
the oligonucleotides used as the primers (corresponding to the 
oligonucleotides PRO-1F, PRO-2R, PRO-2F and PRO-4R, respectively). In the 
amino acid sequences shown by SEQ ID NO 10 and 11, there are sequences 
having homology with amino acid sequences of the hyperthermostable 
protease originating in Pyrococuss furiosus of the present invention as 
well as alkaline serine protease originating in various microorganisms and 
it has been shown that the above DNA fragments amplified by PCR are those 
amplified utilizing the protease gene as the template. 
FIG. 13 illustrates a restriction map of the plasmid p2F-4R. In FIG. 13, 
the thick solid line represents the DNA fragment inserted into the plasmid 
vector pUC18. 
On the other hand, when genomic DNA of Thermobacteroides proteoliticus 
DSM5265 and Staphylothermus marinus DSM3639 are used as templates, 
amplification as observed in case of Thermococcus celer has not been 
recognized. 
It has been known that efficiency of gene amplification by PCR is 
influenced by annealing efficiency of a 3'-terminal portion of a primer 
and a template DNA. Even when amplification of DNA fragment is not 
observed in the above PCR, protease genes can be detected by synthesizing 
oligonucleotides having different sequences but encoding the same amino 
acid sequence and using them as primers. In addition, protease genes can 
also be detected by using these oligonucleotides as probes and carrying 
out Southern hybridization with genomic DNA of various hyperthermophiles. 
Then, the above-described oligonucleotides or amplified DNA fragments 
obtained by the above PCR can be used as probes for screening genomic DNA 
libraries of hyperthermophiles to obtain hyperthermostable protease genes, 
for example, the hyperthermostable protease gene produced by Thermococcus 
celer. 
As an example of genomic DNA libraries of Thermococcus celer, there is a 
library prepared by partially digesting a genomic DNA of Thermococcus 
celer DSM2476 with a restriction enzyme Sau3AI to obtain a DNA fragment, 
ligating the fragment with lambda GEM-11 vector (manufactured by Promega) 
and packaging it into lambda phage particles according to in vitro 
packaging method. Then, the library is transduced into a suitable E. coli, 
for example, E. coli LE392 (manufactured by Promega) to form plaques on a 
plate and then plaque hybridization is carried out by using amplified DNA 
fragments obtained in the above-described PCR. In this manner, phage 
clones containing hyperthermostable protease genes can be obtained. 
Further, the phage DNA prepared from the clone thus obtained is digested 
with suitable restriction enzymes and, after subjecting to agarose 
gel-electrophoresis, DNA fragments in the gel are blotted on a nylon 
membrane. Regarding the membrane thus obtained, hybridization is carried 
out using amplified DNA fragments obtained according to the above PCR as 
probes to detect a DNA fragments containing the protease gene. 
When the above phage DNA is digested with KpnI, a DNA fragment of about 9 
kb is hybridized with the probe and this fragment of about 9 kb can be 
isolated and inserted into KpnI site of the plasmid vector pUC119 to 
obtain a recombinant plasmid. This plasmid has been named as pTC1 and E. 
coli JM109 transformed with this plasmid has been named as Escherichia 
coli JM109/pTC1. 
FIG. 14 illustrates a restriction map of the plasmid pTC1. In FIG. 14, the 
thick solid line represents the DNA fragment inserted into the plasmid 
vector pUC119. 
Furthermore, a DNA fragment of about 4 kb which does not contain the 
hyperthermostable protease gene can be removed from the plasmid pTC1. That 
is, plasmid pTC1 is digested with KpnI and several restriction enzymes 
which cleave the region within the fragment inserted into the plasmid pTC1 
and, after subjecting to agarose gel-electrophoresis, detection of a DNA 
fragment containing the protease gene is carried out according to the same 
manner as that for the above phage DNA. When the plasmid pTC1 is digested 
with KpnI and BamHI, a DNA fragment of about 5 kb is hybridized with the 
probe and this fragment of about 5 kb can be isolated and introduced into 
KpnI-BamH site of the plasmid vector pUC119 to obtain a recombinant 
plasmid. This plasmid has been named as pTC1 and E. coli JM109 transformed 
with this plasmid has been named as Escherichia coli JM109/pTC3. FIG. 15 
illustrates a restriction map of the plasmid pTC3. In FIG. 15, the thick 
solid line represents the DNA fragment inserted into the plasmid vector 
pUC119. 
The nucleotide sequence of the hyperthermostable protease gene contained in 
the DNA fragment inserted into the plasmid pTC3 can be determined by using 
specific primers, i.e., by using suitable oligonucleotides synthesized 
based on the nucleotide sequences as shown by SEQ ID NO 10 and 11 of the 
Sequence Listing as primers. SEQ ID NO 12, 13, 14, 15, 16 and 17 represent 
the nucleotide sequences of the oligonucleotides TCE-2, TCE-4, SEF-3, 
SER-1, SER-3 and TCE-6R which have been used as the primers for 
determination of the nucleotide sequence of the hyperthermostable protease 
gene. In addition, SEQ ID NO 7 of the Sequence Listing represents a part 
of the nucleotide sequence of the hyperthermostable protease gene thus 
obtained. That is, SEQ ID NO 7 is a part of the nucleotide sequence of the 
hyperthermostable protease gene of the present invention. Moreover, SEQ ID 
NO 18 represents an amino acid sequence of an example of the enzyme 
encoded by the hyperthermostable protease gene obtained by the present 
invention. In the DNA fragment inserted into the plasmid pTC3, the 
sequence derived from lambda GEM-11 vector is adjacent to the 5'-end of 
the nucleotide sequence represented sented by SEQ ID NO 7 of the Sequence 
Listing, indicating a defect in a part of the 5'-region of the protease 
gene. In addition, by comparing the nucleotide sequence with those of SEQ 
ID NO 10 and 11 of the Sequence Listing, it has been found that the DNA 
fragment inserted into the plasmid pTC3 contains the 41st and the 
subsequent nucleotides of the nucleotide sequence represented by SEQ ID NO 
10 and the whole nucleotide sequence of SEQ ID NO 11. 
Although the hyperthermostable protease gene obtained from Thermococcus 
celer is defect in a part thereof, as is obvious to a person skilled in 
the art, a DNA fragment containing the whole length of the 
hyperthermostable protease gene can be obtained, for example, (1) by 
repeating screening of a genomic DNA library, (2) by carrying out Southern 
hybridization with genomic DNA, (3) by obtaining a DNA fragment of the 
5'-upstream region by PCR with a cassette (manufactured by Takara Shuzo 
Co., Ltd.) and cassette primers (manufactured by Takara Shuzo Co., Ltd.) 
(Takara Shuzo's Genetic Engineering Products Guide, 1994-1995 ed., pp. 
250-251), and the like. 
A transformant into which a recombinant plasmid containing with the 
hyperthermostable protease gene is transduced, for example, Escherichia 
coli JM109/pTPR13 or Escherichia coli JM109/pTPR36, can be cultured under 
conventional conditions, for example, by culturing the transformant in LB 
medium trypton (10 g/liter), yeast extract (5 g/liter), NaCl (5 g/liter); 
pH 7.2! containing 100 .mu.g/ml of ampicillin at 37.degree. C. to express 
the hyperthermostable protease in the culture. After completion of 
culture, the cultured cells are harvested and the cells are sonicated and 
centrifuged. The supernatant is subjected to heat treatment at 100.degree. 
C. for 5 minutes to denature and remove contaminated proteins. In this 
way, a crude enzyme sample can be obtained. The crude enzyme samples thus 
obtained from Escherichia coli JM109/pTPR13 and Escherichia coli 
JM109/pTPR36 have been named as PF-13 and PF-36. 
Further, a transformant into which a recombinant plasmid containing the 
hyperthermostable protease gene is transduced, Bacillus subtilis 
DB104/pUBP13, can be cultured under conventional conditions, for example, 
by culturing the transformant in LB medium containing 10 .mu.g/ml of 
kanamycin at 37.degree. C. to express the hyperthermostable protease in 
the culture. After completion of culture, the cultured cells are harvested 
and the cells are sonicated and centrifuged. The supernatant is subjected 
to heat treatment at 100.degree. C. for 5 minutes to denature and remove 
contaminated proteins, followed by salting out with ammonium sulfate and 
dialysis. In this way, a partially purified enzyme sample can be obtained. 
The roughly purified enzyme sample thus obtained from Bacillus subtilis 
DB104/pUBP13 has been named as PF-BS13. 
The enzymatic and physicochemical properties of the hyperthermostable 
protease samples produced by the transformants into which the recombinant 
plasmids containing the hyperthermostable protease genes derived from 
Pyrococcus furiosus obtained by the present invention, for example, PF-13, 
PF-36 and PF-BS13 are as follows. 
(1) Activity 
The enzymes obtained by the present invention hydrolyze gelatin to form 
short chain polypeptides. In addition, they hydrolyze casein to form short 
chain polypeptides. 
(2) Method for detecting enzyme activity The detection of enzyme activity 
was carried out by detection of hydrolysis of gelatin with the enzyme on a 
SDS-polyacrylamide gel. Namely, an enzyme sample to be tested was suitably 
diluted and to 10 .mu.l of the sample diluted solution was added 2.5 .mu.l 
of a sample buffer solution (50 mM Tris-HCl pH 7.6, 5% SDS, 5% 
2-mercaptoethanol, 0.005% Bromophenol Blue, 50% glycerol). The mixture was 
subjected to heat treatment at 100.degree. C. for 5 minutes and then 
electrophoresis by using 0.1% SDS-10% polyacrylamide gel containing 0.05% 
gelatin. After completion of electrophoresis, the gel was soaked in 50 mM 
potassium phosphate buffer (pH 7.0) and incubated at 95.degree. C. for 2 
hours to carry out the enzymatic reaction. Then, the gel was stained with 
2.5% Coomassie Brilliant Blue R-250 in 25% ethanol and 10% acetic acid for 
30 minutes and further the gel was transferred in 25% ethanol and 7% 
acetic acid to remove excess dye over 3 to 15 hours. Gelatin hydrolyzed 
with the protease into peptides was diffused outside of the gel during the 
enzymatic reaction and the corresponding position was not stained with 
Coomassie Brilliant Blue, thereby detecting the presence of the protease 
activity. The enzyme samples obtained by the present invention, PF-13, 
PF-36 and PF-BS13, had gelatin hydrolyzing activity at 95.degree. C. 
In addition, the casein hydrolyzing activity was detected according to the 
same manner as described above except that a 0.1% SDS-10% polyacrylamide 
gel containing 0.05% of casein was used. The enzyme samples obtained by 
the present invention, PF-13, PF-36 and PF-BS13, had casein hydrolyzing 
activity at 95.degree. C. 
Moreover, casein hydrolyzing activity of the enzyme sample obtained by the 
present invention, PF-BS13, was determined by the following method. To 100 
.mu.l of 0.1M potassium phosphate buffer (pH 7.0) containing 0.2% casein 
was added 100 .mu.l of a suitably diluted enzyme solution and incubated at 
95.degree. C. for 1 hour. The reaction was stopped by addition of 100 
.mu.l of 15% trichloroacetic acid and the reaction mixture was 
centrifuged. The amount of acid soluble short chain polypeptides contained 
in the supernatant was determined by measuring absorbance at 280 nm and 
the enzyme activity was determined by comparing the absorbance with that 
of an enzyme free control. The enzyme sample obtained by the present 
invention, PF-BS13, had casein hydrolyzing activity under the experimental 
conditions of pH 7.0 at 95.degree. C. 
(3) Stability Stability of the enzyme was examined by detecting remaining 
enzymatic activity of heat treated enzymes by the above-described method 
(2) using SDS-polyacrylamide gel containing gelatin. Namely, the enzyme 
sample was incubated at 95.degree. C. for 3 hours and then a suitable 
amount thereof was subjected to detection of the enzymatic activity to 
compare its activity with that without treatment at 95.degree. C. Although 
the position of enzyme activity on the gel was somewhat changed due to 
incubation at 95.degree. C., lowering of the enzyme activity was scarcely 
observed. The enzyme samples obtained by the present invention, PF-13, 
PF-36 and PF-BS13, were stable to heat treatment at 95.degree. C. for 3 
hours. 
In addition, stability of the enzyme samples obtained by the present 
invention, PF-13 and PF-36, in the presence of surfactants were tested. 
Namely, Triton X-100, SDS or benzalkonium chloride was added to the enzyme 
samples in the final concentration of 0.1%. The mixture was incubated at 
95.degree. C. for 3 hours and a suitable amount thereof was subjected to 
detection of the enzymatic activity. For each surfactant, no substantial 
change in the enzyme activity was found in comparison with that in the 
absence of the surfactant. Then, the enzyme samples obtained by the 
present invention, PF-13 and PF 36, were stable to heat treatment at 
95.degree. C. for 3 hours in the presence of surfactants. 
Moreover, stability of the enzyme sample obtained by the present invention, 
PF-BS13, was tested by the following method. Namely, the enzyme sample as 
such or with addition of SDS in the final concentration of 0.1% was 
incubated at 95.degree. C. for various periods of time and the remaining 
activity was determined by the above-described spectrophotometric method 
(2) based on increase in the amount of acid soluble polypeptides. FIG. 16 
illustrates thermostability of the enzyme sample obtained by the present 
invention, PF-BS13. The ordinate indicates the remaining activity (%) and 
the abscissa indicates incubation time (hr). In FIG. 16, the open circle 
represents the results obtained without addition of SDS and the closed 
circle represents the results in the presence of 0.1% SDS. As seen from 
FIG. 16, PF-BS13 maintained almost 100% activity after incubation at 
95.degree. C. for 4 hours regardless of the presence or absence of 0.1% 
SDS. 
(4) Effect of various reagents 
The enzyme samples were subjected to SDS-polyacrylamide gel containing 
gelatin and then the enzymatic reaction was carried out in 50 mM potassium 
phosphate buffer (pH 7.0) containing 2 mM EDTA or 2 mM 
phenylmethanesulfonyl fluoride (PMSF) to test for effect of both reagents 
on the enzyme activity. No substantial difference in the enzyme activities 
of the enzyme samples obtained by the present invention, PF-13, PF-36 and 
PF-BS13, was observed between the buffer containing 2 mM EDTA and 50 mM 
potassium phosphate buffer alone. On the other hand, when the buffer 
containing 2 mM PMSF was used, the amount of hydrolyzed gelatin in the gel 
was decreased in all the samples, indicating that the activities of the 
enzyme samples were inhibited by PMSF. 
(5) Molecular weight 
The molecular weight of the enzyme sample obtained by the present invention 
on a SDS-polyacrylamide gel containing ing gelatin was estimated. The 
enzyme sample, PF-13, showed plural active bands within the range of 95 
kDa to 51 kDa. Although the migration distance was varied according to the 
amount of a sample applied, etc., the major bands of 84 kDa, 79 kDa, 66 
kDa, 54 kDa and 51 kDa were appeared. When the enzyme sample was subjected 
to electrophoresis after heat treatment at 95.degree. C. for 3 hours in 
the presence of SDS in the final concentration of 0.1%, the bands of 63 
kDa and 51 kDa became intensive. For the enzyme sample, PF-BS13, the same 
results as that of the above with respect to the enzyme sample PF-13 were 
obtained. In case of the enzyme sample PF-36, several minor bands were 
observed in addition to the main bands of 63 kDa and 59 kDa. 
(6) Optimum pH 
The optimum pH of the enzyme samples PF-13 and PF-36 obtained by the 
present invention was tested. After subjecting the enzyme samples to 
electrophoresis on a SDS-polyacrylamide gel containing gelatin, the gel 
was soaked in buffers having different pH and the enzyme reaction was 
carried out to test for the optimum pH. As the buffers, 50 mM sodium 
acetate buffer solution at pH 4.0 to 6.0, 50 mM potassium phosphate buffer 
solution at pH 6.0 to 8.0, 50 mM sodium borate buffer solution at pH 9.0 
to 10.0 were used. Both enzyme samples showed gelatin hydrolyzing activity 
at pH 6.0 to 10.0 and their optimum pH was pH 8.0 to 9.0. 
In addition, the optimum pH of the enzyme sample obtained by the present 
invention, PF-BS13, was determined by the above-described 
spectrophotometric method (2) based on increase in the amount of acid 
soluble polypeptides. 0.2% Casein solutions to be used for the 
determination were prepared by using 0.1M sodium acetate butter solution 
at pH 4.0 to 6.0, 0.1M potassium phosphate buffer solution at pH 6.0 to 
8.0, 0.1M sodium borate buffer solution at pH 9.0 to 10.0 and 0.1M sodium 
phosphate-sodium hydroxide buffer solution at pH 11.0 and they were used 
for the determination. FIG. 17 illustrates the relation between casein 
hydrolyzing activity of the enzyme sample obtained by the present 
invention, PF-BS13 and pH. The ordinate indicates the relative activity 
(%) and the abscissa indicates pH. In Fig. 17, the open circle, the closed 
circle, the open square and the closed square represent the results 
obtained by using the substrate solutions prepared with 0.1M sodium 
acetate buffer solution, 0.1M potassium phosphate buffer solution, 0.1M 
sodium borate buffer solution and 0.1M sodium phosphate-sodeum hydroxide, 
respectively. As seen from FIG. 17, the enzyme sample, PF-BS13, showed 
casein decomposing activity at the pH range of 5.0 to 11.0 and its optimum 
pH was pH 9.0 to 10.0. 
As described hereinabove in detail, according to the present invention, the 
genes encoding the hyperthermostable proteases and the industrial process 
for producing the hyperthermostable proteases using the genes can be 
provided. The enzymes have high thermostability and also show resistance 
to surfactants. Therefore, they are particularly useful for treatment of 
proteins at high temperatures. 
In addition, a DNA fragment obtained by hybridization with the gene 
isolated by the present invention or a part of the nucleotide sequence of 
the isolated gene as a probe can be transduced into a suitable 
microorganism and its heat-treated lysate can be prepared according to the 
same manner as that described with respect to the cosmid protein library. 
Then, a protease activity is tested by an appropriate method. In this 
manner, a hyperthermostable protease gene encoding an enzyme whose 
sequence is not identical with that of the above enzyme but which has a 
similar activity can be obtained. 
The above hybridization can be carried out under the following conditions. 
Namely, DNA fixed on a membrane is incubated in 6.times.SSC containing 
0.5% of SDS, 0.1% of bovine serum albumin, 0.1% of polyvinyl pyrrolidone, 
0.1% of Ficoll 400 and 0.01% denatured salmon sperm DNA (1.times.SSC 
represents 0.15M NaCl and 0.015M sodium citrate, pH 7.0) together with a 
probe at 50.degree. C. for 12 to 20 hours. After completion of incubation, 
the membrane is washed in such a manner that washing is started with 
2.times.SSC containing 0.5% SDS at 37.degree. C., followed by changing SSC 
concentrations within the range to 0.1.times.and varying temperatures up 
to 50.degree. C., until a signal from the fixed DNA can be distinguished 
from the background signal. 
Furthermore, the gene isolated by the present invention, a DNA fragment 
obtained by in vitro gene amplification using a part of the isolated gene 
as a primer, or a DNA fragment obtained by hybridization using the 
fragment obtained by the above amplification as a probe is transduced into 
a suitable microorganism and, according to the same manner as described 
above, a protease activity is determined. In this manner, a 
hyperthermostable protease gene encoding an enzyme whose activity is not 
identical with that of the above enzyme but is similar can be obtained. 
The following examples further illustrates the present invention but are 
not to be construed to limit the scope thereof. In the examples, all the 
"percents" are by weight. 
EXAMPLE 1 
Preparation of genomic DNA of Pyrococcus furiosus 
Pyrococcus furiosus DSM3638 was cultured as follows. 
A culture medium composed of 1% of trypton, 0.5% of yeast extract, 1% of 
soluble starch, 3.5% of Jamarin S.multidot.Solid (manufactured by Jamarin 
Laboratory), 0.5% of Jamarin S.multidot.Liquid (manufactured by Jamarin 
Laboratory), 0.003% of MgSO.sub.4, 0.001% of NaCl, 0.0001% of FeSO.sub.4 
.multidot.7H.sub.2 O, 0.0001% of COSO.sub.4, 0.0001% of CaCl.sub.2 
.multidot.7H.sub.2 O, 0.0001% of ZnSO.sub.4, 0.1 ppm of CuSO.sub.4 
.multidot.5H.sub.2 O, 0.1 ppm of KAl (SO.sub.4).sub.2, 0.1 ppm of H.sub.3 
BO.sub.3, 0.1 ppm of Na.sub.2 MoO.sub.4 .multidot.2H.sub.2 O and 0.25 ppm 
of NiCl.sub.2 .multidot.6H.sub.2 O was placed in a 2 liter-medium bottle 
and sterilized at 120.degree. C. for 20 minutes. Then, nitrogen gas was 
blown into the medium to purge out dissolved oxygen and the above 
bacterial strain was inoculated into the medium, followed by subjecting to 
stationary culture at 95.degree. C. for 16 hours. After completion of 
culture, bacterial cells were collected by centrifugation. 
Then, the collected cells were suspended into 4 ml of 0.05M Tris-HCl (pH 
8.0) containing 25% of sucrose and to this suspension were added 0.8 ml of 
lysozyme 5 mg/ml, 0.25M Tris-HCl (pH 8.0)! and 2 ml of 0.2M EDTA. The 
mixture was incubated at 20.degree. C. for 1 hour. Then, to the mixture 
were added 24 ml of SET solution 150 mM NaCl, 1 mM EDTA and 20 mM 
Tris-HCl (pH 8.0)! and further 4 ml of 5% SDS and 400 .mu.l of Proteinase 
K (10 mg/ml) and the mixture was incubated at 37.degree. C. for 1 hour. 
After completion of the reaction, the reaction mixture was subjected to 
phenol-chloroform extraction and then ethanol precipitation to prepare 
about 3.2 mg of genomic DNA. 
Preparation of cosmid protein library 400 .mu.g of Genomic DNA of 
Pyrococcus furiosus DSM3633 was partially digested with Sau3AI and 
subjected to size-fractionation in size of 35 to 50 kb by density-gradient 
ultra-centrifugation. Then, 1 .mu.g of a triple helix cosmid vector was 
digested with XbaI, dephosphorylated with alkaline phosphatase 
(manufactured by Takara Shuzo Co., Ltd.) and further digested with BamHI. 
The vector was ligated to 140 .mu.g of the above fractionated 35 to 50 kb 
DNA. The genomic DNA fragments of Pyrococcus furiosus were packaged into 
lambda phage particles by in vitro packaging method using Gigapack Gold 
(manufactured by Stratagene) to prepare a library. Then, by using a part 
of the library thus obtained, transduction into E. coli DH5.alpha.MCR was 
carried out and, among transformants obtained, several transformants were 
selected to prepare cosmid DNA. After confirmation of the presence of 
inserted fractions having suitable size, again about 500 transformants 
were selected from the above library and they were independently cultured 
in 150 ml of LB medium (tripton 10 g/liter, yeast extract 5 g/liter, NaCl 
5 g/liter, pH 7.2) containing 100 .mu.g/ml of ampicillin. Each culture was 
centrifuged, the recovered microbial cells were suspended in 1 ml of 20 mM 
Tris-HCl (pH 8.0) and the suspension was subjected heat treatment at 
100.degree. C. for 10 minutes. Then, the suspension was sonicated and 
again subjected heat treatment at 100.degree. C. for 10 minutes. The 
lysates obtained as supernatants after centrifugation were used as the 
cosmid protein library. 
Selection of cosmid containing hyperthermostable protease gene 
The protease activity was detected by testing for hydrolysis gelatin in a 
polyacrylamide gel. 
Namely, 5 .mu.l aliquots of the lysates from the above cosmid protein 
library were taken out and subjected to electrophoresis by using 0.1% 
SDS-10% polyacrylamide gel containing 0.05% of gelatin. After completion 
of electrophoresos, the gel was incubated in 50 mM potassium phosphate 
buffer solution (pH 7.0) at 95.degree. C. for 2 hours. The gel was stained 
in 2.5% Coomassie Brilliant Blue-R-250, 25% ethanol and 10% acetic acid 
for 30 minutes. Then, the gel was transferred to 25% methanol and 7% 
acetic acid to decolorize for 3 to 15 hours. Eight cosmid clones having 
the protease activity, which shows the bands not stained with Coomassie 
Brilliant Blue-R-250 due to hydrolysis of gelatin on the gel were 
selected. 
Preparation of plasmid pTPR1 containing hyperthermostable protease gene 
Among the 8 cosmid clones having the protease activity, one cosmid (cosmid 
No. 304) was selected to prepare cosmid DNA and the cosmid DNA was 
digested with SphI and then ligated to SphI site of the plasmid vector 
pUC119. This recombinant plasmids were transduced into E. coli JM109 and 
the protease activity of the resultant transformants were tested according 
to the same method as that used for screening of the cosmid protein 
library. A plasmid was prepared from the transformant having the protease 
activity and the resultant recombinant plasmid was named as pTPR1. E. coli 
JM109 transformed with the plasmid was named as Escherichia JM109/pTPR1. 
FIG. 1 illustrates a restriction map of the plasmid pTPR1. 
Preparation of plasmid pTPR9 containing 
hyperthermostable protease gene 
The above plasmid pTPR1 was digested with XbaI and subjected to agarose 
gel-electrophoresis to separate three DNA fragments of about 2.5 kb, about 
3.3 kb and about 4.3 kb. Among three fragments thus separated, two 
fragments of about 3.3 kb and about 4.3 kb were recovered. The DNA 
fragment of about 4.3 kb was dephosphorylated with alkaline phosphatase 
(manufactured by Takara Shuzo Co., Ltd.) and then was mixed with the DNA 
fragment of about 3.3 kb to ligate to each other. This was transduced into 
E. coli JM109. The protease activity of the resultant transformants were 
tested by the same method as that used for screening of the cosmid protein 
library. A plasmid was prepared from the transformant having the protease 
activity. The plasmid was named as pTPR 9 and E. coli JM109 transformed 
with the plasmid was named as Escherichia coli JM109/pTPR9. 
FIG. 2 illustrates a restriction map of the plasmid pTPR 9. 
Detection of DNA fragment containing whole length of hyperthermostable 
protease gene 
The cosmid DNA used in the preparation of the above plasmid pTPR1 was 
digested with NotI and then further digested with BamHI, Blnl, EcoT22, 
Nsp(7524)V, PvuII, SalI, SmaI and SpeI, respectively. Then, digested DNA 
was subjected to electrophoresis on a 0.8% agarose gel. After 
electrophoresis, the gel was soaked in 0.5N NaOH containing 1.5M NaCl to 
denature the DNA fragments in the gel and then the gel was neutralized in 
0.5M Tris-HCl (pH 7.5) containing 3M NaCl. The DNA fragments in the gel 
was blotted on a Hybond-N.sup.+ nylon membrane (manufactured by Amasham) 
by Southern blotting. After blotting, the membrane was washed with 
6.times.SSC (1.times.SSC represents 0.15M NaCl, 0.015M sodium citrate, pH 
7.0) and air-dried and DNA was fixed on the membrane by UV irradiation 
using a UV transilluminator for 3 minutes. 
On the other hand, the plasmid pTPR9 was digested with PstI and XbaI and 
subjected to electrophoresis on a 1% agarose gel and the separated DNA 
fragment of about 0.7 kb was recovered. A .sup.32 P-labeled DNA probe was 
prepared by using the DNA fragment as a template and using a random primer 
DNA labeling kit Ver2 (manufactured by Takara Shuzo Co., Ltd.) and 
.alpha.-.sup.32 P!dCTP (manufactured by Amasham). 
The above membrane to which the DNA was fixed was treated in a 
hybridization buffer solution (6.times.SCC containing 0.5% SDS, 0.1% 
bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll 400 and 
0.01% denatured salmon sperm) at 68.degree. C. for 2 hours. Then, it was 
transferred in a similar hybridization buffer solution containing the 
.sup.32 P-labeled DNA probe to allow to hybridize at 68.degree. C. for 14 
hours. After completion of hybridization, the membrane was washed with 
2.times.SSC containing 0.5% of SDS at room temperature and then 
0.1.times.SSC containing 0.5% of SDS at 68.degree. C. After rinsing the 
membrane with 0.1.times.SSC, it was air-dried. A X-ray film was exposed to 
the membrane at -80.degree. C. for 60 hours. The film was developed to 
prepare an autoradiogram. This autoradiogram showed that a protease gene 
was present in the DNA fragment of about 7.5 kb obtained by digestion of 
the cosmid DNA with NotI and PvuII. 
Preparation of plasmid pTPR12 containing whole length of hyperthermostable 
protease gene 
The cosmid DNA used for the preparation of the above plasmid pTPR1 was 
digested with Not I and PvuII and subjected to electrophoresis using a 
0.8% agarose gel to recover DNA fragments of about 7 to 8 kb all together. 
These DNA fragments were mixed with the plasmid vector pUC19 into which a 
Not I linker was introduced at HincII site and which was digested with 
NotI and SmaI. Then, ligation was carried out. The recombinant plasmids 
were transduced into E. coli JM109 and the protease activity of the 
resultant transformants were tested by the same method as that used for 
screening of the cosmid protein library. A plasmid was prepared from the 
transformant having the protease activity. The plasmid was named as pTPR12 
and E. coli JM109 transformed with the plasmid was designated as 
Escherichia coli JM109/pTPR12. 
FIG. 3 illustrates a restriction map of the plasmid pTPR12. 
Preparation of plasmid pTPR 15 containing whole length of hyperthermostable 
protease gene 
The above plasmid pTPR 12 was digested with XbaI and subjected to 
electrophoresis using a 1% agarose gel to recover separated two DNA 
fragments of about 3.3 kb and about 7 kb, respectively. Then, the DNA 
fragments of about 7 kb thus recovered was digested with KpnI and again 
subjected to electrophoresis using a 1% agarose gel to separate two 
fragments of about 3.2 kb and about 3.8 kb. In these fragments, the DNA 
fragment of about 3.2 kb was recovered and ligated to the plasmid vector 
pUC19 digested with XbaI and KpnI. This was transduced into E. coli JM109. 
Plasmids held by the resultant transformants were prepared and the plasmid 
containing only one molecular of the above 3.2 kb fragment was selected. 
This was named as pTPR14. 
FIG. 5 illustrates a restriction map of the plasmid pTPR 14. 
Then, the above plasmid pTPR 14 was digested with XbaI and dephosphorylated 
using alkaline phosphatase. This was mixed with the above fragment of 
about 3.3 kb to carry out ligation and was transduced into E. coli JM109. 
The protease activity of the resultant transformants were tested by using 
the same method as that used for screening of the cosmid protein library. 
A plasmid was prepared from the transformant having the protease activity. 
This plasmid was named as pTPR15 and E. coli JM109 transformed with the 
plasmid was named as Escherichia coli JM109/pTPR15. 
FIG. 6 illustrates a restriction map of the plasmid pTPR 15. 
EXAMPLE 2 
Determination of nucleotide sequence of hyperthermostable protease gene 
For determination of the nucleotide sequence of the hyperthermostable 
protease gene inserted into the above plasmid pTPR 15, deletion mutants 
wherein the DNA fragment portion inserted into the plasmid had been 
deleted in various lengths were prepared by using Kilo sequence deletion 
kit (manufactured by Takara Shuzo Co., Ltd.). Among them, several mutants 
having suitable lengths of deletion were selected and nucleotide sequences 
of respective inserted DNA fragment portions were determined by dideoxy 
method using BcaBEST dideoxy sequencing kit (manufactured by Takara Shuzo 
Co., Ltd.). By putting these results together, nucleotide sequences of the 
inserted DNA fragment contained in the plasmid pTPR15 were determined. 
Among the nucleotide sequences thus obtained, SEQ ID NO 8 of the Sequence 
Listing shows the fragment of 4765 bp between two DraI sites. Furthermore, 
SEQ ID NO 9 shows an amino acid sequence of the hyperthermostable protease 
encoded by the open reading frame contained in the above nucleotide 
sequence. 
Preparation of plasmid pTPR13 containing hyperthermostable protease gene 
The above plasmid pTPR15 was digested with DraI and subjected to 1% agarose 
gel-electrophoresis, followed by recovering the separated DNA fragment of 
about 4.8 kb. Then, the plasmid vector pUC19 was digested with SmaI and, 
after dephosphorylation with alkaline phosphatase, it was mixed with the 
above DNA fragment of about 4.8 kb to carry out ligation and transduced 
into E. coli JM109. The protease activity of the resultant transformants 
were tested by the same method as that used for screening of the cosmid 
protein library. A plasmid was prepared from a transformant having the 
activity. The plasmid was named as pTPR13 and E. coli JM109 transformed 
with the plasmid was named as Escherichia coli JM109/pTPR13. 
FIG. 7 illustrates a restriction map of the plasmid pTPR13. 
Preparation of plasmid pUBP13 containing hyperthermostable protease gene 
for transforming Bacillus subtilis 
The above plasmid pTPR13 was digested with KpnI and BamHI and then 
subjected to 1% agarose gel-electrophoresis, followed by recovering the 
separated DNA fragment of about 4.8 kb. Then, the plasmid vector pUB18-P43 
was digested with KpnI and BamHI and mixed with the above DNA fragment of 
about 4.8 kb to carry out ligation. It was transduced into Bacillus 
subtilis DB104. The protease activity of the resultant transformants 
having kanamycin resistance were tested by the same method as that used 
for screening of the cosmid protein library. A plasmid was prepared from a 
transformant having the activity. The plasmid was named as pUBP13 and 
Bacillus subtilis DB104 transformed with the plasmid was named as Bacillus 
subtilis DB1049/pUBP13. 
FIG. 8 illustrates a restriction map of the plasmid pUBP13. 
Preparation of plasmid pTPR36 containing hyperthermostable protease gene 
defecting in its back half portion 
The above plasmid pTPR13 was digested with EcoRI and the resultant end was 
blunted with a DNA blunting kit (manufactured by Takara Shuzo Co., Ltd.). 
Further, it was digested with KpnI and subjected to 1% agarose 
gel-electrophoresis, followed by recovering the separated DNA fragment of 
about 2.8 kb. Next, the plasmid vector pUC119 was digested with XbaI and 
the resultant end was blunted and further digested with KpnI, followed by 
mixing with the above DNA fragment of 2.8 kb to carry out ligation and 
transducing into E. coli JM109. 
The protease activity of the resultant transformants were tested by the 
same method as that used for screening of the cosmid protein library. A 
plasmid was prepared from a transformant having the activity. The plasmid 
was named as pTPR36 and E. coli JM109 transformed with the plasmid was 
named as Escherichia coli JM109/pTPR36. 
FIG. 9 illustrates a restriction map of the plasmid pTPR36. SEQ ID NO 2 of 
the Sequence Listing shows the nucleotide sequence of the DNA fragment 
inserted into the plasmid pTPR36. Also, SEQ ID NO 1 shows an amino acid 
sequence of the hyperthermostable protease which can be encoded by the 
nucleotide sequence. 
EXAMPLE 3 
Preparation of oligonucleotide for detection of hyperthermostable protease 
gene 
By comparing the estimated amino acid sequence of the hyperthermostable 
protease of the present invention obtained in Example 2 with amino acid 
sequences of known alkaline serine proteases originating in 
microorganisms, it was found that there were homologous amino acid 
sequences commonly present in these enzymes. Among them, three regions 
were selected and oligonucleotides to be used as primers in detection of 
hyperthermostable protease genes by PCR were designed. 
FIGS. 10, 11 and 12 illustrate the relation among the amino acid sequences 
corresponding to the above three regions of the hyperthermostable protease 
of the present invention, nucleotide sequences of the hyperthermostable 
protease of the present invention which encode the above regions, and the 
nucleotide sequences of oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4R 
synthesized based on the above nucleotide sequences. Also, SEQ NO. 3, 4, 5 
and 6 show nucleotide sequences of PRO-1F, PRO-2F, PRO-2R and PRO-4R, 
respectively. 
Preparation of genomic DNA of Thermococcus celer 
Microbial cells were collected from 10 ml of a culture broth of 
Thermococcus celer DSM2476 obtained from Deutsch Sammlung von 
Microorganismen und Zellkulturen GmbH by centrifugation and suspended in 
100 .mu.l of 50 mM Tris-HCl (pH 8.0) containing 25% sucrose. To the 
suspension were added 20 .mu.l of 0.5M EDTA and 10 .mu.l of lysozyme (10 
mg/ml) and the suspension was incubated at 20.degree. C. for 1 hour. To 
this were added 800 .mu.l of SET solution (150 mM NaCl, 1 mM EDTA, 20 mM 
Tris-HCl, pH 8.0), 50 .mu.l of 10% SDS and 10 .mu.l of Proteinase K (20 
mg/ml) and the suspension was further incubated at 37.degree. C. for 1 
hour. Chloroform-phenol extraction was carried out to stop the reaction. 
The reaction mixture was subjected to ethanol precipitation and recovered 
DNA was dissolved in 50 .mu.l of TE buffer solution to obtained a genomic 
DNA solution. 
Detection of hyperthermostable protease by PCR 
A PCR reaction mixture was prepared from the above genomic DNA of 
Thermococcus celer and the oligonucleotides PRO-1F and PRO-2R or the 
oligonucleotides PRO-2F and PRO-4R and a PCR reaction (one cycle: 
94.degree. C. for 1 minute-55.degree. C. for 1 minute-72.degree. C. for 1 
minute, 35 cycles) was carried out. When aliquots of the reaction mixture 
were subjected to agarose gel-electrophoresis, amplification of three DNA 
fragments in case of using the oligonucleotides PRO-1F and PRO-2R and one 
DNA fragment in case of using the oligonucleotides PRO-2F and PRO-4R was 
observed. These amplified fragments were recovered from the agarose gel 
and their DNA ends were blunted by a DNA blunting kit, followed by 
phosphorylating thereof with T4 polynucleotide kinase (manufactured by 
Takara Shuzo Co., Ltd.). Then, the plasmid vector pUC18 was digested with 
HincII and subjected to dephosphorylation with alkaline phosphatase. It 
was mixed with the above PCR amplified DNA fragments to carry out ligation 
and then transduced into E. coli JM109. Plasmids were prepared from the 
resultant transformants and plasmids into which suitable DNA fragments 
were inserted were selected. Nucleotide sequences of the inserted DNA 
fragments were determined by dideoxy method. Among these plasmids, 
regarding a plasmid p1F-2R(2) containing a DNA fragment of about 150 bp 
which was amplified by using the oligonucleotides PRO-1F and PRO-2R and a 
plasmid p2F-4R containing a DNA fragment of about 550 bp which was 
amplified by using the oligonucleotides PRO-2F and PRO-4R, it was found 
that amino acid sequences estimated from the thus-obtained nucleotide 
sequences contained sequences having homology with the amino acid 
sequences of the hyperthermostable protease originating in Pyrococcus 
furiosus of the present invention, subtilisin and the like. 
SEQ NO 10 of the Sequence Listing shows the nucleotide sequence of the DNA 
fragment inserted into the plasmid p1F-2R(2) and an amino acid sequence 
deduced from the nucleotide sequence. Also, SEQ NO 11 of the Sequence 
Listing shows the nucleotide sequence of the DNA fragment inserted into 
the plasmid p2F-4R and an amino acid sequence deduced from the nucleotide 
sequence. In the nucleotide sequence shown by SEQ NO 10 of the Sequence 
Listing, the sequence from the first to 21st nucleotides and that from the 
113th to 145th nucleotides and, in the SEQ NO 11 of the Sequence Listing, 
the sequence from the first to the 32nd nucleotides and that from the 
532nd to the 564th nucleotides are the sequences of the primers used in 
the PCR (corresponding to the oligonucleotides PRO-1F, PRO-2R, PRO-2F and 
PRO-4R, respectively). 
FIG. 13 illustrates a restriction map of the plasmid p2F-4R. 
Screening of protease gene originating in Thermococcus celer 
The above genomic DNA of Thermococcus celer was partially digested with 
Sau3AI and was treated with Klenow fragment (manufactured by Takara Shuzo 
Co., Ltd.) in the presence of dATP and dGTP to partially repair the DNA 
ends. The DNA fragments were mixed with a lambda GEM-11 XhoI half site arm 
vector (manufactured by Promega) to carry out ligation. Then, they were 
subjected to in vitro packaging using Gigapack Gold to prepare a lambda 
phage library containing genomic DNA of Thermococcus celer. A part of the 
library was transduced into E. coli LE392 to form plaques on a plate and 
the plaques were transferred on a Hybond-N.sup.+ -membrane. After 
transfer, the membrane was treated with 0.5 N NaOH containing 1.5M NaCl 
and then 0.5M Tris-HCl (pH 7.5) containing 3M NaCl. Further, it was washed 
with 6.times.SCC, air-dried and irradiated with UV light on a UV 
transilluminator to fix phage DNA on the membrane. 
On the other hand, the plasmid p2F-4R was digested with PmaCI (manufactured 
by Takara Shuzo Co., Ltd.) and StuI (manufactured by Takara Shuzo Co., 
Ltd.) and subjected to 1% agarose gel-electrophoresis to recover the 
separated DNA fragment of about 0.5 kb. By using this fragment as a 
template and using a random primer DNA labeling kit Ver2 and 
.alpha.-.sup.32 P!dCTP, a .sup.32 P-labeled DNA probe was prepared. 
The above membrane having DNA fixed thereon was treated in a hybridization 
buffer solution (6.times.SSC containing 0.5% SDS, 0.1% bovine serum 
albumin, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll 400 and 0.01% denatured 
salmon sperm DNA) at 50.degree. C. for 2 hours. It was transferred to the 
same buffer solution containing the .sup.32 P-labeled DNA prove and 
hybridization was carried out at 50.degree. C. for 15 hours. After 
completion of hybridization, the membrane was washed with 2.times.SSC 
containing 0.5% SDS at room temperature and then 1.times.SSC containing 
0.5% SDS at 50.degree. C. Further, after rinsing the membrane with 
1.times.SCC, it was air-dried and a X-ray film was exposed thereto at 
-80.degree. C. for 6 hours to prepare an autoradiogram. About 4,000 phage 
clones were screened. As a result, one phage clone containing a protease 
gene was obtained. Based on the signal on the autoradiogram, the position 
of this phage clone was found and the plaque corresponding on the plate 
used for transfer to the membrane was isolated into 1 ml of SM buffer 
solution 50 mM Tris-HCl, 0.1M NaCl, 8 mM MgSO.sub.4, 0.01% gelatin (pH 
7.5)! containing 1% of chloroform. 
Detection of phage DNA fragment containing protease gene 
The above phage clone was transduced in to E. coli LE392 and the 
transformant was cultured in NZCYM medium (manufactured by Bio 101) at 
37.degree. C. for 15 hours to obtain a culture broth. A supernatant of the 
culture broth was collected and phage DNA was prepared by using 
QIAGEN-lambda kit (manufactured by DIAGEN). The resultant phage DNA was 
digested with BamHI, EcoRI, EcoRV, HincII, KpnI, NcoI, PstI, SacI, SalI, 
SmaI and SphI (all manufactured by Takara Shuzo Co., Ltd.), respectively, 
and subjected to 1% agarose-electro-phoresis. Then, a membrane on which 
DNA fragments were fixed was prepared by the same method as that used for 
the detection of the DNA fragment containing the whole length of the 
hyperthermostable protease gene of Example 1. The membrane was treated in 
a hybridization buffer solution at 50.degree. C. for 4 hours and then 
transferred to the same hybridization buffer solution containing the same 
.sup.32 P-labeled DNA probe as that used in the above screening of the 
protease gene derived form Thermococcus celer. Then, hybridization was 
carried out at 50.degree. C. for 18 hours. After completion of 
hybridization, the membrane was washed with 1.times.SSC containing 0.5% 
SDS at 50.degree. C. and rinsed with 1.times.SCC. The membrane was 
air-dried and exposed to a X-ray film at -80.degree. C. for 2 hours to 
prepare an autoradiogram. According to this autoradiogram, it was found 
that, in the phage DNA digested with KpnI, the protease gene was contain 
in a DNA fragment of about 9 kb. 
Preparation of plasmid pTC1 containing protease gene 
The above phage DNA containing the protease gene was digested with KpnI and 
subjected to 1% agarose gel-electrophoresis to recover a DNA fragment of 
about 9 kb from the gel. Then, the plasmid vector pUC119 was digested with 
KpnI and dephosphorylated with alkaline phosphatase, followed by mixing 
with the above DNA fragment of about 9 kb to carry out ligation. Then, it 
was transduced into E. coli JM109. Plasmids were prepared from the 
resultant transformants and a plasmid containing only the above DNA 
fragment of about 9 kb was selected. This plasmid was named as pTC1 and E. 
coli JM109 transformed with the plasmid was named with Escherichia coli 
JM109/pTC1. 
FIG. 14 illustrates a restriction map of the plasmid pTC1. 
Preparation of plasmid pTC3 containing hyperthermostable protease gene 
The above plasmid pTC1 was digested with KpnI and further digested with 
BamHI, PstI and SphI, respectively. After subjecting to 1% agarose 
gel-electrophoresis, according to the same operation as that for detecting 
the phage DNA fragment containing the above protease gene, transfer of DNA 
fragments to a membrane and detection of DNA fragments containing the 
hyperthermostable protease gene were carried out. By the signal on the 
resultant autoradiogram, it was shown that a DNA fragment of about 5 kb 
which obtained by digesting the plasmid pTC1 with KpnI and BamHI contained 
the hyperthermostable protease gene. 
Then, the plasmid pTC1 was digested with KpnI and BamHI and then subjected 
to 1% agarose gel-electrophoresis to separate and isolate a DNA fragment 
of about 5 kb. The plasmid vector pUC119 was digested with KpnI and BamHI 
and mixed with the above DNA fragment of about 5 kb to carry out ligation. 
It was transduced into E. coli JM109. Plasmids were prepared form the 
resultant transformants and a plasmid containing the above DNA fragment of 
about 5 kb. This plasmid was named as pTC3 and E. coli JM109 transformed 
with the plasmid was named as Escherichia coli JM109/pTC3. 
FIG. 15 illustrates a restriction map of the plasmid pTC3. 
Determination of nucleotide sequence of hyperthermostable protease gene 
contained in Plasmid pTC3 
For determination of the nucleotide sequence of the hyperthermostable 
protease gene contained in the above plasmid pTC3, 6 oligonucleotides were 
synthesized based on the nucleotide sequences shown by SEQ ID NO 10 and 11 
of the Sequence Listing, respectively. The nucleotide sequences of the 
synthesized oligonucleotides TCE-2, TCE-4, SEF-3, SER-1, SER-3 and TCE-6R 
were shown by SEQ ID NO 12, 13, 14, 15, 16 and 17 of the Sequence Listing. 
The results obtained by dideoxy method using the above oligonucleotides as 
primers and the plasmid pTC3 as a template were summarized to determine 
the nucleotide sequence of the hyperthermostable protease gene. 
SEQ ID NO 7 of the Sequence Listing shows a part of the resultant 
nucleotide sequence. In addition, SEQ ID NO 18 of the Sequence Listing 
shows an deduced amino acid sequence encoded by the nucleotide sequence. 
EXAMPLE 4 
Preparation of enzyme sample Escherichia coli JM109/pTPR36 which was E. 
coli JM109 into which the plasmid pTPR36 containing the hyperthermostable 
protease gene of the present invention obtained in Example 2 was 
transduced was cultured with shaking in 5 ml of LB medium (trypton 10 
g/liter, yeast extract 5 g/liter, NaCl 5 g/liter, pH 7.2) containing 100 
.mu.g/ml of ampicillin at 37.degree. C. for 14 hours. In a 1 
liter-Erlenmeyer flask, 200 ml of the same medium was prepared and 2 ml of 
the above culture broth was inoculated and cultured with shaking at 
37.degree. C. for 10 hours. The culture broth was centrifuged. The 
harvested microbial cells (wet weight 1.6 g) were suspended in 2 ml of 20 
mM Tris-HCl (pH 8.0), sonicated and centrifuged to obtain a supernatant. 
The supernatant was treated at 100.degree. C. for 5 minutes and 
centrifuged again. The resultant supernatant was used as a crude enzyme 
solution (enzyme sample PF-36). 
In addition, according to the same manner, Escherichia coli JM109/pTPR13 
which was E. coli JM109 into which the plasmid pTPR13 containing the 
hyperthermostable protease gene of the present invention was transduced 
was used to prepare a crude enzyme solution (enzyme sample PF-13). 
Moreover, Bacillus subtilis DB104/pUBP13 which was Bacillus subtilis DB104 
into which the plasmid pUBP13 containing the hyperthermostable protease 
gene of the present invention was transduced was cultured with shaking in 
5 ml of LB medium containing 10 .mu.g/ml of kanamycin at 37.degree. C. for 
14 hours. In two 2 liter-Erlenmeyer flasks, respective 600 ml of the same 
mediums were prepared. To each flask was inoculated with 2 ml of the above 
culture broth and cultured with shaking at 37.degree. C. for 26 hours. The 
culture broth was centrifuged. The resultant microbial cells were 
suspended in 15 ml of 20 mM Tris-HCl (pH 8.0), sonicated and centrifuged 
to obtain a supernatant. The supernatant was treated at 100.degree. C. for 
5 minutes and centrifuged again. To the resultant supernatant was added 
ammonium sulfate to 50% saturation and then the resultant precipitate was 
recovered by centrifugation. The recovered precipitate was suspended in 2 
ml of 20 mM Tris-HCl (pH 8.0) and the suspension was dialyzed against the 
same buffer solution. The resultant inner solution was used as a partially 
purified enzyme sample (enzyme sample PF-BS13). 
The protease activity of these enzyme samples and the cosmid clone lysate 
used for preparation of plasmids were tested according to the above method 
for detection of enzyme activity using SDS-polyacrylamide gel containing 
gelatin. 
FIG. 16 illustrates the thermostability of the hyperthermostable protease 
obtained by the present invention. And, FIG. 17 illustrates the optimum pH 
of the hyperthermostable protease obtained by the present invention. 
Further, FIG. 18 illustrates the results of activity staining after 
SDS-polyacrylamide gel electrophoresis of each sample (enzyme samples 
PF-36, PF-13 and PF-BS13 and the lysate). Each sample shows activity at 
95.degree. C. in the presence of SDS. 
As described hereinabove, according to the present invention, genes 
encoding hyperthermostable proteases which show activity at 95.degree. C. 
were obtained. These genes make possible to supply a large amount of a 
hyperthermostable protease having high purity. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 18 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 903 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetAsnLysLysGlyLeuThrValLeuPheIleAlaIleMetLeuLeu 
151015 
SerValValProValHisPheValSerAlaGluThrProProValSer 
202530 
SerGluAsnSerThrThrSerIleLeuProAsnGlnGlnValValThr 
354045 
LysGluValSerGlnAlaAlaLeuAsnAlaIleMetLysGlyGlnPro 
505560 
AsnMetValLeuIleIleLysThrLysGluGlyLysLeuGluGluAla 
65707580 
LysThrGluLeuGluLysLeuGlyAlaGluIleLeuAspGluAsnArg 
859095 
ValLeuAsnMetLeuLeuValLysIleLysProGluLysValLysGlu 
100105110 
LeuAsnTyrIleSerSerLeuGluLysAlaTrpLeuAsnArgGluVal 
115120125 
LysLeuSerProProIleValGluLysAspValLysThrLysGluPro 
130135140 
SerLeuGluProLysMetTyrAsnSerThrTrpValIleAsnAlaLeu 
145150155160 
GlnPheIleGlnGluPheGlyTyrAspGlySerGlyValValValAla 
165170175 
ValLeuAspThrGlyValAspProAsnHisProPheLeuSerIleThr 
180185190 
ProAspGlyArgArgLysIleIleGluTrpLysAspPheThrAspGlu 
195200205 
GlyPheValAspThrSerPheSerPheSerLysValValAsnGlyThr 
210215220 
LeuIleIleAsnThrThrPheGlnValAlaSerGlyLeuThrLeuAsn 
225230235240 
GluSerThrGlyLeuMetGluTyrValValLysThrValTyrValSer 
245250255 
AsnValThrIleGlyAsnIleThrSerAlaAsnGlyIleTyrHisPhe 
260265270 
GlyLeuLeuProGluArgTyrPheAspLeuAsnPheAspGlyAspGln 
275280285 
GluAspPheTyrProValLeuLeuValAsnSerThrGlyAsnGlyTyr 
290295300 
AspIleAlaTyrValAspThrAspLeuAspTyrAspPheThrAspGlu 
305310315320 
ValProLeuGlyGlnTyrAsnValThrTyrAspValAlaValPheSer 
325330335 
TyrTyrTyrGlyProLeuAsnTyrValLeuAlaGluIleAspProAsn 
340345350 
GlyGluTyrAlaValPheGlyTrpAspGlyHisGlyHisGlyThrHis 
355360365 
ValAlaGlyThrValAlaGlyTyrAspSerAsnAsnAspAlaTrpAsp 
370375380 
TrpLeuSerMetTyrSerGlyGluTrpGluValPheSerArgLeuTyr 
385390395400 
GlyTrpAspTyrThrAsnValThrThrAspThrValGlnGlyValAla 
405410415 
ProGlyAlaGlnIleMetAlaIleArgValLeuArgSerAspGlyArg 
420425430 
GlySerMetTrpAspIleIleGluGlyMetThrTyrAlaAlaThrHis 
435440445 
GlyAlaAspValIleSerMetSerLeuGlyGlyAsnAlaProTyrLeu 
450455460 
AspGlyThrAspProGluSerValAlaValAspGluLeuThrGluLys 
465470475480 
TyrGlyValValPheValIleAlaAlaGlyAsnGluGlyProGlyIle 
485490495 
AsnIleValGlySerProGlyValAlaThrLysAlaIleThrValGly 
500505510 
AlaAlaAlaValProIleAsnValGlyValTyrValSerGlnAlaLeu 
515520525 
GlyTyrProAspTyrTyrGlyPheTyrTyrPheProAlaTyrThrAsn 
530535540 
ValArgIleAlaPhePheSerSerArgGlyProArgIleAspGlyGlu 
545550555560 
IleLysProAsnValValAlaProGlyTyrGlyIleTyrSerSerLeu 
565570575 
ProMetTrpIleGlyGlyAlaAspPheMetSerGlyThrSerMetAla 
580585590 
ThrProHisValSerGlyValValAlaLeuLeuIleSerGlyAlaLys 
595600605 
AlaGluGlyIleTyrTyrAsnProAspIleIleLysLysValLeuGlu 
610615620 
SerGlyAlaThrTrpLeuGluGlyAspProTyrThrGlyGlnLysTyr 
625630635640 
ThrGluLeuAspGlnGlyHisGlyLeuValAsnValThrLysSerTrp 
645650655 
GluIleLeuLysAlaIleAsnGlyThrThrLeuProIleValAspHis 
660665670 
TrpAlaAspLysSerTyrSerAspPheAlaGluTyrLeuGlyValAsp 
675680685 
ValIleArgGlyLeuTyrAlaArgAsnSerIleProAspIleValGlu 
690695700 
TrpHisIleLysTyrValGlyAspThrGluTyrArgThrPheGluIle 
705710715720 
TyrAlaThrGluProTrpIleLysProPheValSerGlySerValIle 
725730735 
LeuGluAsnAsnThrGluPheValLeuArgValLysTyrAspValGlu 
740745750 
GlyLeuGluProGlyLeuTyrValGlyArgIleIleIleAspAspPro 
755760765 
ThrThrProValIleGluAspGluIleLeuAsnThrIleValIlePro 
770775780 
GluLysPheThrProGluAsnAsnTyrThrLeuThrTrpTyrAspIle 
785790795800 
AsnGlyProGluMetValThrHisHisPhePheThrValProGluGly 
805810815 
ValAspValLeuTyrAlaMetThrThrTyrTrpAspTyrGlyLeuTyr 
820825830 
ArgProAspGlyMetPheValPheProTyrGlnLeuAspTyrLeuPro 
835840845 
AlaAlaValSerAsnProMetProGlyAsnTrpGluLeuValTrpThr 
850855860 
GlyPheAsnPheAlaProLeuTyrGluSerGlyPheLeuValArgIle 
865870875880 
TyrGlyValGluIleThrProSerValTrpTyrIleAsnArgThrTyr 
885890895 
LeuAspThrAsnThrGluPhe 
900 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2835 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TTTAAATTATAAGATATAATCACTCCGAGTGATGAGTAAGATACATCATTACAGTCCCAA60 
AATGTTTATAATTGGAACGCAGTGAATATACAAAATGAATATAACCTCGGAGGTGACTGT120 
AGAATGAATAAGAAGGGACTTACTGTGCTATTTATAGCGATAATGCTCCTTTCAGTAGTT180 
CCAGTGCACTTTGTGTCCGCAGAAACACCACCGGTTAGTTCAGAAAATTCAACAACTTCT240 
ATACTCCCTAACCAACAAGTTGTGACAAAAGAAGTTTCACAAGCGGCGCTTAATGCTATA300 
ATGAAAGGACAACCCAACATGGTTCTTATAATCAAGACTAAGGAAGGCAAACTTGAAGAG360 
GCAAAAACCGAGCTTGAAAAGCTAGGTGCAGAGATTCTTGACGAAAATAGAGTTCTTAAC420 
ATGTTGCTAGTTAAGATTAAGCCTGAGAAAGTTAAAGAGCTCAACTATATCTCATCTCTT480 
GAAAAAGCCTGGCTTAACAGAGAAGTTAAGCTTTCCCCTCCAATTGTCGAAAAGGACGTC540 
AAGACTAAGGAGCCCTCCCTAGAACCAAAAATGTATAACAGCACCTGGGTAATTAATGCT600 
CTCCAGTTCATCCAGGAATTTGGATATGATGGTAGTGGTGTTGTTGTTGCAGTACTTGAC660 
ACGGGAGTTGATCCGAACCATCCTTTCTTGAGCATAACTCCAGATGGACGCAGGAAAATT720 
ATAGAATGGAAGGATTTTACAGACGAGGGATTCGTGGATACATCATTCAGCTTTAGCAAG780 
GTTGTAAATGGGACTCTTATAATTAACACAACATTCCAAGTGGCCTCAGGTCTCACGCTG840 
AATGAATCGACAGGACTTATGGAATACGTTGTTAAGACTGTTTACGTGAGCAATGTGACC900 
ATTGGAAATATCACTTCTGCTAATGGCATCTATCACTTCGGCCTGCTCCCAGAAAGATAC960 
TTCGACTTAAACTTCGATGGTGATCAAGAGGACTTCTATCCTGTCTTATTAGTTAACTCC1020 
ACTGGCAATGGTTATGACATTGCATATGTGGATACTGACCTTGACTACGACTTCACCGAC1080 
GAAGTTCCACTTGGCCAGTACAACGTTACTTATGATGTTGCTGTTTTTAGCTACTACTAC1140 
GGTCCTCTCAACTACGTGCTTGCAGAAATAGATCCTAACGGAGAATATGCAGTATTTGGG1200 
TGGGATGGTCACGGTCACGGAACTCACGTAGCTGGAACTGTTGCTGGTTACGACAGCAAC1260 
AATGATGCTTGGGATTGGCTCAGTATGTACTCTGGTGAATGGGAAGTGTTCTCAAGACTC1320 
TATGGTTGGGATTATACGAACGTTACCACAGACACCGTGCAGGGTGTTGCTCCAGGTGCC1380 
CAAATAATGGCAATAAGAGTTCTTAGGAGTGATGGACGGGGTAGCATGTGGGATATTATA1440 
GAAGGTATGACATACGCAGCAACCCATGGTGCAGACGTTATAAGCATGAGTCTCGGTGGA1500 
AATGCTCCATACTTAGATGGTACTGATCCAGAAAGCGTTGCTGTGGATGAGCTTACCGAA1560 
AAGTACGGTGTTGTATTCGTAATAGCTGCAGGAAATGAAGGTCCTGGCATTAACATCGTT1620 
GGAAGTCCTGGTGTTGCAACAAAGGCAATAACTGTTGGAGCTGCTGCAGTGCCCATTAAC1680 
GTTGGAGTTTATGTTTCCCAAGCACTTGGATATCCTGATTACTATGGATTCTATTACTTC1740 
CCCGCCTACACAAACGTTAGAATAGCATTCTTCTCAAGCAGAGGGCCGAGAATAGATGGT1800 
GAAATAAAACCCAATGTAGTGGCTCCAGGTTACGGAATTTACTCATCCCTGCCGATGTGG1860 
ATTGGCGGAGCTGACTTCATGTCTGGAACTTCGATGGCTACTCCACATGTCAGCGGTGTC1920 
GTTGCACTCCTCATAAGCGGGGCAAAGGCCGAGGGAATATACTACAATCCAGATATAATT1980 
AAGAAGGTTCTTGAGAGCGGTGCAACCTGGCTTGAGGGAGATCCATATACTGGGCAGAAG2040 
TACACTGAGCTTGACCAAGGTCATGGTCTTGTTAACGTTACCAAGTCCTGGGAAATCCTT2100 
AAGGCTATAAACGGCACCACTCTCCCAATTGTTGATCACTGGGCAGACAAGTCCTACAGC2160 
GACTTTGCGGAGTACTTGGGTGTGGACGTTATAAGAGGTCTCTACGCAAGGAACTCTATA2220 
CCTGACATTGTCGAGTGGCACATTAAGTACGTAGGGGACACGGAGTACAGAACTTTTGAG2280 
ATCTATGCAACTGAGCCATGGATTAAGCCTTTTGTCAGTGGAAGTGTAATTCTAGAGAAC2340 
AATACCGAGTTTGTCCTTAGGGTGAAATATGATGTAGAGGGTCTTGAGCCAGGTCTCTAT2400 
GTTGGAAGGATAATCATTGATGATCCAACAACGCCAGTTATTGAAGACGAGATCTTGAAC2460 
ACAATTGTTATTCCCGAGAAGTTCACTCCTGAGAACAATTACACCCTCACCTGGTATGAT2520 
ATTAATGGTCCAGAAATGGTGACTCACCACTTCTTCACTGTGCCTGAGGGAGTGGACGTT2580 
CTCTACGCGATGACCACATACTGGGACTACGGTCTGTACAGACCAGATGGAATGTTTGTG2640 
TTCCCATACCAGCTAGATTATCTTCCCGCTGCAGTCTCAAATCCAATGCCTGGAAACTGG2700 
GAGCTAGTATGGACTGGATTTAACTTTGCACCCCTCTATGAGTCGGGCTTCCTTGTAAGG2760 
ATTTACGGAGTAGAGATAACTCCAAGCGTTTGGTACATTAACAGGACATACCTTGACACT2820 
AACACTGAATTCTAG2835 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGWWSDRRTGTTRRHGTHGCDGTDMTYGACACSGG35 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
KSTCACGGAACTCACGTDGCBGGMACDGTTGC32 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ASCMGCAACHGTKCCVGCHACGTGAGTTCCGTG33 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CHCCGSYVACRTGBGGAGWDGCCATBGAVGTDCC34 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 898 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GATCTGAAGGGCAAGGTCATAGGCTGGTACGACGCCGTCAACGGCAGGTCGACCCCCTAC60 
GATGACCAGGGACACGGAACCCACGTTGCGGGTATCGTTGCCGGAACCGGCAGCGTTAAC120 
TCCCAGTACATAGGCGTCGCCCCCGGCGCGAAGCTCGTCGGCGTCAAGGTTCTCGGTGCC180 
GACGGTTCGGGAAGCGTCTCCACCATCATCGCGGGTGTTGACTGGGTCGTCCAGAACAAG240 
GACAAGTACGGGATAAGGGTCATCAACCTCTCCCTCGGCTCCTCCCAGAGCTCCGACGGA300 
ACCGACTCCCTCAGTCAGGCCGTCAACAACGCCTGGGACGCCGGTATAGTAGTCTGCGTC360 
GCCGCCGGCAACAGCGGGCCGAACACCTACACCGTCGGCTCACCCGCCGCCGCGAGCAAG420 
GTCATAACCGTCGGTGCAGTTGACAGCAACGACAACATCGCCAGCTTCTCCAGCAGGGGA480 
CCGACCGCGGACGGAAGGCTCAAGCCGGAAGTCGTCGCCCCCGGCGTTGACATCATAGCC540 
CCGCGCGCCAGCGGAACCAGCATGGGCACCCCGATAAACGACTACTNCAACAAGGGCTCT600 
GGATCCAGCATGGACACCCCGCACGTTTCGGGCGTTGGCGGGCTCATCCTCCAGGCCCAC660 
CCGAGCTGGACCCCGGACAAGGTGAAGACGCCCTCATCGAGACCGCCGACATAGTCGNCC720 
CCAAGGAGATAGCGGACATCGCCTACGGTGCGGGTAGGGTGAACGTCTTCAAGGGCATCA780 
AGTNCGACGACTACGNCAAGNTCACCTTCACCGGNTCCGTCGGCGACAAGGGAAGGGGCA840 
CCACACCTTCGACGTCAGNGGGGGCACTTCGTGAACGNCACCCTCTNCTNGGACANGG898 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4765 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TTTAAATTATAAGATATAATCACTCCGAGTGATGAGTAAGATACATCATTACAGTCCCAA60 
AATGTTTATAATTGGAACGCAGTGAATATACAAAATGAATATAACCTCGGAGGTGACTGT120 
AGAATGAATAAGAAGGGACTTACTGTGCTATTTATAGCGATAATGCTCCTTTCAGTAGTT180 
CCAGTGCACTTTGTGTCCGCAGAAACACCACCGGTTAGTTCAGAAAATTCAACAACTTCT240 
ATACTCCCTAACCAACAAGTTGTGACAAAAGAAGTTTCACAAGCGGCGCTTAATGCTATA300 
ATGAAAGGACAACCCAACATGGTTCTTATAATCAAGACTAAGGAAGGCAAACTTGAAGAG360 
GCAAAAACCGAGCTTGAAAAGCTAGGTGCAGAGATTCTTGACGAAAATAGAGTTCTTAAC420 
ATGTTGCTAGTTAAGATTAAGCCTGAGAAAGTTAAAGAGCTCAACTATATCTCATCTCTT480 
GAAAAAGCCTGGCTTAACAGAGAAGTTAAGCTTTCCCCTCCAATTGTCGAAAAGGACGTC540 
AAGACTAAGGAGCCCTCCCTAGAACCAAAAATGTATAACAGCACCTGGGTAATTAATGCT600 
CTCCAGTTCATCCAGGAATTTGGATATGATGGTAGTGGTGTTGTTGTTGCAGTACTTGAC660 
ACGGGAGTTGATCCGAACCATCCTTTCTTGAGCATAACTCCAGATGGACGCAGGAAAATT720 
ATAGAATGGAAGGATTTTACAGACGAGGGATTCGTGGATACATCATTCAGCTTTAGCAAG780 
GTTGTAAATGGGACTCTTATAATTAACACAACATTCCAAGTGGCCTCAGGTCTCACGCTG840 
AATGAATCGACAGGACTTATGGAATACGTTGTTAAGACTGTTTACGTGAGCAATGTGACC900 
ATTGGAAATATCACTTCTGCTAATGGCATCTATCACTTCGGCCTGCTCCCAGAAAGATAC960 
TTCGACTTAAACTTCGATGGTGATCAAGAGGACTTCTATCCTGTCTTATTAGTTAACTCC1020 
ACTGGCAATGGTTATGACATTGCATATGTGGATACTGACCTTGACTACGACTTCACCGAC1080 
GAAGTTCCACTTGGCCAGTACAACGTTACTTATGATGTTGCTGTTTTTAGCTACTACTAC1140 
GGTCCTCTCAACTACGTGCTTGCAGAAATAGATCCTAACGGAGAATATGCAGTATTTGGG1200 
TGGGATGGTCACGGTCACGGAACTCACGTAGCTGGAACTGTTGCTGGTTACGACAGCAAC1260 
AATGATGCTTGGGATTGGCTCAGTATGTACTCTGGTGAATGGGAAGTGTTCTCAAGACTC1320 
TATGGTTGGGATTATACGAACGTTACCACAGACACCGTGCAGGGTGTTGCTCCAGGTGCC1380 
CAAATAATGGCAATAAGAGTTCTTAGGAGTGATGGACGGGGTAGCATGTGGGATATTATA1440 
GAAGGTATGACATACGCAGCAACCCATGGTGCAGACGTTATAAGCATGAGTCTCGGTGGA1500 
AATGCTCCATACTTAGATGGTACTGATCCAGAAAGCGTTGCTGTGGATGAGCTTACCGAA1560 
AAGTACGGTGTTGTATTCGTAATAGCTGCAGGAAATGAAGGTCCTGGCATTAACATCGTT1620 
GGAAGTCCTGGTGTTGCAACAAAGGCAATAACTGTTGGAGCTGCTGCAGTGCCCATTAAC1680 
GTTGGAGTTTATGTTTCCCAAGCACTTGGATATCCTGATTACTATGGATTCTATTACTTC1740 
CCCGCCTACACAAACGTTAGAATAGCATTCTTCTCAAGCAGAGGGCCGAGAATAGATGGT1800 
GAAATAAAACCCAATGTAGTGGCTCCAGGTTACGGAATTTACTCATCCCTGCCGATGTGG1860 
ATTGGCGGAGCTGACTTCATGTCTGGAACTTCGATGGCTACTCCACATGTCAGCGGTGTC1920 
GTTGCACTCCTCATAAGCGGGGCAAAGGCCGAGGGAATATACTACAATCCAGATATAATT1980 
AAGAAGGTTCTTGAGAGCGGTGCAACCTGGCTTGAGGGAGATCCATATACTGGGCAGAAG2040 
TACACTGAGCTTGACCAAGGTCATGGTCTTGTTAACGTTACCAAGTCCTGGGAAATCCTT2100 
AAGGCTATAAACGGCACCACTCTCCCAATTGTTGATCACTGGGCAGACAAGTCCTACAGC2160 
GACTTTGCGGAGTACTTGGGTGTGGACGTTATAAGAGGTCTCTACGCAAGGAACTCTATA2220 
CCTGACATTGTCGAGTGGCACATTAAGTACGTAGGGGACACGGAGTACAGAACTTTTGAG2280 
ATCTATGCAACTGAGCCATGGATTAAGCCTTTTGTCAGTGGAAGTGTAATTCTAGAGAAC2340 
AATACCGAGTTTGTCCTTAGGGTGAAATATGATGTAGAGGGTCTTGAGCCAGGTCTCTAT2400 
GTTGGAAGGATAATCATTGATGATCCAACAACGCCAGTTATTGAAGACGAGATCTTGAAC2460 
ACAATTGTTATTCCCGAGAAGTTCACTCCTGAGAACAATTACACCCTCACCTGGTATGAT2520 
ATTAATGGTCCAGAAATGGTGACTCACCACTTCTTCACTGTGCCTGAGGGAGTGGACGTT2580 
CTCTACGCGATGACCACATACTGGGACTACGGTCTGTACAGACCAGATGGAATGTTTGTG2640 
TTCCCATACCAGCTAGATTATCTTCCCGCTGCAGTCTCAAATCCAATGCCTGGAAACTGG2700 
GAGCTAGTATGGACTGGATTTAACTTTGCACCCCTCTATGAGTCGGGCTTCCTTGTAAGG2760 
ATTTACGGAGTAGAGATAACTCCAAGCGTTTGGTACATTAACAGGACATACCTTGACACT2820 
AACACTGAATTCTCAATTGAATTCAATATTACTAACATCTATGCCCCAATTAATGCAACT2880 
CTAATCCCCATTGGCCTTGGAACCTACAATGCGAGCGTTGAAAGCGTTGGTGATGGAGAG2940 
TTCTTCATAAAGGGCATTGAAGTTCCTGAAGGCACCGCAGAGTTGAAGATTAGGATAGGC3000 
AACCCAAGTGTTCCGAATTCAGATCTAGACTTGTACCTTTATGACAGTAAAGGCAATTTA3060 
GTGGCCTTAGATGGAAACCCAACAGCAGAAGAAGAGGTTGTAGTTGAGTATCCTAAGCCT3120 
GGAGTTTATTCAATAGTAGTACATGGTTACAGCGTCAGGGACGAAAATGGTAATCCAACG3180 
ACAACCACCTTTGACTTAGTTGTTCAAATGACCCTTGATAATGGAAACATAAAGCTTGAC3240 
AAAGACTCGATTATTCTTGGAAGCAATGAAAGCGTAGTTGTAACTGCAAACATAACAATT3300 
GATAGAGATCATCCTACAGGAGTATACTCTGGTATCATAGAGATTAGAGATAATGAGGTC3360 
TACCAGGATACAAATACTTCAATTGCGAAAATACCCATAACTTTGGTAATTGACAAGGCG3420 
GACTTTGCCGTTGGTCTCACACCAGCAGAGGGAGTACTTGGAGAGGCTAGAAATTACACT3480 
CTAATTGTAAAGCATGCCCTAACACTAGAGCCTGTGCCAAATGCTACAGTGATTATAGGA3540 
AACTACACCTACCTCACAGACGAAAACGGTACAGTGACATTCACGTATGCTCCAACTAAG3600 
TTAGGCAGTGATGAAATCACAGTCATAGTTAAGAAAGAGAACTTCAACACATTAGAGAAG3660 
ACCTTCCAAATCACAGTATCAGAGCCTGAAATAACTGAAGAGGACATAAATGAGCCCAAG3720 
CTTGCAATGTCATCACCAGAAGCAAATGCTACCATAGTATCAGTTGAGATGGAGAGTGAG3780 
GGTGGCGTTAAAAAGACAGTGACAGTGGAAATAACTATAAACGGAACCGCTAATGAGACT3840 
GCAACAATAGTGGTTCCTGTTCCTAAGAAGGCCGAAAACATCGAGGTAAGTGGAGACCAC3900 
GTAATTTCCTATAGTATAGAGGAAGGAGAGTACGCCAAGTACGTTATAATTACAGTGAAG3960 
TTTGCATCACCTGTAACAGTAACTGTTACTTACACTATCTATGCTGGCCCAAGAGTCTCA4020 
ATCTTGACACTTAACTTCCTTGGCTACTCATGGTACAGACTATATTCACAGAAGTTTGAC4080 
GAATTGTACCAAAAGGCCCTTGAATTGGGAGTGGACAACGAGACATTAGCTTTAGCCCTC4140 
AGCTACCATGAAAAAGCCAAAGAGTACTACGAAAAGGCCCTTGAGCTTAGCGAGGGTAAC4200 
ATAATCCAATACCTTGGAGACATAAGACTATTACCTCCATTAAGACAGGCATACATCAAT4260 
GAAATGAAGGCAGTTAAGATACTGGAAAAGGCCATAGAAGAATTAGAGGGTGAAGAGTAA4320 
TCTCCAATTTTTCCCACTTTTTCTTTTATAACATTCCAAGCCTTTTCTTAGCTTCTTCGC4380 
TCATTCTATCAGGAGTCCATGGAGGATCAAAGGTAAGTTCAACCTCCACATCTCTTACTC4440 
CTGGGATTTCGAGTACTTTCTCCTCTACAGCTCTAAGAAGCCAGAGAGTTAAAGGACACC4500 
CAGGAGTTGTCATTGTCATCTTTATATATACCGTTTTGTCAGGATTAATCTTTAGCTCAT4560 
AAATTAATCCAAGGTTTACAACATCCATCCCAATTTCTGGGTCGATAACCTCCTTTAGCT4620 
TTTCCAGAATCATTTCTTCAGTAATTTCAAGGTTCTCATCTTTGGTTTCTCTCACAAACC4680 
CAATTTCAACCTGCCTGATACCTTCTAACTCCCTAAGCTTGTTATATATCTCCAAAAGAG4740 
TGGCATCATCAATTTTCTCTTTAAA4765 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1398 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetAsnLysLysGlyLeuThrValLeuPheIleAlaIleMetLeuLeu 
151015 
SerValValProValHisPheValSerAlaGluThrProProValSer 
202530 
SerGluAsnSerThrThrSerIleLeuProAsnGlnGlnValValThr 
354045 
LysGluValSerGlnAlaAlaLeuAsnAlaIleMetLysGlyGlnPro 
505560 
AsnMetValLeuIleIleLysThrLysGluGlyLysLeuGluGluAla 
65707580 
LysThrGluLeuGluLysLeuGlyAlaGluIleLeuAspGluAsnArg 
859095 
ValLeuAsnMetLeuLeuValLysIleLysProGluLysValLysGlu 
100105110 
LeuAsnTyrIleSerSerLeuGluLysAlaTrpLeuAsnArgGluVal 
115120125 
LysLeuSerProProIleValGluLysAspValLysThrLysGluPro 
130135140 
SerLeuGluProLysMetTyrAsnSerThrTrpValIleAsnAlaLeu 
145150155160 
GlnPheIleGlnGluPheGlyTyrAspGlySerGlyValValValAla 
165170175 
ValLeuAspThrGlyValAspProAsnHisProPheLeuSerIleThr 
180185190 
ProAspGlyArgArgLysIleIleGluTrpLysAspPheThrAspGlu 
195200205 
GlyPheValAspThrSerPheSerPheSerLysValValAsnGlyThr 
210215220 
LeuIleIleAsnThrThrPheGlnValAlaSerGlyLeuThrLeuAsn 
225230235240 
GluSerThrGlyLeuMetGluTyrValValLysThrValTyrValSer 
245250255 
AsnValThrIleGlyAsnIleThrSerAlaAsnGlyIleTyrHisPhe 
260265270 
GlyLeuLeuProGluArgTyrPheAspLeuAsnPheAspGlyAspGln 
275280285 
GluAspPheTyrProValLeuLeuValAsnSerThrGlyAsnGlyTyr 
290295300 
AspIleAlaTyrValAspThrAspLeuAspTyrAspPheThrAspGlu 
305310315320 
ValProLeuGlyGlnTyrAsnValThrTyrAspValAlaValPheSer 
325330335 
TyrTyrTyrGlyProLeuAsnTyrValLeuAlaGluIleAspProAsn 
340345350 
GlyGluTyrAlaValPheGlyTrpAspGlyHisGlyHisGlyThrHis 
355360365 
ValAlaGlyThrValAlaGlyTyrAspSerAsnAsnAspAlaTrpAsp 
370375380 
TrpLeuSerMetTyrSerGlyGluTrpGluValPheSerArgLeuTyr 
385390395400 
GlyTrpAspTyrThrAsnValThrThrAspThrValGlnGlyValAla 
405410415 
ProGlyAlaGlnIleMetAlaIleArgValLeuArgSerAspGlyArg 
420425430 
GlySerMetTrpAspIleIleGluGlyMetThrTyrAlaAlaThrHis 
435440445 
GlyAlaAspValIleSerMetSerLeuGlyGlyAsnAlaProTyrLeu 
450455460 
AspGlyThrAspProGluSerValAlaValAspGluLeuThrGluLys 
465470475480 
TyrGlyValValPheValIleAlaAlaGlyAsnGluGlyProGlyIle 
485490495 
AsnIleValGlySerProGlyValAlaThrLysAlaIleThrValGly 
500505510 
AlaAlaAlaValProIleAsnValGlyValTyrValSerGlnAlaLeu 
515520525 
GlyTyrProAspTyrTyrGlyPheTyrTyrPheProAlaTyrThrAsn 
530535540 
ValArgIleAlaPhePheSerSerArgGlyProArgIleAspGlyGlu 
545550555560 
IleLysProAsnValValAlaProGlyTyrGlyIleTyrSerSerLeu 
565570575 
ProMetTrpIleGlyGlyAlaAspPheMetSerGlyThrSerMetAla 
580585590 
ThrProHisValSerGlyValValAlaLeuLeuIleSerGlyAlaLys 
595600605 
AlaGluGlyIleTyrTyrAsnProAspIleIleLysLysValLeuGlu 
610615620 
SerGlyAlaThrTrpLeuGluGlyAspProTyrThrGlyGlnLysTyr 
625630635640 
ThrGluLeuAspGlnGlyHisGlyLeuValAsnValThrLysSerTrp 
645650655 
GluIleLeuLysAlaIleAsnGlyThrThrLeuProIleValAspHis 
660665670 
TrpAlaAspLysSerTyrSerAspPheAlaGluTyrLeuGlyValAsp 
675680685 
ValIleArgGlyLeuTyrAlaArgAsnSerIleProAspIleValGlu 
690695700 
TrpHisIleLysTyrValGlyAspThrGluTyrArgThrPheGluIle 
705710715720 
TyrAlaThrGluProTrpIleLysProPheValSerGlySerValIle 
725730735 
LeuGluAsnAsnThrGluPheValLeuArgValLysTyrAspValGlu 
740745750 
GlyLeuGluProGlyLeuTyrValGlyArgIleIleIleAspAspPro 
755760765 
ThrThrProValIleGluAspGluIleLeuAsnThrIleValIlePro 
770775780 
GluLysPheThrProGluAsnAsnTyrThrLeuThrTrpTyrAspIle 
785790795800 
AsnGlyProGluMetValThrHisHisPhePheThrValProGluGly 
805810815 
ValAspValLeuTyrAlaMetThrThrTyrTrpAspTyrGlyLeuTyr 
820825830 
ArgProAspGlyMetPheValPheProTyrGlnLeuAspTyrLeuPro 
835840845 
AlaAlaValSerAsnProMetProGlyAsnTrpGluLeuValTrpThr 
850855860 
GlyPheAsnPheAlaProLeuTyrGluSerGlyPheLeuValArgIle 
865870875880 
TyrGlyValGluIleThrProSerValTrpTyrIleAsnArgThrTyr 
885890895 
LeuAspThrAsnThrGluPheSerIleGluPheAsnIleThrAsnIle 
900905910 
TyrAlaProIleAsnAlaThrLeuIleProIleGlyLeuGlyThrTyr 
915920925 
AsnAlaSerValGluSerValGlyAspGlyGluPhePheIleLysGly 
930935940 
IleGluValProGluGlyThrAlaGluLeuLysIleArgIleGlyAsn 
945950955960 
ProSerValProAsnSerAspLeuAspLeuTyrLeuTyrAspSerLys 
965970975 
GlyAsnLeuValAlaLeuAspGlyAsnProThrAlaGluGluGluVal 
980985990 
ValValGluTyrProLysProGlyValTyrSerIleValValHisGly 
99510001005 
TyrSerValArgAspGluAsnGlyAsnProThrThrThrThrPheAsp 
101010151020 
LeuValValGlnMetThrLeuAspAsnGlyAsnIleLysLeuAspLys 
1025103010351040 
AspSerIleIleLeuGlySerAsnGluSerValValValThrAlaAsn 
104510501055 
IleThrIleAspArgAspHisProThrGlyValTyrSerGlyIleIle 
106010651070 
GluIleArgAspAsnGluValTyrGlnAspThrAsnThrSerIleAla 
107510801085 
LysIleProIleThrLeuValIleAspLysAlaAspPheAlaValGly 
109010951100 
LeuThrProAlaGluGlyValLeuGlyGluAlaArgAsnTyrThrLeu 
1105111011151120 
IleValLysHisAlaLeuThrLeuGluProValProAsnAlaThrVal 
112511301135 
IleIleGlyAsnTyrThrTyrLeuThrAspGluAsnGlyThrValThr 
114011451150 
PheThrTyrAlaProThrLysLeuGlySerAspGluIleThrValIle 
115511601165 
ValLysLysGluAsnPheAsnThrLeuGluLysThrPheGlnIleThr 
117011751180 
ValSerGluProGluIleThrGluGluAspIleAsnGluProLysLeu 
1185119011951200 
AlaMetSerSerProGluAlaAsnAlaThrIleValSerValGluMet 
120512101215 
GluSerGluGlyGlyValLysLysThrValThrValGluIleThrIle 
122012251230 
AsnGlyThrAlaAsnGluThrAlaThrIleValValProValProLys 
123512401245 
LysAlaGluAsnIleGluValSerGlyAspHisValIleSerTyrSer 
125012551260 
IleGluGluGlyGluTyrAlaLysTyrValIleIleThrValLysPhe 
1265127012751280 
AlaSerProValThrValThrValThrTyrThrIleTyrAlaGlyPro 
128512901295 
ArgValSerIleLeuThrLeuAsnPheLeuGlyTyrSerTrpTyrArg 
130013051310 
LeuTyrSerGlnLysPheAspGluLeuTyrGlnLysAlaLeuGluLeu 
131513201325 
GlyValAspAsnGluThrLeuAlaLeuAlaLeuSerTyrHisGluLys 
133013351340 
AlaLysGluTyrTyrGluLysAlaLeuGluLeuSerGluGlyAsnIle 
1345135013551360 
IleGlnTyrLeuGlyAspIleArgLeuLeuProProLeuArgGlnAla 
136513701375 
TyrIleAsnGluMetLysAlaValLysIleLeuGluLysAlaIleGlu 
138013851390 
GluLeuGluGlyGluGlu 
1395 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 145 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 2..145 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AGTTGCGGTAATTGACACGGGTATAGACGCGAACCACCCCGATCTG46 
ValAlaValIleAspThrGlyIleAspAlaAsnHisProAspLeu 
151015 
AAGGGCAAGGTCATAGGCTGGTACGACGCCGTCAACGGCAGGTCGACC94 
LysGlyLysValIleGlyTrpTyrAspAlaValAsnGlyArgSerThr 
202530 
CCCTACGATGACCAGGGACACGGAACTCACGTNGCNGGAACNGTTGCT142 
ProTyrAspAspGlnGlyHisGlyThrHisValAlaGlyThrValAla 
354045 
GGT145 
Gly 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 564 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..564 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TCTCACGGAACTCACGTGGCGGGAACAGTTGCCGGAACAGGCAGCGTT48 
SerHisGlyThrHisValAlaGlyThrValAlaGlyThrGlySerVal 
505560 
AACTCCCAGTACATAGGCGTCGCCCCCGGCGCGAAGCTCGTCGGTGTC96 
AsnSerGlnTyrIleGlyValAlaProGlyAlaLysLeuValGlyVal 
65707580 
AAGGTTCTCGGTGCCGACGGTTCGGGAAGCGTCTCCACCATCATCGCG144 
LysValLeuGlyAlaAspGlySerGlySerValSerThrIleIleAla 
859095 
GGTGTTGACTGGGTCGTCCAGAACAAGGATAAGTACGGGATAAGGGTC192 
GlyValAspTrpValValGlnAsnLysAspLysTyrGlyIleArgVal 
100105110 
ATCAACCTCTCCCTCGGCTCCTCCCAGAGCTCCGACGGAGCCGACTCC240 
IleAsnLeuSerLeuGlySerSerGlnSerSerAspGlyAlaAspSer 
115120125 
CTCAGTCAGGCCGTCAACAACGCCTGGGACGCCGGTATAGTAGTCTGC288 
LeuSerGlnAlaValAsnAsnAlaTrpAspAlaGlyIleValValCys 
130135140 
GTCGCCGCCGGCAACAGCGGGCCGAACACCTACACCGTCGGCTCACCC336 
ValAlaAlaGlyAsnSerGlyProAsnThrTyrThrValGlySerPro 
145150155160 
GCCGCCGCGAGCAAGGTCATAACCGTCGGTGCAGTTGACAGCAACGAC384 
AlaAlaAlaSerLysValIleThrValGlyAlaValAspSerAsnAsp 
165170175 
AACATCGCCAGCTTCTCCAGCAGGGGACCGACCGCGGACGGAAGGCTC432 
AsnIleAlaSerPheSerSerArgGlyProThrAlaAspGlyArgLeu 
180185190 
AAGCCGGAAGTCGTCGCCCCCGGCGTTGACATCATAGCCCCGCGCGCC480 
LysProGluValValAlaProGlyValAspIleIleAlaProArgAla 
195200205 
AGCGGAACCAGCATGGGCACCCCGATAAACGACTACTACACCAAGGCC528 
SerGlyThrSerMetGlyThrProIleAsnAspTyrTyrThrLysAla 
210215220 
TCTGGAACCTCAATGGCCACTCCCCATGTTACCGGT564 
SerGlyThrSerMetAlaThrProHisValThrGly 
225230235 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GGCAAGGTCATAGGCTGGTA20 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CCAGAACAAGGATAAGTACG20 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GGCACCCCGATAAACGACTA20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ACGCCTATGTACTGGGAGTT20 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CGTACTTATCCTTGTTCTGG20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
TGTAGTAGTCGTTTATCGGG20 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 237 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AspLeuLysGlyLysValIleGlyTrpTyrAspAlaValAsnGlyArg 
151015 
SerThrProTyrAspAspGlnGlyHisGlyThrHisValAlaGlyIle 
202530 
ValAlaGlyThrGlySerValAsnSerGlnTyrIleGlyValAlaPro 
354045 
GlyAlaLysLeuValGlyValLysValLeuGlyAlaAspGlySerGly 
505560 
SerValSerThrIleIleAlaGlyValAspTrpValValGlnAsnLys 
65707580 
AspXaaTyrGlyIleArgValIleAsnLeuSerLeuGlySerSerGln 
859095 
SerSerAspGlyThrAspSerLeuSerGlnAlaValAsnAsnAlaTrp 
100105110 
AspAlaGlyIleValValCysValAlaAlaGlyAsnSerGlyProAsn 
115120125 
ThrTyrThrValGlySerProAlaAlaAlaSerLysValIleThrVal 
130135140 
GlyAlaValAspSerAsnAspAsnIleAlaSerPheSerSerArgGly 
145150155160 
ProThrAlaAspGlyArgLeuLysProGluValValAlaProGlyVal 
165170175 
AspIleIleAlaProArgAlaSerGlyThrSerMetGlyThrProIle 
180185190 
AsnAspTyrXaaAsnLysGlySerGlySerSerMetAspThrProHis 
195200205 
ValSerGlyValGlyGlyLeuIleLeuGlnAlaHisProSerTrpThr 
210215220 
ProAspLysValLysThrProSerSerArgProProThr 
225230235 
__________________________________________________________________________