JPH09149790A - New serine protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new serine protease derived from a human colonic cancer cell and available from genetic engineering, having a specific amino acid sequence capable of establishing a screening system of an inhibitor specific to the enzyme and utilizing for screening, etc., of treating agents for various kinds of diseases. SOLUTION: This new serine protease or its partially peptide contains an amino acid sequence (leucine of amino acid No. 1 may be lost) of amino acid Nos. 1-223 represented by formula 1, an amino acid sequence of an amino acid Nos. 1-233 represented by formula II and an amino acid sequence of amino acid Nos. 1-241 represented by formula III, and can be utilized for screening, etc., of a treating agent for various kinds of diseases by screening system of an inhibitor which is specific to the enzyme. The serine protease is obtained by extracting mRNA from COLO 201 cell derived from human colonic cancer and preparing cDNA library using the mRNA and screening cDNA library, integrating the resultant serine protease gene into a vector and expressing the gene in a host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel serine protease, a gene encoding the same, a method for producing the serine protease, and a method for screening an inhibitor using the serine protease.

[0002]

BACKGROUND OF THE INVENTION Serine proteases are widely present in animals, plants and microorganisms, and especially in higher animals, digestion of food,
It is known to be involved in numerous biological reactions such as blood coagulation / fibrinolysis, complement activation, hormone production, ovulation / fertilization, phagocytosis, cell proliferation, development / differentiation, aging, and cancer metastasis (Neurath , H. Science, 224, 350-357, 1984). Further, serine proteases in higher animals are roughly classified into the chymotrypsin family and the subtilisin family based on the primary structure of their active centers. In the chymotrypsin family, a histidine residue is essential in addition to the serine residue at the active center for its activity expression, and the amino acid sequences near the serine residue and histidine residue are well conserved. It has been known.

Therefore, using these saved areas, the PC
Attempts have also been made to clone the serine protease gene by the R method. That is, alanine-alanine-histidine-in the vicinity of histidine residues, which are often conserved in serine proteases, are used as PCR primers.
Aspartic acid-serine-glycine-glycine-proline (DSGG) near cysteine (AAHC) and serine residues
It has been reported that P) was used to isolate a novel serine protease gene.

For example, Sakanari et al. Isolated a serine protease gene from nematodes and protozoa that has 67% similarity to rat trypsin II (Sakanari, JA, Stau.
nton, CE, Eakin, AE, Craik, CS and McKerr
ow, JH, Proc. Natl. Acad. Sci. USA, 86, 4863-48
67, 1989). The Mueller-Hill group also analyzed rat pancreas to rat trypsin V and rat elastase.
Isolate IV (Kang, J., Wiegand, U. and Mueller-Hil
l, B., Gene, 110, 181-187, 1992), and human trypsin IV was isolated from human brain in the same group (Wiega
nd, U., Corbach, S., Minn, A., Kang, J. and Muelle
r-Hill, B., Gene, 136, 167-175, 1993).

However, in these prior art documents, isolation was carried out based on cDNAs derived from nematodes and protozoa as well as pancreatic and brain tissues. Moreover, since the serine protease gene isolated using such PCR primers exists as a zymogen, it has not yet been confirmed whether it is a gene encoding a protein having serine protease activity. Furthermore, it is not difficult to imagine that the isolation of the serine protease gene will become easier if the cDNAs of not only nematodes and protozoa and organs but also various cancer cell lines that can be propagated by culture can be used. However, the reality is that serine protease activity cannot be measured in the supernatant of cultured cells to which serum is added.

[0006]

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel serine protease and a novel serine protease gene encoding the same. Another object of the present invention is to provide a method for producing a large amount of the protease using the gene and a method for screening a specific inhibitory substance using the enzyme.

[0007]

The present inventors have focused on human colon cancer COLO 201 cells as a starting material for the isolation of a novel serine protease gene. That is,
The present inventors recognized serine protease enzyme activity in the culture supernatant obtained by culturing COLO 201 cells in a protein-free medium,
For the isolation of such a novel serine protease gene, it was discovered that it is effective to use cDNA prepared from cancer cells such as COLO 201 cells. Moreover, in order to confirm whether or not the isolated gene truly encodes an enzyme activity, the gene was successfully expressed as a mature protein, and the present invention was completed.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION COLO 201 cells derived from human colon cancer
(ATCC CCL-224) can be cultured by any method commonly used for culturing animal cells. It is also possible to carry out static culture using a medium containing no protein. A specific example is described in Example 1.

The enzyme activity in the culture supernatant can be easily measured by using a commercially available synthetic substrate to which 7-amino-4-methylcoumarin or p-nitroanilide is bound. A specific example is described in Example 2. As a result, serine protease enzyme activity was clearly observed in the culture supernatant of human colon cancer-derived COLO 201 cells. Therefore, the following experiment was carried out for the purpose of isolating all serine protease genes expressed in COLO 201 cells derived from human colon cancer, including this enzyme activity. That is, mRNA was isolated and purified from human colon cancer-derived COLO 201 cells, and cDNA was isolated.
A library was made. PC designed from the prepared cDNA library based on the serine protease motif
Screening by PCR was performed using the R primer, and the obtained PCR product was subcloned.

As a result, a clone was confirmed that contained a nucleotide sequence encoding an amino acid conserved in serine protease between the active residues serine and histidine. Using the gene thus obtained as a probe, a full-length gene was cloned by a conventional method. As a result, SP59 gene, SP60 gene and SP67 gene were isolated and a novel serine protease could be confirmed. A specific example is described in Example 3.

[0011] As a result of the above, the inventors of the present invention have found that from the cDNA of human colon cancer-derived COLO 201 cells, novel serine protease genes (SP59 gene, SP60 gene and SP67 gene) having a similarity of less than 30% with known serine proteases. )
Was successfully isolated. Moreover, when the expression of mRNA in human organs was confirmed using the isolated novel serine protease gene as a probe, SP59 gene, SP60 gene and SP67 gene were found.
Expression of both genes was found in human organs, and the SP59 gene was particularly strongly expressed in the brain and had a size of about 1.4 kb. A specific example is described in Example 4. From this,
It was confirmed that the isolated novel serine protease gene was also expressed in human organs.

From the structure of the isolated novel serine protease gene, a method for expressing it as a mature protein in animal cells was devised. That is, trypsin, which is a typical serine protease, is known to exist as a mature active protein having isoleucine as the N-terminal amino acid by the action of the enzyme enterokinase distributed in the duodenal mucosa after being expressed as a pro-form, trypsinogen. ing.

Therefore, a chimeric gene (Trp59 gene) was prepared in which the gene encoding the mature protein of the SP59 gene was connected to the signal sequence of the trypsin gene and the gene encoding the enterokinase recognition sequence. The Trp59 gene, which is a chimeric gene, was
After transfection into S-1 cells, enterokinase was allowed to act on the culture supernatant of COS-1 cells, and as a result, serine protease enzyme activity was confirmed. A specific method is described in Example 5.

From the above results, not only was it revealed that the serine protease gene isolated this time was a novel serine protease gene due to its primary structure, but it was also revealed that the serine protease gene expressed activity as a mature protein. . In the present invention, the nucleotide sequences shown in SEQ ID NOs: 3, 4 and 5 are disclosed as the nucleotide sequences of the gene encoding the novel serine protease, but the serine protease gene of the present invention is not limited thereto. Once the amino acid sequence of the natural serine protease has been determined, it is possible to design and prepare various nucleotide sequences encoding the same amino acid sequence, based on the degeneracy of the codons. In this case, it is preferable to use codons that are frequently used depending on the host to be used.

In order to obtain the gene encoding the natural serine protease of the present invention, cD is prepared as described in Example 3.
NA can be obtained, but is not limited to. That is, if one nucleotide sequence encoding the amino acid sequence of the natural serine protease is determined, the gene encoding the natural serine protease is a cDNA according to a strategy different from the strategy specifically disclosed in the present invention.
It can also be cloned from the genome of the cell that produces it.

When cloning from the genome, the various primer or probe nucleotides used in Example 3 can be used as probes for the selection of genomic DNA fragments. Also, other probes designed based on the nucleotide sequences shown in SEQ ID NOs: 3, 4 or 5 can be used. General methods for cloning the desired DNA from the genome are well known in the art (Current Pr
otocols In Molecular Biology, John Wiley & Sons
Company, Chapters 5 and 6).

The gene encoding the natural serine protease of the present invention can also be prepared by chemical synthesis. Chemical synthesis of DNA is automated DNA in the industry
Synthesizer, eg Applied Biosystems 396DN
It is easy to adopt an A / RNA synthesizer or the like. Therefore, those skilled in the art can easily synthesize the DNA having the nucleotide sequences shown in SEQ ID NOs: 3, 4 and 5.

The gene encoding the natural serine protease of the present invention with a codon different from the natural codon is
It can also be prepared by chemical synthesis as described above, or is a site-directed mutagenesis method using DNA or RNA having the nucleotide sequence shown in SEQ ID NO: 3, 4 or 5 as a template together with a mutagenesis primer.
It can also be obtained according to a conventional method such as cted mutagenesis (eg, Current Protocols In Molec).
ular Biology, John Wiley & Sons, Chapter 8).

When the serine protease gene of the present invention is obtained as described above, it can be used to produce a recombinant serine protease by a conventional gene recombination method. That is, the DNA encoding the serine protease of the present invention is inserted into an appropriate expression vector, the expression vector is introduced into an appropriate host cell, the host cell is cultured, and the obtained culture (cell or medium) From the target serine protease. The serine protease of the present invention may be obtained in a form that is biochemically or chemically modified, for example, N-terminally acylated, for example, C _1-6 acylated such as formylated, acetylated or deleted. Good. The expression system can be improved in secretion efficiency and expression level by adding or improving a signal sequence or selecting a host. As a means for adding and improving a signal sequence, a gene encoding a signal peptide of another structural peptide may be inserted upstream of the structural gene of the serine protease of the present invention on the 5 ′ side via a gene encoding a cleavable partial peptide. The method of connecting is mentioned. A specific example is the method using the gene encoding the signal sequence of the trypsin gene and the enterokinase recognition sequence described in Example 5.

Prokaryote or eukaryote can be used as the host. As the prokaryote, bacteria, particularly Escherichia coli , Bacillus bacterium, for example, Bacillus subtilis ( B. subtilis ) and the like can be used.
Eukaryotes include yeasts such as Saccharomyces ( Sac
charomyces ), eukaryotic microorganisms such as S. cerevisiae , insect cells such as yoga cells ( Spodo)
ptera frugiperda ), cabbage looper cells ( Trichoplusia ni ), silkworm cells ( Bombyx mori ), animal cells such as human cells, monkey cells, mouse cells and the like, specifically, COS-
1 cell, Vero cell, CHO cell, L cell, myeloma cell, C127 cell, BALB / c3T3 cell, Sp
-2 / O cells and the like can be used. In the present invention, the organism itself, for example, insects such as silkworms and cabbage loopers can also be used.

As the expression vector, plasmid, phage, phagemid, virus (baculo (insect), vaccinia (animal cell)) and the like can be used. The promoter in the expression vector is selected depending on the host cell, and examples of the promoter for bacteria include lac promoter and t promoter.
The rp promoter and the like are used, and examples of the yeast promoter include adhl promoter, pqk promoter and the like. Further, as a promoter for insects, a baculovirus polyhedrin promoter, etc., and as an animal cell, an ear of Simian Virus 40.
Examples include ly or late promoter, CMV promoter, HSV-TK promoter, SRα promoter and the like. In addition to the above, the expression vector includes an enhancer, a splicing signal, a poly A addition signal, a selection marker (for example, dihydrofolate reductase gene (methotrexate resistance), neo gene (G4
18 resistance) etc.) and the like are preferably used. When using an enhancer, for example, SV4
0 enhancer or the like is inserted upstream or downstream of the gene.

Transformation of the host with the expression vector can be carried out by conventional methods well known in the art, and these methods include, for example, Current Protocols in Mol.
ecular Biology, John Wiley & Sons, Inc. Culture of the transformant can also be performed according to a conventional method. Purification of serine protease from the culture can be performed according to a conventional method for isolating and purifying a protein, for example, ultrafiltration, various column chromatography, for example, chromatography using sepharose.

Since the serine protease of the present invention thus obtained is a functional protein, it is possible to screen the present enzyme-specific inhibitory substance using the present enzyme. Useful for exploratory research. Specific examples of the screening method include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermented products, or natural components obtained from various cell culture supernatants and artificial components such as various synthetic compounds. The enzyme inhibiting activity of the test sample can be measured by measuring the enzyme activity in the same manner as in Example 2. Further, the above-mentioned enzyme activity measurement, binding affinity measurement, etc. using a host transformed with the partial peptide of the serine protease of the present invention or the above-mentioned gene of the present enzyme or the DNA encoding the partial peptide thereof or a cell membrane fraction thereof, etc. It is a preferred embodiment of the screening method of the present invention.

That is, the serine protease of the present invention can be used in the screening method of the present invention as its partial peptide. Moreover, in the screening method of the present invention, DN encoding the gene encoding the serine protease of the present invention or a partial peptide thereof is used.
A host cell transformed with a recombinant vector comprising A and expressing the serine protease of the present invention or a partial peptide thereof or a cell membrane fraction thereof may be used.

In this case, the partial peptide includes a peptide fragment existing near the serine residue in the active site and a recognition site of an antibody specific to the serine protease of the present invention as used in Example 3 (6). Peptide fragments consisting of a region unique to the serine protease of the present invention, such as a possible peptide fragment, can be mentioned. The partial peptide can be prepared by the method described above for the serine protease of the present invention, a peptide synthesis method known per se, or the cleavage of the serine protease with an appropriate protease.

The above-mentioned cell membrane fraction is D which encodes the serine protease of the present invention or its partial peptide.
After culturing a host cell capable of expressing NA under conditions capable of expressing and crushing the host cell containing the obtained serine protease or its partial peptide by a method known per se, many cell membranes obtained are contained. It refers to a fraction.

The screening method for an inhibitor using the serine protease or its partial peptide of the present invention is as follows:
Using the serine protease of the present invention or a partial peptide thereof or a host cell containing the serine protease or a partial peptide thereof or a cell membrane fraction thereof,
It is performed by screening a test sample.
As a specific method, it is carried out by an enzyme activity measuring method or a binding affinity measuring method using a substrate of the serine protease of the present invention and its partial peptide, for example, a synthetic substrate such as a chromogenic substrate, a substrate labeled with a radionuclide, and the like. . When using a host cell containing a serine protease, the cell can be immobilized (with glutaraldehyde, formaldehyde, etc.) by a method known per se and used.

[0028]

EXAMPLES The present invention will be described below based on examples. Embodiment 1 FIG. Culture of human colon cancer-derived COLO 201 cells
Preparation of culture supernatant 80 human colon cancer-derived COLO 201 cells (ATCC CCL-224)
It was cultured in a T-flask (Nunc) having a culture area of cm ² . That is, 2 × 10 ⁶ cells were seeded per T flask and 10
RPMI-1640 containing% fetal bovine serum (FBS, GIBCO BRL)
It culture | cultivated until it became confluent using the culture medium (Nissui Pharmaceutical). Next, 10 ^-8 M sodium selenite (Sigma
) The medium was replaced with RPMI-1640 medium containing no added protein. 2
After culturing for a week, the culture supernatant was recovered, sterilized by filtration with a 0.22 μm sterilizing filter (Millipore), and then used as a material for measuring the enzyme activity in the culture supernatant.

Embodiment 2 FIG . COLO 201 derived from human colon cancer
Measurement of enzyme activity in cell culture supernatant The serine protease enzyme activity in the culture supernatant obtained in Example 1 was measured by the test team chromogenic substrate S-2251 (HD-Vallyl-L).
-Leucyl-L-lysyl-p-nitroanilide dihydrochloride,
(1st chemical). That is, with purified water
Test team chromogenic substrate S-2251 50 μ dissolved in 1 mg / ml
l, 0.1 M Tris / HCl (pH 7.5) 40 μl and COLO 201
Add 10 μl of cell culture supernatant and leave at room temperature for 60 minutes.
The absorbance at 05 nm was measured.

When the absorbance when 10 μl of medium was added instead of the culture supernatant was used as a blank, the absorbance of the culture supernatant of COLO 201 cells was 0.42. Also, another substrate HD-
Valyl-L-leucyl-L-arginyl-p-nitroanilide dihydrochloride (Daiichi Pure Chemicals) showed similar activity. Further, as a result of observing the effects of various protease inhibitors in this measurement system, it was confirmed that the culture supernatant of COLO 201 cells clearly had serine protease enzyme activity (Table 1).

[0031]

[Table 1]

Embodiment 3 FIG . Novel serine protease inheritance
Cloning of Offspring and Identification of Protein (1) Isolation and Purification of COLO 201 Cell mRNA COLO 201 cell mRNA was prepared using Isogen (Nippon Gene) according to the attached document. That is, CO
LO 201 cells were grown to confluence in T-flasks (Nunc, 80 cm ² ) and then lysed by adding 1 ml of isogen per T-flask. Further, add 200 μl of chloroform and stir to give 15,000 rp.
Centrifugation was performed for 15 minutes at m and 4 ° C.

After centrifugation, the aqueous phase was recovered, and the recovered aqueous phase was
Add 00 μl of isopropanol and stir to 15,000 rp
Centrifugation was carried out for 30 minutes at 4 ° C. Precipitate the total RNA obtained to 400
Dissolve in μl of diethylpyrocarbonate (DEPC) -treated distilled water and add 400 μl of 2x elution buffer (20 mM Tris-HC
pH 7.5, 2 mM EDTA, 0.2% SDS) was added and mixed. Further, 500 μl of Oligotex-dT30 (Nippon Roche) suspension was added and mixed, and the mixture was heated at 65 ° C. for 5 minutes. After quenching in ice, 1
30 μl of 5 M NaCl was added and the mixture was heated at 37 ° C. for 10 minutes.

After heating, the mixture was centrifuged at 15,000 rpm and 4 ° C. for 3 minutes, the supernatant was removed, and the precipitate was washed with 500 μl of washing buffer.
(10mM Tris-HCl pH7.5, 1mM EDTA, 0.1% SDS, 0.1M Na
It was suspended in Cl) and further centrifuged at 15,000 rpm and 4 ° C. for 3 minutes. After removing the supernatant, the precipitate was suspended in 400 μl of DEPC-treated distilled water. After heating at 65 ° C for 5 minutes, 15,0
The supernatant was collected by centrifugation at 00 rpm and 4 ° C for 3 minutes.

20 μl of 5M NaCl and 1 ml of ethanol were added to this supernatant, and the mixture was stirred and then centrifuged at 15,000 rpm and 4 ° C. for 20 minutes. The precipitate was washed with 500 μl of 70% ethanol, lightly air-dried, and then dissolved in 10 μl of DEPC-treated distilled water. As a result, about 12 μg of polyA ⁺ RNA was obtained from 16 T-flasks.
I got (2) Preparation of cDNA library For preparation of the cDNA library, use the Super Script Plasmid System (L
ife Technologies).

Step 1. cDNA synthesis 5 μl (approximately 6 μg) of COLO 201 cell mRNA was added to oligo dT Not.
2 μl (1 μg) of I primer was added, heated at 70 ° C. for 10 minutes, and then rapidly cooled in ice. To this heat-denatured mRNA, add 4 μl of 5
× First strand buffer (250mM Tris-HCl pH8.3, 375mM
KCl, 15 mM MgCl ₂ ), 1 μl of 10 mM dNTP, 2 μl of 0.1 M
DTT, DEPC treated distilled water and 5 μl (1000 U) of Super
Script II RT was added and reacted at 37 ° C for 1 hour.

Next, 91 μl of DEPC-treated distilled water, 30 μl of 5 × Second strand buffer (100 mM T
ris-HCl pH6.9, 450mM KCl, 23mM MgCl ₂ , 0.75mM β-N
AD ⁺ , 50 mM (NH ₄ ) ₂ SO ₄ ), 3 μl of 10 mM dNTP, 1 μl (10U)
E.Coli DNA ligase, 4 μl (40 U) E.Coli DNA pol
Add imerase and 1 μl (2 U) E. Coli RNase H
After reacting for 2 hours at ℃, 2 μl (10 U) of T4 DNA polymerase was added and reacted at 16 ℃ for 5 minutes.

Further, 10 μl of 0.5 M EDTA was added to this solution and mixed, and then 150 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added, and after stirring, 1
The supernatant was recovered by centrifugation at 5,000 rpm for 5 minutes. To the obtained supernatant, 10 μl of 5M KOAc and 400 μl of ethanol were added, and the mixture was stirred and centrifuged at 15,000 rpm for 10 minutes. The precipitate obtained by centrifugation was washed with 500 μl of 70% ethanol, lightly air-dried, and then dissolved in 25 μl of DEPC-treated distilled water.

Step 2. Addition of Sal I adapter To 25 μl of double-stranded cDNA obtained in step 1, 10 μl of 5 × T
4 DNA ligase buffer (250mM Tris-HCl pH7.6, 50mM MgC
l ₂ , 5mM ATP, 5mM DTT, 25% (w / v), PEG 8000) , Sal I
Adapter solution 10 μl (10 μg) and 5 μl (5 U) T4
After adding DNA ligase and reacting at 16 ° C for 16 hours, 50 μl of phenol: chloroform: isoamyl alcohol (25:
24: 1) was added, and the mixture was stirred and then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant. Add 5 μl of 5M KOAc, 125 μl to the collected supernatant.
l Add ethanol and stir, cool at -80 ° C for 20 minutes, then
It was centrifuged at 15,000 rpm for 10 minutes. The precipitate obtained by centrifugation was washed with 200 μl of 70% ethanol, lightly air-dried, and then 40 μl.
l dissolved in DEPC treated distilled water.

Step 3. Digestion with the restriction enzyme Not I To the reaction mixture from step 2 (20 μl), add Not I (4 μl, 60 U)
After reacting at ℃ for 3 hours, phenol: chloroform: isoamyl alcohol (25: 24: 1) extraction was performed and the supernatant was collected. The supernatant was fractionated with Chromaspin-1000 column (Clontech) to a size of 1 kilobase pair or more to obtain 50 μl of eluate.

Step 4. Ligation with pSPORT vector 1 μl (50 ng; Life Technologie) of pSPORT vector digested with Sal I and Not I into 3 μl of size fractionated cDNA solution
s), and 11 μl of DEPC-treated distilled water, 4 μl
5 x T4 DNA ligase buffer and 1 μl 5 x T4 DNA
Ligase was added, and the mixture was reacted at room temperature for 3 hours.

After the reaction, extraction with phenol: chloroform: isoamyl alcohol (25: 24: 1) was performed, and 5 μl (5 μl
g) Yeast tRNA, 5 μl of 5M KOAc and 125 μl of ethanol were added and stirred, and after cooling at -80 ° C for 20 minutes, 15,0
It was centrifuged at 00 rpm for 10 minutes. Precipitate obtained by centrifugation is 20
Wash with 0 μl 70% ethanol, air dry briefly, and then add 5 μl
It was dissolved in TE (10 mM Tris-HCl pH8.0, 1 mM EDTA).

Step 5. Transformation into Escherichia coli DH10B The ligated cDNA obtained in step 4 was used as Escherichia coli Electr
o MAX DH10B (F ', mcrA, φ 80dlacZ ΔM15, Δ (mrr-hsdRM
S-mcrBC), ΔlacX74, deoR, recA1, endA1, araD139, Δ
(ara, leu) 7697, galU, galK, λ-, rpsL, nupG: Life T
echnologies) by the electroporation method. That is, 2 μl of ligated cDNA was added to 50 μl of DH10B cells to give a final volume of 26 μl × 2,
It was carried out on an electroporator (Bio-Rad) under the conditions of 400 V and 330 μF.

Next, 4 ml of SOC medium (2% bacto tryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KC
E. coli was collected with l, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose), shake-cultured at 37 ° C. for 1 hour, and then LB plate containing 50 mg / ml ampicillin (1% Bact tryptone,
The cells were soaked in 0.5% Bacto yeast extract, 0.5% NaCl, 0.1% glucose, 1.5% Bacto agar) and cultured overnight at 37 ° C. As a result, a cDNA library containing about 1.1 × 10 ⁶ clones was obtained.

(3) PCR using serine protease conserved region The oligomer KY185 shown in SEQ ID NO: 1 was synthesized based on the amino acid conserved region near the active residue (His). In addition, based on the amino acid conserved region near the active residue (Ser), SEQ ID NO: 2
The oligomer KY189 shown below was synthesized. Example 3 (2) Step 3. Using the cDNA obtained in
PCR was performed using Ampli-Taq polymerase (Perkin Elmer) with 5-KY189 as a primer. This PCR
The reaction solution was subcloned into pCR II vector (Invitrogen) to obtain a clone having a DNA fragment of 431 base pairs. As a result of sequencing these clones, 2
It was confirmed to contain a nucleotide sequence encoding an amino acid sequence conserved in serine protease between two active residues (His, Ser).

(4) Screening for serine protease A fluorescent labeled probe was prepared by PCR using the plasmid obtained in Example 3. (3) above as a template. Using this probe, Step 3 of Example 3 (2) The cDNA library of about 1.1 million clones obtained in 1. was screened by a conventional method. As a result, approximately 200,000 to 6 positive clones were obtained. Examine the size of the inserted DNA fragment, select the longest clone pSPORT / SP59- # 3 (about 1.4 kilobase pairs),
The sequence of this gene is Taq Dye Deoxy Terminator C
It was determined by ycle Sequencing Kit (Applied Biosystems).

(5) Features of nucleotide sequence The nucleotide sequence of the cDNA of pSPORT / SP59- # 3 is shown in SEQ ID NO: 3. As a result, the total cDNA length of pSPORT / SP59- # 3 is 1,438.
It consists of 155 base pairs of 5'untranslated region, 732 base pairs of translated region, and 551 base pairs of 3'untranslated region. It was revealed that the translation region encodes 244 amino acid residues.

(6) Peptide fragment of SP59 protein
Preparation of Antibodies Against Agents Among the amino acid sequences of SP59, partial peptide of SEQ ID NO: 6 (amino acid number 56 to 67 of SEQ ID NO: 3 with Cys added), partial peptide of SEQ ID NO: 7 (SEQ ID NO: 3
Amino acid numbers 96 to 110) and the partial peptide of SEQ ID NO: 8 (amino acid numbers 210 to 223 of SEQ ID NO: 3)
Was synthesized to obtain partial peptides having a purity of 90% or more.

Each peptide fragment is a bovine serum albumin (BSA) activated with N- (m-maleimidobenzoyloxy) succinimide (MBS, Nacalai Tesque).
, Nacalai Tesque) and immunized. That is, 5 mg of BSA was dissolved in 50 mM phosphate buffer (pH 8.0), 1.25 mg of MBS dissolved in DMSO was added, and the mixture was stirred at room temperature for 30 minutes to obtain MBS activated BSA. Next, MBS activated BS
5 mg of each peptide fragment dissolved in 50 mM phosphate buffer (pH 7.0) was added to A, and the mixture was stirred by stirring at room temperature for 3 hours. Each coupled peptide fragment was mixed with Freund's complete adjuvant (Nacalai Tesque) and an antiserum was prepared by a conventional method.

(7) Purification of SP59 Protein from Human Pancreatic Cancer-Derived HPC-Y3 Cell Culture Supernatant 10 mg of freeze-dried HPC-Y3 cell culture supernatant obtained in the same manner as in Example 1 was supplemented with 0.1 M NaCl. Contains 10 mM Tris / HCl, pH
Dissolve as in 1 mg / ml in 7.4 and at a flow rate of 4 ml / min
It was subjected to gel filtration chromatography using Superose 6 (Pharmacia). SP obtained in (6) for each fraction
Western blot using 59 partial peptide antibody and synthetic substrate (Boc-Phe-Ser-Arg-4-methyl-coumaryl-7
-The enzyme activity was measured using amide (hereinafter, MCA) and Boc-Gln-Ala-Arg-MCA. As a result, the fraction
Activity was observed in the fraction eluted at 63-70, and the same fraction was directly applied to ion exchange chromatography using a Mono Q column (Pharmacia).

Next, the fraction not bound to the Mono Q column was left as it was.
After applying to a hydroxyapatite column (PENTAX) pre-equilibrated with 10 mM phosphate buffer, pH 6.8, elution was performed with a linear gradient of phosphate buffer, and the active fraction was 150 mM phosphate buffer. Elute with liquid. Then, apply to a Mono S column pre-equilibrated with 20 mM phosphate buffer, pH 6.8, and add 0.1 M
The active fraction was eluted as a single peak at the NaCl concentration.
The eluted fraction was desalted on a C4 column and then subjected to N-terminal amino acid analysis.

(8) Analysis of N-terminal amino acid sequence The N-terminal amino acid sequence of SP59 protein was analyzed as follows. That is, SDS-polyacrylamide electrophoresis was performed from the HPC-Y3 cell protein-free culture supernatant using the SP59 protein purified by the method described above. After electrophoresis, Matsudaira (Matsudaira, P. (1987) J. Biol. Ch.
It was transferred to a PVDF membrane according to the method of Em. 262, 10035-10038). In addition, Speicher (Speicher, DW (1989) "Techni
ques in Protein Chemistry "(Hugli, TEed.), pp.24-
SP59 protein was detected by Coomassie blue staining according to the method of 35. Academic Press, San Diego). The SP59 protein-stained fraction was cut out, washed thoroughly, air-dried, and used as a sample for N-terminal amino acid sequence analysis. For analysis, Applied Biosystems
ystems) 477 A gas phase sequencer.

The phenylthiohydantoin derivative was applied to reversed phase HPLC (Hewick, RM, Hunkapiller, MW, Hoo) of Applied Biosystems 120A online system.
d, LE, & Dreyer, WJ (1981) J. Biol. Chem. 256, 799
0-7997). As a result, it was confirmed that the mature N-terminal amino acid sequence of SP59 protein was the amino acid sequence (LVHG) as predicted. Also mature
It was revealed that an amino acid sequence (VHG) in which one N-terminal amino acid leucine of SP59 protein was deleted was also present.

(9) Cloning of SP60 and SP67 genes and identification of proteins From the COLO201 cells, cloning of SP60 and SP67 genes and identification of proteins were carried out in the same manner as described above, and a catalytic triad specific to serine protease was left. SP60 (SEQ ID NO: 4) and SP67 (SEQ ID NO: 5) having a group (catalytic triad) were obtained. These DNA are SP
Expression can be carried out in the same manner as in 59 to obtain serine protease.

Example 4. Northern block of the SP59 gene
Expression in human organs by digestion pSPORT / SP59- # 3 was digested with the restriction enzyme Mlu I to isolate and purify a DNA fragment of approximately 1.4 kilobase pairs, and α- ³² P dCTP (Amer
sham) and labeled as a probe. This probe was reacted with a membrane filter (Clontech) on which mRNA prepared from 16 kinds of organs was blotted at 65 ° C for 2 hours.

Next, this membrane filter was washed with 0.1% S.
2 × SSC containing DS (150 mM NaCl, 15 mM sodium citrate) at room temperature for 20 minutes, then washed twice with 1 × SSC, 0.1% SDS at 65 ° C for 30 minutes, and then washed with BAS2000 imaging plate (Fuji Photo The film) was exposed for 30 minutes and analyzed. The result is shown in FIG. The expression of mRNA of SP59 gene in human organs was particularly strong in the brain, and the size was about 1.4 kb. Also, SP60 gene and SP
When 67 genes were tested in the same manner as the SP59 gene, strong expression was observed in the colon, prostate, small intestine and kidney, and colon, small intestine, prostate and pancreas, respectively.

Example 5. Novel encoded by SP59 gene
Measurement of Enzymatic Activity of Serine Protease Mature Protein (1) Construction of Expression Plasmid After digesting pSPORT / SP59- # 3 with Mlu I, a DNA fragment of about 1.4 kilobase pairs was isolated and purified and dissolved in TE. Similarly, SV
PdKCR vector with 40 promoters (Nikaido, T. et al.,
Nature, 311 , 631-635 (1984): pKCR vector in which pBR322 part was replaced with pBR327 ) was also digested with Mlu I,
It was dephosphorylated with alkaline phosphatase, extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1), precipitated with ethanol and dissolved in TE.

The pSPORT / SP59- # 3 DNA fragment and the pdKCR vector DNA fragment were ligated according to a conventional method, and
JM109 was transformed, and the resulting colonies were analyzed by PCR to obtain the desired serine protease SP59 expression plasmid pdKCR / SP59. Next, the gene encoding the signal sequence following the initiation methionine of trypsin II and the enterokinase recognition sequence was amplified, and Eco RI was added upstream of 5'side, and Bsp MI restriction enzyme recognition site was added downstream of 3'side. The primer was designed. KY239 and KY24
0 is shown in SEQ ID NOs: 9 and 10.

Using these primers KY239 and KY240, pCR II / Trypsin II plasmid (two specific primers (Emi, M., Nakamura et al., Gene, 41. 305-31) was prepared.
0, 1986) to obtain the cDNA obtained in Step 5 of Example 3 (2).
PCR was performed using the template amplified from the library and subcloned into the pCRII vector as a template. After digesting the product with restriction enzymes ( Eco RI and Bsp MI),
A 75 bp DNA fragment was isolated and purified.

Similarly, primers KY241 and KY207 were designed to add a Bsp MI restriction enzyme recognition site upstream of the gene encoding the mature protein of SP59 gene. KY241 and KY207 are SEQ ID NOs: 11 and 12
It was shown to. Using these primers KY241 and KY207, the pSPORT / SP59- # 3 plasmid was used as a template.
PCR is performed and the product is digested with restriction enzymes ( Bsp MI and Bpu 11
After digestion with 02I), the DNA fragment was isolated and purified. Next, the DNA fragment encoding the trypsin II signal sequence and enterokinase recognition sequence and the DNA fragment encoding the mature protein of the SP59 gene were pre-digested with restriction enzymes ( Eco RI and Bpu 1102I) according to a conventional method. PdKCR / SP59
The vector was ligated and E. coli JM109 was transformed. Among the transformed colonies, colonies containing the desired chimeric gene were confirmed by PCR to obtain the expression plasmid (pdKCR / Trp59) of the desired chimeric gene (Trp59).

(2) Expression in COS-1 cells Using the expression plasmid of the chimeric gene (Trp59) prepared in Example 5 (1), expression in animal cells was tried. COS-1 cells were used as animal cells for expression, and pdKCR / Trp59 and pdKCR were respectively transfected as expression plasmids by the lipofectin method. Ie 1 diameter
COS-1 cells were inoculated with 1 × 10 ⁶ cells in a 0 cm culture dish (Corning, 430167). As a medium, 10%
Dulbecco's minimum essential med containing fetal bovine serum
ium (DMEM, Nissui Pharmaceutical) was used.

The next day, Opti-MEM medium (Life Technologie
s) After rinsing the cells with 5 ml, add an additional 5 ml Opti-ME.
M medium was added and the cells were cultured at 37 ° C for 2 hours. After culturing, a mixed solution of 1 μg of the above-mentioned plasmid and 10 μg of lipofectin (Pharmacia) was added per dish, and the mixture was further incubated at 37 ° C. for 5 hours. After culturing, 5 ml of Opti-MEM medium was added to make a total of 10 ml, and culturing was carried out at 37 ° C. for 72 hours. After culturing, the culture supernatant was collected by centrifugation and used as a measurement sample of enzyme activity.

(3) Measurement of enzyme activity The enzyme activity in the cell culture supernatant obtained in Example 5 (2) was measured. That is, 50 μl of the culture supernatant of COS-1 cells was added to 10 μl of enterokinase (1 mg / ml, Biozyme Laboratories).
μl was mixed and reacted at room temperature for 15 minutes. Next, add 50 μl of 0.2 M substrate solution prepared by diluting the synthetic substrate Boc-Phe-Ser-Arg-MCA (Peptide Institute) in DMSO with 0.1 M Tris / HCl, pH 8.0, and then at room temperature for 60 minutes. It was made to react. After the reaction, fluorescence at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm was measured.

As a result, as shown in FIG. 2, enzymatic activity was observed by adding enterokinase to the culture supernatant of COS-1 cells expressing the Trp59 gene. As a result, SP
The novel serine protease mature protein encoded by the 59 gene was revealed to exhibit enzymatic activity. From the above results, it was revealed not only that the serine protease gene isolated this time is a novel serine protease gene due to its primary structure, but also that it expresses activity as a mature protein.

[0065]

INDUSTRIAL APPLICABILITY The present inventors have developed COLO 2 derived from human colon cancer.
A novel serine protease gene was isolated from 01 cells, and it was revealed that the isolated gene has serine protease enzyme activity as a mature protein. Moreover, it was revealed that the SP59 gene was strongly expressed in the human brain, although the novel serine protease gene obtained for the first time was derived from colon cancer.

Thus, it is clear that the isolation of the serine protease gene using cancer cells is useful as a resource for novel genes. Furthermore, even if a novel serine protease gene is isolated or mRNA expression is examined using the isolated gene, whether the translated protein is functionally expressed locally in the organ , Not guaranteed.

The fact that the novel serine protease gene was expressed by the above-mentioned method and clarified that it codes for a functional protein proves the usefulness of the gene. Further, since the protein expressed using the gene is a functional protein, it is possible for the first time to screen various disease therapeutic agents by establishing a screening system for this enzyme-specific inhibitor. .

[0068]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GTGCTCACNG CNGCBCAYTG 20

SEQ ID NO: 2 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence AGCGGNCCNC CDGARTCVCC 20

SEQ ID NO: 3 Sequence length: 1438 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear sequence type: cDNA to mRNA sequence GGACACACGC TGTAGCTGTC TCCCCGGCTG GCTGGCTCGC TCTCTCCTGG GGACACAGAG 60 GTCGGCAGGC AGCACACAGA GGGACCTACTT GGCACGCATT ACTCAAGAAT 120

CCCCGGAGGC CCGGAGGCCT GCAGCAGGAG CGGCC ATG AAG AAG CTG ATG GTG 173 Met Lys Lys Leu Met Val -20 GTG CTG AGT CTG ATT GCT GCA GCC TGG GCA GAG GAG CAG AAT AAG TTG 221 Val Leu Ser Leu Ile Ala Ala Ala Ala Ala Ala Ala Gla Tla Glu Gln Asn Lys Leu -15 -10 -5 -1 -1 GTG CAT GGC GGA CCC TGC GAC AAG ACA TCT CAC CCC TAC CAA GCT GCC 269 Val His Gly Gly Pro Cys Asp Lys Thr Ser His Pro Tyr Gln Ala Ala 5 10 15 CTC TAC ACC TCG GGC CAC TTG CTC TGT GGT GGG GTC CTT ATC CAT CCA 317 Leu Tyr Thr Ser Gly His Leu Leu Cys Gly Gly Val Leu Ile His Pro 20 25 30 CTG TGG GTC CTC ACA GCT GCC CAC TGC AAA AAA CCG AAT CTT CAG GTC 365 Leu Trp Val Leu Thr Ala Ala His Cys Lys Lys Pro Asn Leu Gln Val 35 40 45 TTC CTG GGG AAG CAT AAC CTT CGG CAA AGG GAG AGT TCC CAG GAG CAG 413 Phe Leu Gly Lys His Asn Leu Arg Gln Arg Glu Ser Ser Gln Glu Gln 50 55 60 65 AGT TCT GTT GTC CGG GCT GTG ATC CAC CCT GAC TAT GAT GCC GCC AGC 461 Ser Ser Val Val Arg Ala Val Ile His Pro Asp Tyr Asp Ala Ala Ser 70 75 80 CAT GAC CAG GAC ATC ATG CTG TTG CGC CTG GCA CGC CCA GCC AAA CTC 509 His Asp Gln Asp Ile Met Leu Leu Arg Leu Ala Arg Pro Ala Lys Leu 85 90 95 TCT GAA CTC ATC CAG CCC CTT CCC CTG GAG AGG GAC TGC TCA GCC AAC 557 Ser Glu Leu Ile Gln Pro Leu Pro Leu Glu Arg Asp Cys Ser Ala Asn 100 105 110

ACC ACC AGC TGC CAC ATC CTG GGC TGG GGC AAG ACA GCA GAT GGT GAT 605 Thr Thr Ser Cys His Ile Leu Gly Trp Gly Lys Thr Ala Asp Gly Asp 115 120 125 TTC CCT GAC ACC ATC CAG TGT GCA TAC ATC CAC CTG GTG TCC CGT GAG 653 Phe Pro Asp Thr Ile Gln Cys Ala Tyr Ile His Leu Val Ser Arg Glu 130 135 140 145 GAG TGT GAG CAT GCC TAC CCT GGC CAG ATC ACC CAG AAC ATG TTG TGT 701 Glu Cys Glu His Ala Tyr Pro Gly Gln Ile Thr Gln Asn Met Leu Cys 150 155 160 GCT GGG GAT GAG AAG TAC GGG AAG GAT TCC TGC CAG GGT GAT TCT GGG 749 Ala Gly Asp Glu Lys Tyr Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly 165 170 175 GGT CCG CTG GTA TGT GGA GAC CAC CTC CGA GGC CTT GTG TCA TGG GGT 797 Gly Pro Leu Val Cys Gly Asp His Leu Arg Gly Leu Val Ser Trp Gly 180 185 190 AAC ATC CCC TGT GGA TCA AAG GAG AAG CCA GGA GTC TAC ACC AAC GTC 845 Asn Ile Pro Cys Gly Ser Lys Glu Lys Pro Gly Val Tyr Thr Asn Val 195 200 205 TGC AGA TAC ACG AAC TGG ATC CAA AAA ACC ATT CAG GCC AAG 887 Cys Arg Tyr Thr Asn Trp Ile Gln Lys Thr Ile Gln Ala Lys 210 215 220 TGACCCTGAC ATGTGACATC TACCTCCCGA CCTACCACCC CACTGGCTGG TTCCAGAACG 947 TCTCTCACCT AGACCTTGCC TCCCCTCCTC TCCTGCCCAG CTCTGACCCT GATGCTTAAT 1007 AAACGCAGCG ACGTGAGGGT CCTGATTCTC CCTGGTTTTA CCCCAGCTCC ATCCTTGCAT 1067 CACTGGGGAG GACGTGATGA GTGAGGACTT GGGTCCTCGG TCTTACCCCC ACCACTAAGA 1127 GAATACAGGA AAATCCCTTC TAGGCATCTC CTCTCCCCAA CCCTTCCACA CGTTTGATTT 1187 CTTCCTGCAG AGGCCCAGCC ACGTGTCTGG AATCCCAGCT CCGCTGCTTA CTGTCGGTGT 1247 CCCCTTGGGA TGTACCTTTC TTCACTGCAG ATTTCTCACC TGTAAGATGA AGATAAGGAT 1307 GATACAGTCT CCATAAGGCA GTGGCTGTTG GAAAGATTTA AGGTTTCACA CCTATGACAT 1367 ACATGGAATA GCACCTGGGC CACCATGCAC TCAATAAAGA ATGAATTTTA TTAAAAAAAA 1427 AAAAAAAAAA A 1438

SEQ ID NO: 4 Sequence length: 699 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence GTG GTG GGT GGG GAG GAG GCC TCT GTG GAT TCT TGG CCT TGG CAG GTC 48 Val Val Gly Gly Glu Glu Ala Ser Val Asp Ser Trp Pro Trp Gln Val 1 5 10 15 AGC ATC CAG TAC GAC AAA CAG CAC GTC TGT GGA GGG AGC ATC CTG GAC 96 Ser Ile Gln Tyr Asp Lys Gln His Val Cys Gly Gly Ser Ile Leu Asp 20 25 30 CCC CAC TGG GTC CTC ACG GCA GCC CAC TGC TTC AGG AAA CAT ACC GAT 144 Pro His Trp Val Leu Thr Ala Ala His Cys Phe Arg Lys His Thr Asp 35 40 45 GTG TTC AAC TGG AAG GTG CGG GCA GGC TCA GAC AAA CTG GGC AGC TTC 192 Val Phe Asn Trp Lys Val Arg Ala Gly Ser Asp Lys Leu Gly Ser Phe 50 55 60 CCA TCC CTG GCT GTG GCC AAG ATC ATC ATC ATT GAA TTC AAC CCC ATG 240 Pro Ser Leu Ala Val Ala Lys Ile Ile Ile Ile Glu Phe Asn Pro Met 65 70 75 80 TAC CCC AAA GAC AAT GAC ATC GCC CTC ATG AAG CTG CAG TTC CCA CTC 288 Tyr Pro Lys Asp Asn Asp Ile Al a Leu Met Lys Leu Gln Phe Pro Leu 85 90 95

ACT TTC TCA GGC ACA GTC AGG CCC ATC TGT CTG CCC TTC TTT GAT GAG 336 Thr Phe Ser Gly Thr Val Arg Pro Ile Cys Leu Pro Phe Phe Asp Glu 100 105 110 GAG CTC ACT CCA GCC ACC CCA CTC TGG ATC ATT GGA TGG GGC TTT ACG 384 Glu Leu Thr Pro Ala Thr Pro Leu Trp Ile Ile Gly Trp Gly Phe Thr 115 120 125 AAG CAG AAT GGA GGG AAG ATG TCT GAC ATA CTG CTG CAG GCG TCA GTC 432 Lys Gln Asn Gly Gly Lys Met Ser Asp Ile Leu Leu Gln Ala Ser Val 130 135 140 CAG GTC ATT GAC AGC ACA CGG TGC AAT GCA GAC GAT GCG TAC CAG GGG 480 Gln Val Ile Asp Ser Thr Arg Cys Asn Ala Asp Asp Ala Tyr Gln Gly 145 150 155 160 GAA GTC ACC GAG AAG ATG ATG TGT GCA GGC ATC CCG GAA GGG GGT GTG 528 Glu Val Thr Glu Lys Met Met Cys Ala Gly Ile Pro Glu Gly Gly Val 165 170 175 GAC ACC TGC CAG GGT GAC AGT GGT GGG CCC CTG ATG TAC CAA TCT GAC 576 Asp Thr Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Tyr Gln Ser Asp 180 185 190 CAG TGG CAT GTG GTG GGC ATC GTT AGC TGG GGC TAT GGC TGC GGG GGC 624 Gln Trp His Val Val Gly Ile Val Ser Trp Gly T yr Gly Cys Gly Gly 195 200 205 CCG AGC ACC CCA GGA GTA TAC ACC AAG GTC TCA GCC TAT CTC AAC TGG 672 Pro Ser Thr Pro Gly Val Tyr Thr Lys Val Ser Ala Tyr Leu Asn Trp 210 215 220 ATC TAC AAT GTC TGG AAG GCT GAG CTG 699 Ile Tyr Asn Val Trp Lys Ala Glu Leu 225 230

SEQ ID NO: 5 Sequence length: 723 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA sequence GTT GTT GGG GGC ACG GAT GCG GAT GAG GGC GAG TGG CCC TGG CAG GTA 48 Val Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp Pro Trp Gln Val 1 5 10 15 AGC CTG CAT GCT CTG GGC CAG GGC CAC ATC TGC GGT GCT TCC CTC ATC 96 Ser Leu His Ala Leu Gly Gln Gly His Ile Cys Gly Ala Ser Leu Ile 20 25 30 TCT CCC AAC TGG CTG GTC TCT GCC GCA CAC TGC TAC ATC GAT GAC AGA 144 Ser Pro Asn Trp Leu Val Ser Ala Ala His Cys Tyr Ile Asp Asp Arg 35 40 45 GGA TTC AGG TAC TCA GAC CCC ACG CAG TGG ACG GTC TTC CTG GGC TTG 192 Gly Phe Arg Tyr Ser Asp Pro Thr Gln Trp Thr Val Phe Leu Gly Leu 50 55 60 CAC GAC CAG AGC CAG CGC AGC GCC CCT GGG GTG CAG GAG CGC AGG CTC 240 His Asp Gln Ser Gln Arg Ser Ala Pro Gly Val Gln Glu Arg Arg Leu 65 70 75 80 AAG CGC ATC ATC TCC CAC CCC TTC TTC AAT GAC TTC ACC TTC GAC TAT 288 Lys Arg Ile Ile Ser His Pro Ph e Phe Asn Asp Phe Thr Phe Asp Tyr 85 90 95 GAC ATC GCG CTG CTG GAG CTG GAG AAA CCG GCA GAG TAC AGC TCC ATG 336 Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro Ala Glu Tyr Ser Ser Met 100 105 110

GTG CGG CCC ATC TGC CTG CCG GAC GCC TCC CAT GTC TTC CCT GCC GGC 384 Val Arg Pro Ile Cys Leu Pro Asp Ala Ser His Val Phe Pro Ala Gly 115 120 125 AAG GCC ATC TGG GTC ACG GGC TGG GGA CAC ACC CAG TAT GGA GGC ACT 432 Lys Ala Ile Trp Val Thr Gly Trp Gly His Thr Gln Tyr Gly Gly Thr 130 135 140 GGC GCG CTG ATC CTG CAA AAG GGT GAG ATC CGC GTC ATC AAC CAG ACC 480 Gly Ala Leu Ile Leu Gln Lys Gly Glu Ile Arg Val Ile Asn Gln Thr 145 150 155 160 ACC TGC GAG AAC CTC CTG CCG CAG CAG ATC ACG CCG CGC ATG ATG TGC 528 Thr Cys Glu Asn Leu Leu Pro Gln Gln Ile Thr Pro Arg Met Met Cys 165 170 175 GTG GGC TTC CTC AGC GGC GGC GTG GAC TCC TGC CAG GGT GAT TCC GGG 576 Val Gly Phe Leu Ser Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly 180 185 190 GGA CCC CTG TCC AGC GTG GAG GCG GAT GGG CGG ATC TTC CAG GCC GGT 624 Gly Pro Leu Ser Ser Val Glu Ala Asp Gly Arg Ile Phe Gln Ala Gly 195 200 205 GTG GTG AGC TGG GGA GAC GGC TGC GCT CAG AGG AAC AAG CCA GGC GTG 672 Val Val Ser Trp Gly Asp Gly Cys Ala Gln Arg A sn Lys Pro Gly Val 210 215 220 TAC ACA AGG CTC CCT CTG TTT CGG GAC TGG ATC AAA GAG AAC ACT GGG 720 Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp Ile Lys Glu Asn Thr Gly 225 230 235 240 GTA 723 Val

SEQ ID NO: 6 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Sequence Leu Arg Gln Arg Glu Ser Ser Gln Glu Gln Ser Ser Cys 1 5 10

SEQ ID NO: 7 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Sequence Lys Leu Ser Glu Leu Ile Gln Pro Leu Pro Leu Glu Arg Asp Cys 1 5 10 15

SEQ ID NO: 8 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Sequence Cys Arg Tyr Thr Asn Trp Ile Gln Lys Thr Ile Gln Ala Lys 1 5 10

SEQ ID NO: 9 Sequence Length: 26 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence CACAGAATTC CACCATGAAT CTACTT 26

SEQ ID NO: 10 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TAGCACCTGC CGATCTTGTC ATCATCA 27

SEQ ID NO: 11 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GCAGACCTGC AGAACAAGTT GGTGCATG 28

SEQ ID NO: 12 Sequence Length: 18 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence AAAACCAGGG AGAATCAG 18

[Brief description of the drawings]

FIG. 1 is a photograph replacing a drawing of a nitrocellulose membrane showing the results of the expression of SP59 gene in various human organs by Northern Blotting. PBL: peripheral blood lymph
ocyte (peripheral lymphocyte)

FIG. 2 is a diagram showing the results of examining the enzymatic activity of a mature protein encoded by the SP59 gene expressed in COS-1 cells. The white column shows the case where enterokinase was added, and the shaded column shows the case where enterokinase was not added. In addition, pdKCR indicates the culture supernatant of COS-1 cells transfected with only the expression vector used.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12N 9/52 C12N 5/00 B // A61K 38/46 A61K 37/54 (C12N 15/09 ZNA (C12R 1:91) (C12N 1/21 C12R 1:19) (C12N 9/52 C12R 1:19) (C12N 9/52 C12R 1:91) (72) Inventor Nozomi Yamaguchi Street in Kuramaguchi, Kita-ku, Kyoto City, Kyoto Prefecture Teramachi Nishiiri Le Shin Ryoukoucho 285-79

Claims (7)

[Claims] 1. An amino acid sequence of amino acid numbers 1 to 223 shown in SEQ ID NO: 3 (however, leucine of amino acid number 1 may be deleted), amino acid numbers 1 to 233 shown in SEQ ID NO: 4. A serine protease or a partial peptide thereof comprising the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of amino acid numbers 1 to 241 shown in SEQ ID NO: 5. 2. An amino acid sequence of amino acid numbers 1 to 223 shown in SEQ ID NO: 3 (however, leucine of amino acid number 1 may be deleted), amino acid numbers 1 to 233 shown in SEQ ID NO: 4. DNA encoding a serine protease or a partial peptide thereof comprising the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of amino acid numbers 1 to 241 shown in SEQ ID NO: 5. 3. The nucleotide number 219 of SEQ ID NO: 3.
No. 887, No. 222-887, nucleotide Nos. 1-699 of SEQ ID NO: 4 or nucleotide Nos. 1-723 of SEQ ID NO: 5 or partial nucleotide sequences thereof.
A. 4. A recombinant vector comprising the DNA according to claim 2 or 3. 5. A host transformed with the recombinant vector according to claim 4. 6. The method for producing a serine protease or a partial peptide thereof according to claim 1, wherein the host according to claim 5 is cultured, and the serine protease or a partial peptide thereof is collected from the culture. how to. 7. A method for screening an inhibitor using the serine protease or the partial peptide thereof according to claim 1.