KR100291101B1 - Hcv protease gene and expression thereof in yeast - Google Patents

Abstract

PURPOSE: Provided are an HCV protease, its coding DNA, an expression vector containing the DNA sequence, a transformant transformed with the vector and a manufacturing method of HCV protease by culturing the transformed yeast. CONSTITUTION: An HCV protease or a fused protein fused with ubiquitin includes amino acid sequence as described in fig. An expression vector pYLBC-A/G-UB-PRO contains DNA including the HCV protease coding nucleotide sequence. A transformant, Saccharomyces cerevisiae DCO4-UB-PRO(ATCC 74116), is prepared by transformation with the expression vector and cultured to manufacture HCV protease.

Description

Protease Genes of Hepatitis C Virus and Expression in Yeast

Figure 1 shows the nucleotide sequence and amino acid sequence of the protease gene of the Korean hepatitis C virus,

Figure 2 shows the strategy of linking the protease gene of the Korean hepatitis C virus and the prepared expression vector,

3A and 3B show the results of SDS polyacrylamide gel electrophoresis and Western blotting after expressing protease of Korean hepatitis C virus using the yeast expression vector of the present invention, respectively. .

The present invention provides a protein of the hepatitis C virus by culturing a protease protein of the Korean hepatitis C virus, a DNA encoding the same, an expression vector comprising the DNA sequence, a yeast transformed with the expression vector and a transformed yeast. It relates to a method for producing a degrading enzyme.

Until recently, viral hepatitis has been known to be caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV), and epsin baba (EBV). Unlike the above hepatitis, non-AB hepatitis B virus (NANBH) virus, which accounts for 90% of hepatitis developed after blood transfusion, has been identified (Alter, HJ, et al., Lancet 2, 838-841 (1975)). It is estimated that about 1 million people worldwide are infected with this non-A hepatitis B virus each year (Alter, HJ, in AJ Zuckerman (ed.), Viral Hepatitis and Liver Disease, 537-542, Alan R. Liss, New York (1988), of which about 40-50% develop chronic hepatitis and about 20% develop liver cirrhosis and liver cancer (Dienstag, JL, et al., Seminar Liver Dis. 6, 67-81 (986)).

Bradley et al. Reported that non-A non-B-type virus (hereinafter referred to as HCV) can be isolated and recovered by infecting plasma from NANBH patients with chimpanzees (Bradley, DW et al., Gastroenterology 88, 773-779). (1985)), Chu et al., Said the virus is a positive-stranded RNA virus, consisting of about 10,000 bases and one open encoding a polyprotein precursor of about 3010-3011 amino acids. Reported as having an open reading frame (ORF) (Choo, QL, et al., Science 244, 359-362 (1989); Choo, QL, et al., Proc. Natl. Acad. Sci. USA 88) , 2451-2455 (1991).

The genetic structure of hepatitis C virus has similarities to those of flavivirus and pestivirus, and due to their flexibility, the polyprotein of hepatitis C virus is nucleus from the amino terminus. (core) -envelope 1-envelope 2 / unstructured 1 (E2 / NS1) -unstructured 2 (NS2) -unstructured 3 (NS3) -unstructured 4 (NS4) -unstructured 5 (NS5) (Choo, QL, et al., Proc. Natl. Aca. Sci. USA 88, 2451-2455 (1991); Takamizawa, A., et al., J. Virol. 65). 1105-1113 (1991); Kato, N., et al., Proc. Natl. Acad. Sci. USA 87, 6524-6528 (1990)).

Envelope 1 and envelope 2 / non-structural 1 proteins (hereinafter referred to as envelope 2 proteins) were found to be glycoproteins with molecular weights of 33,000 and 72,000 Daltons, respectively, in vitro (Houghton, M., et al., Hepatology 14, 381-388 (1991); Hijkata, M., et al., Proc. Natl. Acad. Sci. USA 88, 5547-5551 (1991)), presumably vaccines of flaviviruses Significant structural similarities to the non-structural 1 protein and gp53 / 55 protein, presumed to be the vaccine of pestiviruses, have been the primary targets for vaccine development and treatment of hepatitis C virus (Spete, RR et al., Virology 188, 819-830 (1992).

On the other hand, structural proteins encoded by structural genes are transduced during polyprotein transfer and are cleaved by signalases in host cells to produce nuclear proteins, coat 1, coat 2 or non-structural 1 proteins (L. Markoff). , J. Virol. 63, 3345-3352 (1989)), nonstructural proteins encoded by nonstructural genes are cleaved by viral proteolytic enzymes encoded by some genes of nonstructural 3 to produce individual nonstructural proteins. (TJ Chambers, Annu. Rev. Microbiol. 44, 649-688 (1980)).

In the case of flaviviruses, the nonstructural 3 (NS3) protein has been shown to be a viral protease and a serine type protease that cleaves between lysine or arginine and glycine, serine or alanine (TJ Chambers, Proc. Natl.Acad. Sci., 87, 8902-8998 (1990)).

Such viral proteases can provide a treatment for viral infections by developing inhibitors of these enzymes. In the case of human immunodeficiency virus, for example, the development of an inhibitor against HIV protease has become an important electricity for the treatment of AIDS (Z. Hostomsky, Biochem. Biophys. Res. Comm. 161, 1056-1063 (1989). HIV protease is thought to cleave p55 gag precursors in the order of nuclear structural proteins p17, p24, p8, and p7, and McQuade et al. Are synthetic peptides designed as inhibitors of HIV protease. Has been reported to reduce the production of p24 protein by up to 70% as a result of adding HIV to blood cells infected with HIV (TJ McQuade, et. Al. Science 247, 454-456 (1990)).

Ubiquitin, a protein of 5,500 Daltons discovered in 1975, is found throughout eukaryotic cells and is absent in prokaryotic cells (Goldstein, et al., Proc. Natl. Acad. Sci. 72, 11). 1975)). Ubiquitin from animal cells has been found to be the same, and ubiquitin has many important roles, but among these, proteolysis is known to be important (Finley, et al., Trends in Biochem. Sci. 16, 343 (1985). ))

In the yeast ubiquitin system, ubiquitin, consisting of 76 amino acids, is known to have a much faster intracellular cleavage process than other forms of ubiquitin, and it is known to efficiently cleave arginine-glycine following (Ozkaynak, et al., Nature 312, 663-666 (1987). Also, Bachmair et al reported that foreign proteins bound to ubiquitin are cleaved very precisely after arginine-glycine-glycine by enzymes present in cells (Bachmair, et al., Science 234, 178-). 186 (1986).

Therefore, when the protease gene is combined with the ubiquitin gene and expressed in yeast, mass expression of the protease protein is induced by the ubiquitin gene, and the protease gene product is separated from the ubiquitin protein by the enzyme present in the cell. You can get a large quantity.

An object of the present invention is to provide a protease of the Korean hepatitis C virus having the amino acid sequence of FIG. 1, and to encode the DNA, the expression vector comprising the DNA sequence, the microorganism transformed with the expression vector and It provides a method for producing a Korean hepatitis C virus protease, including culturing a microorganism.

In addition, by using the ubiquitin system in yeast in order to provide a large amount of HCV protease, ubiquitin fused proteolytic enzyme encoding the DNA, an expression vector comprising the same, yeast transformed with the vector and the yeast To provide a Korean type hepatitis C protease protein, including incubation.

Furthermore, the development of inhibitors of this protease contributes to the prevention and treatment of hepatitis C.

The present invention is described in detail as follows.

The Korean hepatitis C virus protease according to the present invention has an amino acid sequence as shown in FIG. 1 (see Korean Patent Application No. 91-9510, filed June 10, 1991).

The amino acid sequence of the proteolytic enzyme of the present invention may be synthesized according to a conventional peptide synthesis method, but as described in the following examples, an expression vector including a DNA sequence encoding the same by a genetic engineering method is prepared and transformed with the expression vector. The converted microorganisms can be cultured and produced.

The amino acid sequence of the protease of the present invention also includes an amino acid sequence substituted with amino acids that do not affect the activity and function of the protein.

In addition, the proteolytic enzymes of the present invention can be prepared in the form of a synthetic protein with a specific protein that makes the expression and separation-purification effective. In particular, a preparative protein with ubiquitin prepared according to the present invention is preferable.

DNA encoding the protease of the present invention can be obtained directly from the cDNA library or synthesized chemically according to known methods. For example, any of a variety of known methods may be used, such as a phosphoamidite solid support method such as Matteucci (J. Am. Chem. Soc. 103: 3185 (1981)).

In general, due to the degeneracy of the codons, a large number of codons corresponding to one amino acid exist, so that the DNA sequence encoding the amino acid sequence of the present invention may be partially different.

In order to clone the protease gene of the present invention, a primer for PCR was designed and synthesized from the nucleotide sequence information of the cDNA fragment of the Korean hepatitis C virus, and the Korean hepatitis C virus cDNA (ATCC 75008, hereinafter KHCV) was prepared using these primers. -LBCI DNA) is used as a template to amplify the protease gene by polymerase chain reaction.

On the other hand, by combining the artificially synthesized ubiquitin gene and the proteolytic enzyme gene can be amplified a large amount of the synthetic protein gene.

The DNA encoding the recombinant protein gene thus prepared can be expressed in a suitable known prokaryotic or eukaryotic cell expression system using appropriate vectors and host cells. Using, for example, pYLBC-A / G-HGH (KFCC 10669, deposited on December 27, 1988) or pYLBC-A / G-UB-HGH (ATCC 74071, deposited on June 18, 1991) devised by the applicant. Expressed in yeast.

Transformation of expression vectors into host cells was performed and obtained according to known techniques (Beggs., Nature 275, 104 (1978) and Hinnen et al., Proc. Natl. Acad. Sci. USA 75, 1929 (1978)). The transformed microorganism is cultured under appropriate conditions to obtain the protease protein of the present invention. This was confirmed by electrophoresis on polyacrylamide gel and Westing blotting with immunoglobulins isolated from serum of hepatitis C patients (Towbin, et al., Proc. Natl. Acad. Sci. USA, 76. 4350 (1979) Gene product specifically reacts with the antibodies of the Korean hepatitis C virus.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the invention and do not limit the scope of the invention. Experimental methods and procedures used in the following examples were carried out according to the following Reference Examples 1) -6) unless otherwise specified.

REFERENCE EXAMPLE 1 Cleavage of DNA using restriction enzymes.

A restriction enzyme and a reaction complete solution were added to a sterile 1.5 ml eppendorf tube to a reaction volume of 50 µl to 100 µl and reacted at 37 ° C. for 1 to 2 hours. After the reaction was completed, heat treatment was performed at 65 ° C. for 15 minutes to inactivate the restriction enzyme, and phenol extraction and ethanol precipitation were used for the restriction enzyme which was heat resistant. Restriction enzymes and reaction buffers used were purchased from New England Biolabs, MA, USA. The composition of the 10-fold reaction buffer is as follows.

10-fold NEB buffer 1: 100 mM bis tris propane-HCl, 100 mM MgCl ₂ , 10 mM Dithiothreitol (DTT), pH 7.0

10-fold NEB buffer 2: 100 mM Tris-HCl, 100 mM MgCl ₂ , 500 mM NaCl, 10 mM DTT, pH 7.0

10-fold NEB buffer 3: 100 mM Tris-HCl, 100 mM MgCl ₂ , 500 mM NaCl, 10 mM DTT, pH 7.0

10-fold NEB buffer 4: 200 mM tris-acetate, 100 mM magnesium acetate, 500 mM potassium acetate, 10 mM DTT, pH 7.0

REFERENCE EXAMPLE 2 Phenol Extraction and Ethanol Precipitation

Phenol extraction was used to deactivate the restriction enzyme or extract the nucleic acid after the restriction enzyme reaction was completed. Phenol was used after saturation with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution.

Mix the sample and phenol at a ratio of 1: 1 (v / v), shake vigorously to mix, and centrifuge (15,000xG, 5 minutes) to transfer the supernatant to a new tube. The same procedure was repeated three times, followed by extracting the upper layer with an equal volume of chloroform solution (chloroform to dibutanol ratio of 24: 1 (v / v)) to 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol. . This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least 2 hours, followed by centrifugation (15,000xG, 20 minutes, 4 ° C) to precipitate the nucleic acid.

Reference Example 3 Ligation

T4 DNA ligase (T4 DNA ligase, New England Biolabs, Inc.) and 10-fold ligation buffer (0.5M Tris-HCl, pH7.8, 0.1M MgCl ₂ , 0.2M dithiothreitol, 10 mM ATP) , 0.5 mg / ml BSA) was used to perform the ligation reaction. Usually, 10 units of ligase is used in a volume of 20 μl, and 100 units of ligase are used for ligation of DNA having blunt ends. At least 5 hours at 16 ° C. or 14 hours at 4 ° C. After completion of the reaction, the ligase was inactivated by heating at 65 ° C. for 15 minutes.

Reference Example 4 E. coli Transformation

E. coli host cells (E. coli HB101, W3110 or JM105) were inoculated into 100 ml of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 0.5% sodium chloride) and the optical density (OD) value at 650 nm was 0.25. Incubate at 37 ° C. until reaching 0.5. Cells are then separated by centrifugation (2,500 × G, 10 minutes) and then washed by adding 50 ml of 0.1 M MgCl ₂ . It was centrifuged again (2,500 × G, 10 minutes) and 50 ml 0.1M CaCl ₂ , 0.05M MgCl ₂ solution was added thereto and left to stand on ice for 30 minutes. This was again centrifuged (2,500 × G, 10 minutes) and uniformly dispersed in 5 ml of the same solution (0.1M CaCl ₂ , 0.05M MgCl ₂ ). All solutions and vessels used above were used after cooling at 0 ° C. 10 μl of the coupling reaction solution was added to 0.2 ml of the solution obtained above, and the mixture was left at 0 ° C. for 40 minutes and then heat-treated at 42 ° C. for 1 minute. This was added to 2.0 ml of liquid LB medium and incubated at 37 ° C. for 1 hour, and then the culture solution was plated on solid LB medium containing 50 μg / ml ampicillin and then incubated at 37 ° C. overnight. M13 vector DNA was heat treated and then 100 μl of JM105 cells, 15 μl of 100 mM IPTG (isopropylthiogalactoside), 25 μl of 4% X-Gal and 3 ml of 2 × soft solid YT preheated to 48 ° C. Add medium (1% NaCl, 1% yeast extract, 1.6% Bakto tryptone, 8.7% Bakto agar) and mix well, then double solid YT medium (1% NaCl, 1% yeast extract, 1.6% Bakto tryptone, 1.5 % Bacto agar).

Reference Example 5 Synthesis of Oligomer

The oligomers were synthesized by a DNA synthesizer (Applied Biosystems, Inc., model 380 B, USA) using automated solid phase phosphoamidite chemistry and then modified polyacrylamide gel (2M urea, 12% acrylic). Amide, bis (29: 1 w / w) in 50 mM Tris, 50 mM boric acid, 1 mM EDTA-Na ₂ ) was separated by electrophoresis. Next, it was attached to SEP-PAK (waters Inc., USA), a C18 column, and purified using pure acetonitrile: water (50:50) as an eluent. The concentration was determined by measuring absorbance at 260 mm.

Reference Example 6 Polymerase Chain Reaction (PCR)

Template DNA (10 ng to 100 ng), 10 μg of 10-fold Taq polymerase buffer solution (10 mM Tris-Cl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.1% (w / v) gelatin), 10 μl of dNTP mixed solution ( 10 g each of dGTP, dATP, dTTP, and dCTP), 2 µg of primer, and 0.5 µl of Ampli Taq DNA polymerase (Perkin Elmer Cetus, USA) were added to distilled water to make a total volume of 100 µl. To this was added 50 μl of mineral oil to prevent evaporation of the solution. The temperature cycler (Perkin Elmer Cetus, USA) was used, and the cycle program was repeated for 1 minute at 95 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C, and finally further reacted at 72 ° C for 10 minutes.

Example 1 Amplification of Korean Hepatitis C Virus Protease Gene and Cloning into Expression Vector

(Step 1)

Proteolytic enzyme gene of previously cloned Korean HCV cDNA (KHCV-LBCl, Applicant's Korean Patent Application No. 91-9510, Accession No. ATCC 75008, Deposit Date May 14, 1991) was combined with synthetic ubiquitin gene (Hereinafter, Korean HCV bound to ubiquitin is referred to as 'UB-PRO'). The following primers were synthesized to clone them into yeast expression vectors.

Probe PPROUBI 5 '-CTTGGTGTTGAGACTCCGCGGTGGTGGGAAGGAGATACTTCTGGG-3'

(25 bases of the 5′-end overlap each other with 25 bases of the 3′-end of the ubiquitin gene, with the remainder comprising 3333 bases to 3380 bases of KHCV-LBCl);

Primer PPROSAL 5 '-GACTAGTGTAAGTGGGCCACTTGGAAT-3'

(With terminating codon after 4029 base of KHCV-LBCl and at the same time with restriction enzyme SalI recognition site).

(Step 2)

Into the reaction tube, 50ng of KHCV-LBCl DNA was used as a template, and 2µg of the primers PPROUBI and 2µg of PPROSAL were added. After the volume was adjusted to 100 µl, the polymerase chain reaction was carried out 25 times as in Reference Example 6.

(Step 3)

The PCR product obtained in step 2 was isolated from 5% polyacrylamide gel, and it was confirmed that 670 base pairs of DNA was amplified and separated and purified from polyacrylamide gel under the same conditions. This DNA fragment was named fragment PRO.

(Step 4)

2 μg of plasmid pYLBC-A / G-UB-HGH (ATCC 74071) was completely cleaved with restriction enzymes PstI and SaII under NEB buffer 3 under the conditions of 2 μg of the same plasmid with restriction enzymes PstI and restriction enzyme SacII. Under the conditions, complete cleavage and separation with 0.7% agarose gel separated and purified about 9.8 kb and 3.4 kb fragments, respectively. These fragments are hereinafter referred to as fragment PL2 and fragment PT2, respectively. The fragment PRO obtained in step 3 was completely digested with restriction enzyme SaII and restriction enzyme SacII under the conditions of buffer 3, and then extracted with phenol / chloroform and dissolved in 20 µl of TE solution. The resulting fragment was named fragment PRO-T2 / L, which was then conjugated as follows.

100 ng fragment PRO-T2 / L and 100 ng fragment PL2, about 100 ng fragment PT2, 2 μl of 10-fold conjugation solution, and 10 units of T4 DNA ligase were added to the reaction tube. Distilled water was added to make it react, and reaction was carried out at 16 ° C for 12 hours. E. coli HB101 (ATCC 33694) was transformed with the generated vector after the reaction to obtain pYLBC-A / G-UB-PRO containing fragment UBPRO (FIG. 2).

Example 2 Induction of Protease Gene Expression of Hepatitis C Virus

Recombinant plasmids were extracted in bulk by alkaline lysis (Ish-Horowicz, D., et. Al., Nucl. Acid Res., 9, 2989 (1981)) and used for yeast transformation. Transformation was based on Beggs (Nature 275, 104 (1978)) and Hinnen et al. (Hinnen et. Al., Proc. Natl. Acad. Sci. USA 75, 1929 (1978)). 12 ml of overnight yeast (S. cerevisiae DC04, Yeast Genetic Stock Center, University of Califonia, Berkeley, Calif., USA) was inoculated into 50 ml of fresh YEPD (2% peptone, 1% yeast extract, 2% glucose). Shaking was incubated at 30 ° C. until the OD value at 650 nm became 0.8 to 1. The cell precipitate obtained by centrifuging the culture at 5000 rpm for 5 minutes was shaken in 20 ml of water, and then centrifuged again (25,000 rpm). The cell precipitate was shaken again with 20 ml of 1M sorbitol solution and re-centrifuged (25,000 rpm). The precipitated cells were dissolved in 5 ml of SP (1 M sorbitol, 50 mM potassium phosphate, pH 7.5) and 5 l of 1 M DTT, followed by 50 l of zymolase (10 mg / ml in 50% glycerol / 50% SP). After 30 minutes of incubation at 30 ° C. in a gentle shaking condition (250rpm), the cell solution was extracted from time to time and treated with 1/10 volume of 0.1% SDS to investigate the degree of spheroplast formation under an optical microscope. . After culturing about 90% or more of yeast cells to spiroplast (usually about 30 minutes), centrifugation (15,000 rpm 3 minutes) to obtain spiroplast, 5 ml of 1M sorbitol and 5 ml of STC (1M sorbitol) , 10mM Tris HCl, pH7.5), respectively, and finally dissolved in 1ml of STC was used for transformation. After mixing 12.5 μl of 10 μg plasmid and 12.5 μl of 2 × STC (double concentration of STC), 50 μl of the spiroplast was added thereto and incubated at room temperature for 10 minutes. Spiroplast precipitate by adding 500 μl of PEG / TC solution (40% PEG-4000, 10 mM CaCl ₂ , 10 mM Tris HCl, pH 7.5) for 10 minutes and then centrifuging (Dynac Centrifuge; 3 minutes at 25000 rpm). After dissolving in 200 μl of 1M sorbitol, 7 ml of regeneration agar, 3 g of bactoa per 100 ml, 50 ml of 2M sorbitol, 0.07 g of amino acid without yeast, 0.1 g lacking leucine And an amino acid mixture of 0.4 ml of 50% glucose and 45 ml of water), which were applied onto a plate and incubated at 30 ° C. for 3 to 5 days to obtain a yeast colony transformed with the recombinant plasmid. Yeast Sacchaomyces cerevisiae DC04-UB-PRO transformed with pYLBC-A / G-UP-PRO was deposited with ATCC on December 11, 1991 (ATCC 74116).

Transformed yeast strains were cultured without 3 ml of leucine (yeast nitrogen base without amino acids (Difco, USA) per 1 liter of culture) and 0.25 g of leucine deficient amino acid mixture. And 5% glucose), incubated at 30 ° C. for 24 hours, followed by centrifugation (15000 rpm, 3 minutes) to obtain yeast cell concentrate, and then suspended in glucose-deficient YEP (peptone 2%, yeast extract 1%). After culturing for hours, 2 hours, 3 hours, 4 hours, 5 hours and overnight, each culture was centrifuged to remove 400 μl of buffer (10 mM Tris HCl, pH7.5, 1 mM PMSF (phenylmethylsulfonyl fluoride) , 8M urea), glass beads having a diameter of 0.4 mm were added in the same volume, and shaken vigorously to destroy cells to obtain a yeast extract. 10 μl of the yeast extract was electrophoresed with 15% SDS-polyacrylamide gel according to the method of Laemmli, et al., Nature 227, 680 (1970), followed by staining with Coomassie brilliant blue R250. It was confirmed that protease protein was produced (see also FIG. 3-A).

The protein isolated on the gel was then blotted to a nitro cellulose filter according to Towin's method (Towin, Proc. Nat'l. Acad. Sci. U.S.A. 76, 4750 (1979)). The filter was placed in PBS (10 mM phosphoric acid, pH 7.0, 0.15 M sodium chloride) containing 0.5% gelatin, 0.2% Tween 20, and shaken for 2 hours at room temperature.

Thereafter, immunoglobulin (IgG, 8.2mg / ml) isolated from serum of Korean hepatitis C patient was diluted 1: 200 in PBS containing 0.5% gelatin and 0.05% Tween, and gently shaken at room temperature for 1 hour. After the reaction, the mixture was washed four times for 5 minutes with PBS containing 0.2% Tween 20. In this filter, anti-human immunoglobulin (Bio-Rad Lab., Anti-Human IgG-HRP) labeled with horseradish peroxidase in PBS containing 0.5% gelatin and 0.05% Tween 20: After dilution to 200, the reaction was performed by shaking for 1 hour at room temperature, followed by 4 times of washing with PBS containing 0.2% Tween 20 for 5 minutes, followed by washing with 50 mM Tris buffer (pH 7.0). The filter was developed by adding 50 mM Tris buffer containing 400 μg / ml 4-chloro-1-naphthol and 0.03% hydrogen peroxide water (see also Figure 3-B). As a result, it was confirmed that the expressed protein responds specifically to the antibody of the patient infected with Korean hepatitis C.

In Figures 3-A and 3-B, the first row is yeast containing no plasmid and the second row is yeast containing pYLBC-A / G-UB-PRO, which is a sample taken after 1 hour of culture, and Heat is a sample taken after 2 hours of culture as yeast containing pYLBC-A / G-UB-PRO, and column 4 is a sample taken after 3 hours of culture as yeast containing pYLBC-A / G-UB-PRO. Row 5 is a yeast containing pYLBC-A / G-UB-PRO and samples taken after 4 hours of culture, and row 6 is a yeast containing pYLBC-A / G-UB-PRO and 5 hours of culture The sample was taken later, column 7 is a yeast containing pYLBC-A / G-UB-PRO and collected overnight after culture, column 8 is a standard protein molecular weight from 200, 97, 72, 43, 29, 18, 14 kilo Daltons.

Claims (7)

A proteolytic enzyme of a Korean-type hepatitis C virus comprising the following amino acid sequence or a fusion recombinant protein with ubiquitin comprising the same. DNA comprising a nucleotide sequence encoding the protein of claim 1. An expression vector comprising the DNA of claim 2. The expression vector according to claim 3, which is pYLBC-A / G-UB-PRO. Yeast transformed with the expression vector of claim 3 or 4. The method of claim 5, wherein Saccharomyces cerevisiae DCO4-UB-PRO (ATCC 74116). A method for producing a protease of the Korean-type hepatitis C virus comprising culturing the yeast of claim 5.