WO2002010396A9 - Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom - Google Patents

Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom

Info

Publication number
WO2002010396A9
WO2002010396A9 PCT/KR2000/000825 KR0000825W WO0210396A9 WO 2002010396 A9 WO2002010396 A9 WO 2002010396A9 KR 0000825 W KR0000825 W KR 0000825W WO 0210396 A9 WO0210396 A9 WO 0210396A9
Authority
WO
WIPO (PCT)
Prior art keywords
hcv
rna polymerase
protein
ns5bδ
ns5b
Prior art date
Application number
PCT/KR2000/000825
Other languages
French (fr)
Other versions
WO2002010396A1 (en
Inventor
Mi-Kyung Min
Ha-Jung Kim
Soo-Il Chung
Seung-Won Jin
Seong Il Choi
Baik-Lin Seong
Original Assignee
Mogam Biotech Res Inst
Hanmi Pharm Ind Co Ltd
Protheon Co Ltd
Mi-Kyung Min
Ha-Jung Kim
Soo-Il Chung
Seung-Won Jin
Seong Il Choi
Baik-Lin Seong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mogam Biotech Res Inst, Hanmi Pharm Ind Co Ltd, Protheon Co Ltd, Mi-Kyung Min, Ha-Jung Kim, Soo-Il Chung, Seung-Won Jin, Seong Il Choi, Baik-Lin Seong filed Critical Mogam Biotech Res Inst
Priority to PCT/KR2000/000825 priority Critical patent/WO2002010396A1/en
Priority to AU2000261861A priority patent/AU2000261861A1/en
Publication of WO2002010396A1 publication Critical patent/WO2002010396A1/en
Publication of WO2002010396A9 publication Critical patent/WO2002010396A9/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to a gene expression system for mass production of hepatitis C viral (HCV) RNZ -dependent RNA polymerase and an enzyme assay using fusion protein expressed therefrom.
  • HCV hepatitis C viral
  • this invention relates to the gene expression system which uses N-terminal domain of E . coli lysyl-tRNA synthetase as a fusion partner and produces HCV RNA-dependent RNA polymerase (NS5B) in water-soluble form on a large scale, and the in vi tro enzyme assay for measuring the polymerase activity.
  • HCV RNA-dependent RNA polymerase
  • the gene expression system of the present invention can produce the HCV RNA polymerase in water- soluble form on a large scale, so it is useful for a construction of in vitro enzyme assay system for HCV enzyme proteins ' and a screening of antiviral agents.
  • the in vi tro enzyme assay for measuring the HCV RNA polymerase of the present invention can be used to develop HCV inhibitory agents.
  • Hepatitis C virus (as referred to be ⁇ HCV ) is the major cause of nonA and nonB type hepatitis.
  • HCV Hepatitis C virus
  • 10-20% of the inflected person have progressed in acute hepatitis, and the rest of them in chronic infection state (Alter, H. J. et al . , N. Eng. J. Med. , 321:1494-1500, 1989). It has been reported that one hundred million seven thousands of people are infected with HCV universally, especially in Africa, Japan and Korea, 15-20% of total population is in chronic infection state, and the number of people newly infected with HCV runs into a million annually.
  • the major characteristic which distinguishes the infection with HCV from the infection with HBV is the point that the possibility of chronic hepatitis is higher than that of HBV. It has been found that the patient suffered from cirrhosis of the liver or liver cancer without infection with HCV, shows a remarkably higher formation • rate of anti-HCV antibody than that of control (Ikeda et al . , Hepatology , 18:47-53, 1993). According to this report, 75% of the infected person with HCV has developed in liver cancer 15 years later. This is very higher than compared with the possibility to be developed in liver cancer is 27% in case of HBV infection, and represents that the HCV infection and the outbreak of cancer are mutually related rather than any other carcinogens .
  • ⁇ -interferon has been widely used. However, it has been reported that about 20% of the patient shows only a curative effect of ⁇ -interferon when ⁇ -interferon is administered into the patient suffering from hepatitis ' (Hoofnagal, J. H., And. Intern . Med. , 39:24-275, 1994).
  • ⁇ -interferon is major type of HCV therapeutic agents which is the most widely used in the world, but it has the problem to cure the hepatitis by HCV infection since the curative effect of ⁇ -interferon is low for HCV-lb type which has a high transfer rate from hepatitis to liver cirrhosis or liver cancer.
  • ⁇ -interferon has no effect on HCV having interferon resistance sequence. So it is keenly necessary to develop new therapeutic agents or remedy for hepatitis by HCV infection.
  • HCV is belong to flavivirus family, and HCV genome is composed of positive single strand RNA having 9.5 kb in size.
  • HCV RNA is polyprotein which is composed of a single ORF (open reading frame) and about 3,000 amino acids, and contains nucleic acid sequences encoding different kinds of about 10 proteins.
  • HCV RNA also contains about 340 nucleotides of 5' untranslated region (UTR) that are important for RNA synthesis and 98 nucleotides of 3' UTR that are a short variable region-polypyrimidine tract. Especially, the 98
  • ⁇ nucleotides of 3' UTR are a highly conserved sequence in various HCV .
  • the polyprotein of HCV is composed of NH 3 -C-E1-E2- p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH in order. That is, a viral nucleocapside protein and structural proteins of Core (C) , El and E2 composed of an envelope, are existed in amino terminal of the polyprotein, and nonstructural proteins of NS2, NS3, NS4A, NS4B and NS5B essential for viral proliferation, are existed in carboxyl terminal in the polyprotein. 9 matured viral proteins are generated by acting host signal peptides and viral protease of NS2-3 and NS3 to the precusor polyproteins and cutting the specific site of precusor polyproteins .
  • the host protease cuts between NH-C-El-E2-p7-NS
  • metailoprotease composed of one-third amino terminal of NS2 and NS3 cuts between NS2-NS3 by autocleavage
  • NS3 serin protease cuts the rest of sites .
  • NS5B protein of the HCV polyproteins has a conserved GOD motif of RNA-dependent RNA polymerase (RdRp) , it is important for viral proliferation that the minus strand is synthesized from the positive strand, and the virus genome is synthesized by using the minus strand as template.
  • RdRp RNA-dependent RNA polymerase
  • NS5B protein has no specificity to the RNA genome and performs primer-dependent RNA polymerization or primer-independent RNA polymerization using monopolymeric RNA as template (Behren, S. E. et al . , EMBO J. , 15:12-22, 1996; Luo, G. et al . , J. Virol . , 74:851-863, 2000) .
  • As considered the structure of HCV it can be easily expected that inhibitory agents to the protease protein or RNA polymerase protein is the most effective for the new type of HCV therapeutic agents or remedy in the same manner of RNA virus like AIDS virus.
  • NS5B protein activity has been performed by using a recombinant NS5B protein.
  • the recombinant NS5B protein is expressed in insect cell using Baculovirus vector system, and the protein has RdRp activity (Behrn, S. E. et al . , EMBO J. , 15:12-22, 1996)
  • the NS5B protein is expressed in E . coli in the form of inclusion body using GST fusion (Yuan, Z. M. et al . , BBRC, 232:231-235, 1997) .
  • the NS5B protein which is deleted with highly hydrophobic 21 amino acids at the carboxyl terminal to increase the solubility of protein is fused with GST, and the soluble NS5B-GST fusion protein is expressed by culturing the recombinant E, coli at 30 ° C and purified in 1 mg/L of production rate (Yamacita. T. et al. r J. Biol . Chem . f 273:15479-15486, 1998).
  • the NS5B protein in the form of inclusion body is ⁇ refolded by direct expression of NS5B in E .
  • NS5B protein coli and the active form of NS5B protein is obtained, or little amount of soluble NS5B protein is purified (Al, R. H.,' et al . , Virus Res . , 53:141-149, 1998) .
  • the existing recombinant NS5B protein expression system has problems that its expression level is very low in case of using E. coli expression system which is widely used for the production of recombinant protein, and the final production yield of the recombinant protein is not much in case of refolding. Therefore, the recombinant NS5B protein expression system using E. col i expression system is not appropriate for in vi tro study of the protein structure analysis or the screening system construction.
  • the NS5B protein which plays an essential role for HCV replication is the objective material to develop antiviral agents with serine protease.
  • HCV is not possible for in vitro cell culture and has no proper animal model, it is of great significance to develop the in vitro assay system or the screening system by using the biochemical property of the NS5B protein.
  • HCV is very important to secure the active form of NS5B protein in quantity.
  • the present inventors have developed that the expression system expresses the protein in the high active form by making up for shortcomings of the existing recombinant protein expression vector, minimizing the protein expression in the inactive form of inclusion body and increasing the solubility.
  • the expression vector pGE-lysN
  • the expression vector is constructed by using 154 amino terminal residues of
  • E . coli lysyl-tRNA synthetase (lysS) , and many proteins are expressed from E . coli in the soluble form by using the expression vector (KR 96-44010; WO PCT/KR97/00186)
  • the present inventors have constructed the gene expression system for mass production of HCV NS5B and developed the in vi tro assay system for measuring the protein activity using the fusion protein expressed therefrom.
  • the present invention provides a gene construct which contains a gene for amino terminal of E. coli lysyl-tRNA. polymerase and the whole or part of HCV NS5B
  • This invention also provides a recombinant expression vector which contains the gene construct.
  • this invention provides a transformant strain which is transformed with the recombinant expression vector.
  • This invention also provides a fusion protein which is expressed by culturing the transformant strain.
  • the present invention provides an enzyme assay for measuring the activity of RNA-dependent RNA polymerase by using the expressed fusion protein. Further features of the present invention will appear hereinafter.
  • FIG. 1 shows a preparing procedure of recombinant expression vector plysN ⁇ -NS5B ⁇ .
  • FIG.2 shows an enzyme restriction map of plysN ⁇ - NS5B ⁇ , wherein LysN: amino terminal of E. coli lysyl-tRNA polymerase (154 amino acids) ; GSGSGS: linker peptid composed of glycine and serine; H : histidine; D4K: enterokinase recognition site; NS5B ⁇ : HCV NS5B of which is removed 20 amino acid residue of carboxyl terminal.
  • FIG.3 shows a SDS-PAGE result of cell debris and supernatant that are obtained by centrifuging the cell lysate of the cultured recombinant strain, wherein Lane 1: protein size marker; Lane 2: HMS174 (plysN ⁇ -NS5B ⁇ ) IPTG induction, total cell lysate; Lane 3: HMS174 (plysN ⁇ -NS5B ⁇ ) IPTG non-induction, total cell lysate; Lane 4: HMS174 (plysN ⁇ -NS5B ⁇ ) IPTG induction, supernatant; Lane 5: purified plysB ⁇ -NS5B ⁇ .
  • FIG.4 shows a result which measures the polyA- dependent .
  • FIG.5 shows a result which analyzes the effect of divalent cation g 2+ and Mn 2+ concentration to the polyA-dependent polyuridylylase activity.
  • FIG.6 shows a result which analyzes the effect of temperature to the polyA-dependent polyuridylylase activity.
  • FIG.7 shows the polyA-dependent polyuridylylase activity of LysN ⁇ -NS5B ⁇ according to the amount of protein .
  • FIG.8 shows the polyA-dependent polyuridylylase activity of LysN ⁇ -NS5B ⁇ according to the time course.
  • FIG.9 shows a result of reaction kinetics to the polyA-dependent polyuridylylase activity of LysN ⁇ -NS5B ⁇ protein, wherein A: Lineweaver-Burk graph to substrate UTP which is obtained from the reaction fixed template/primer into saturated concentration at the fixed value of enzyme concentration; B: a measured value of the early velocity at the various concentration of UTP.
  • the present invention provides a gene construct which contains a gene for amino terminal of E . coli lysyl-tRNA polymerase and the whole or part of HCV NS5B.
  • the gene construct of the present invention is composed of conjugate protein, linker peptide, histidine tagging sequence, recognition site of protein hydrolase, recognition site of restriction enzyme and the whole or part of HCV RNA-dependent RNA-polymerase (NS5B, 1773 base pairs) .
  • the conjugate protein of the present invention uses amino terminal domain of E.
  • the linker peptide of the present invention is GSGSGS represented by the SEQ. ID NO.l following lysN, wherein GSGSGS is oligopeptide of glycine (G) and serine (S). It can be possible to regulate the length of linker peptide more longer.
  • the gene construction ' of the present invention contains the tagging sequence at the rear of the linker peptide sequence to make easy for purification and isolation of fusion protein.
  • the gene construct of the present invention contains the restriction site recognized by sequence specific protease to easily separate the only foreign protein from the expressed fusion protein.
  • the gene construct of the present invention contains the recognition site of enteropeptidase represented by the SEQ. ID MO .2 (DDDDK, D4K) at the rear of histidine tagging sequence. The enteropeptidase cuts the carboxyl terminal of the recognition site.
  • the gene construct of the present invention also contains the recognition site of restriction enzyme for simply cloning of the foreign protein at the rear of the protein restriction site.
  • the recognition site of restriction enzyme in the present invention can be used all kinds of the recognition site of restriction enzyme, particularly, it is preferable to use the recognition site of restriction enzyme composed of Kpnl-BamHI- EcoRV-Sall-Hindlll at the rear of the protein restriction site.
  • the present invention provides a recombinant expression vector " (plysN ⁇ -NS5B) which contains the gene construct composed of the whole NS5B.
  • the present invention also provides a recombinant expression vector (plysN ⁇ -NS5B ⁇ ) which contains the gene construct composed of the deleted NS5B ⁇ , wherein the NS5B ⁇ is removed highly hydrophobic 20 amino acids at the carboxyl terminal of HCV NS5B (see FIG .1 and FIG.2) .
  • the recombinant expression vector is constructed by inserting the gene fragment containing the gene construct into the plasmid pGE-lysN (KR 96-44020) .
  • the ⁇ : plasmid pGE-lysN is the recombinant plasmid which carries the cassette composed of T7 promoter-lysN- histidine tag-enterokinase recognition site- multicloning site.
  • the NS5B and its variant are inserted into the modified pGE-lysN to yield plysN ⁇ - NS5B and plysN ⁇ -NS5B ⁇ respectively.) (see FIG.l).
  • the recombinant strain is prepared by transforming the recombinant expression vector plysN ⁇ -NS5B ⁇ of the present invention into E . coli HMS174 (DE3) plysE, and is named as HMS174 (plysN ⁇ -NS5B ⁇ ) .
  • the recombinant strain has been deposited at Korean Culture Center of Microorganism on July 5, 2000 (Accession No.; KCCM 10193) .
  • the soluble fusion protein LysN ⁇ -NS5B ⁇ is expressed by culturing the recombinant strain (see FIG.3), and the NS5B protein is isolated by protease treatment from the expressed LysN ⁇ -NS5B ⁇ .
  • RNA-dependent RNA polymerase (RdRp) activity of the LysN ⁇ -NS5B ⁇ fusion protein or NS5B protein the activity of polyuridylylase has been analyzed by using the mixture containing polyA as template and oilgo dT as primer (see FIG.4) .
  • divalent cation essential for the enzyme activity it shows an optimal activity at 5 mM Mg 2+ , and 10 mM Mn 2+ .
  • the optimal activity at Mn 2+ only corresponds to 62% of that at Mg 2+ (see FIG.5).
  • the optimal temperature is 30 ° C (see FIG.6), and the amount of enzyme reaction product increases proportional to the amount of enzyme (see FIG.7) and the reaction time (see FIG.8) .
  • the enzyme does not represent any specificity Ao RNA as primers.
  • Km value of the enzyme is 4.5 uM (see FIG.9) .
  • the present invention can be used for a screening of antiviral agents as the inhibitor of the RNA polymerase by using the in vitro enzyme assay of RNA- dependent RNA polymerase activity which uses the mass- produced fusion protein or NS5B protein.
  • Example 1 Construction of plysB ⁇ -NS5B ⁇
  • DNA fragment containing 5' -H4-DK4-NS5B-BamHI-3' was obtained by polymerase chain reaction (as referred to be ⁇ PCR' ) using the primer 1 represented by the SEQ. ID NO.3 and the primer 2 represented by the SEQ. ID NO.4 with plasmid pTrcHisA+NS5B as template, which contains NS5B (1773 base pairs) of HCV-3a at the 5'BamHI-3'EcoRI recognition site of pTrcHisA vector.
  • pGE-lysN ⁇ using 154 amino acid residue of amino terminal of E.
  • DNA fragment (fragment B) containing 5' -Ndel-lysN- GSGSG-H10-D4K-3' was obtained by PCR using the primer 3 represented by the SEQ. ID NO.5 and the primer 4 represented by the SEQ. ID NO.6 with the pGE-lysN ⁇ as template. After the DNA fragment A and B were purified from the agarose gel, fusion PCR amplification was performed by using the primer 3 represented by the SEQ.
  • the primer 2 represented by the SEQ. ID NO .4 with the purified fragment A and B as template.
  • the PCR product was isolated and digested with the restriction enzyme Ndel and BamHI, it was cloned into the recognition site Ndel and BamHI of pGE-lysN.
  • plysN ⁇ -NS5B plasmid was transformed into E. coli HMS174 (DE3) pLysE, and the expression level was measured. As a result, the expression level was low. So, in order to obtain the higher expression yield, the expression vector which carries the C-terminal deleted NS5B was constructed as follows .
  • the expression vector encoding the 20 carboxyl residues deleted NS5B was constructed using the primer 3 represented by the SEQ. ID NO.3 and the primer 5 represented by the SEQ. ID NO.7 with the recombinant plsmid plysN ⁇ -NS5B as template.
  • the expression vector plysN ⁇ -NS5B ⁇ was constructed by inserting the digested DNA fragment into the Ndel and BamHI restriction site of pGE-lysN.
  • the recombinant strain was prepared by transforming the recombinant plasmid plysN ⁇ -NS5B ⁇ into E .
  • HMS174 col i HMS174 (DE3) ⁇ lysE and named as HMS174 (plysN ⁇ - NS5B ⁇ ) .
  • the recombinant strain HMS174 (plysN ⁇ -NS5B ⁇ ) was deposited at Korean Culture Center of Microorganisms on July 5, 2000 (Accession No.; KCCM 10193) .
  • Single colony of the recombinant strain HMS174 (plysN ⁇ -NS5B ⁇ ) was inoculated into LB medium containing amphicilin 50 ug/ml and chloramphenicol 30 ug/ml, and cultured overnight.
  • the culture solution was . diluted into 1/10 to 1/15 using the same composition of LB medium. After adding 1 mM IPTG when the absorbance of OD 6 oo at 37 ° C is 1, the diluted culture solution was incubated at the same temperature for 4 hours . After that, cells were collected by centrifugation, stored in ice box for 30 min, and lysed by sonication.
  • the cell lysate was centrifuged at 12,000 g, 4 ° C for 25 min and separated into supernatant and precipitate.
  • the part of cell lysate, supernatant and precipitate was taken respectively, and mixed with 2X SDS buffer solution. After the each reaction mixture was heated at 100 ° C for 2 min and electrophoresed on 6% SDS-PAGE, the gel was stained with Coomassie blue. As illustrated in the FIG.3, the expressed protein was soluble, cultured at 37 ° C, and the expression level was approximately 5 % of the total soluble protein.
  • 30% ammonium sulfate was added to the supernatant for precipitating the other proteins.
  • the supernatant obtained by centrifugation of the above mixture, and the precipitate was dialyzed with bl buffer solution composed of 50 mM NaP0 4 (pH 6.8), 100 mM NaCl, 0.25 M sucrose, 10% glycerol, 10 mM DTT, 1 mM EDTA, 0,1 mM sucrose monolaurate, 0.02% sodium azaide (NaAzaide) and protease inhibitor, and isolated its protein fraction using UNO S6 column (Biorad) .
  • the obtained NS5B protein was dissolved with 50% ammonium sulfate in bl buffer and then passed on gelfiltration column of Superdex 75 column.
  • Example 3 Enzyme activity analysis of LysN-NS5B ⁇ fusion protein
  • RNA-dependent RNA polymerase (RdRp) of LysN ⁇ -NS5B ⁇
  • the amount of n incorporated [ P] UTP was measured by the assay system of polyuridylylase activity using polyA (Pharmacia) as template and oligo dT 12-18 (Pharmacia) as primer.
  • the standard reaction condition for measuring the enzyme activity was composed as following; 1) reacting 20 ul reaction solution at 30 ° C for 60 min, wherein the reaction solution was composed of 50 mM HEPES (pH 8.0), 25 mM KC1, 5 mM MgCl 2 , 1 mM DTT, 1 mM EDTA, 10 uM UTP, 2.0 uCi [ 32 P]UTP, 50 ng/ml actinomycin D (Sigma) 20 ug/ml rifamycin (Sigma), 20 U RNasin (Giboco-BRL) , 0.5 ug/ml purified LysN ⁇ -NS5B ⁇ , 100 ug/ml polyA and 25 ug/ml oligo dT 12-18, 2) stopping the reaction by adding EDTA into the reaction solution to be 10 mM in final concentration, 3) putting the total reaction solution on DE 81 filter disc (Whatman) and drying in the air, 4) washing the filter disc with
  • the reaction solution substituted oligo dT 12-18 with oligo dA 12-18, and as a positive control to the activity of RNA- dependent RNA polymerase, the RdRp activity of E . coli RNA polymerase were used.
  • actinomycin D as inhibitor of the DNA-dependent RNA polymerase
  • rifamycin as inhibitor of the E . col RNA polymerase were added to the enzyme reaction solution.
  • the reaction solution substituted the primers with oligo dA 12-18 was separately prepared from the protein having any other UTP incorporating activity except RdRp.
  • the expression system of NS5B protein in the present invention produces the NS5B protein in the active soluble form on a large scale and is used for in vi tro assay system.
  • the in vi tro assay system of the present invention can be used for: screening inhibitory agents of HCV protein, especially the NS5B protein as antiviral agents, so it may be used to develop therapeutic agents or remedy for hepatitis treatment and prevention by hepatitis C virus.

Abstract

The present invention relates to gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom. Particularly, the present invention relates to the gene expression system which uses N-terminal domain of E.coli lysyl-tRNA synthetase as fusion partner and mass-produces HCV RNA-depedent RNA polymerase(NS5B) in water-soluble type, and the in vitro enzyme assay for measuring the polymerase activity. The gene expression system of the present invention can mass-produce the HCV RNA polymerase in water-soluble type, so it is useful for a construction of in vitro assay system for HCV enzyme proteins and a screening of antiviral agents. In addition, the in vitro assay for measuring the HCV RNA polymerase of the present invention can be used to develop HCV inhibitory agents.

Description

GEKIE EXPRESSION SYSTEM FOR MASS PRODUCTION OF HEPATITIS C VIRAL RNA-DEPENDENT RNA POLYMERASE AND ENZYME ASSAY USING FUSION PROTEIN EXPRESSED THEREFROM FIELD OF THE INVENTION
The present invention relates to a gene expression system for mass production of hepatitis C viral (HCV) RNZ -dependent RNA polymerase and an enzyme assay using fusion protein expressed therefrom. Particularly, this invention relates to the gene expression system which uses N-terminal domain of E . coli lysyl-tRNA synthetase as a fusion partner and produces HCV RNA-dependent RNA polymerase (NS5B) in water-soluble form on a large scale, and the in vi tro enzyme assay for measuring the polymerase activity. The gene expression system of the present invention can produce the HCV RNA polymerase in water- soluble form on a large scale, so it is useful for a construction of in vitro enzyme assay system for HCV enzyme proteins' and a screening of antiviral agents. In addition, the in vi tro enzyme assay for measuring the HCV RNA polymerase of the present invention can be used to develop HCV inhibitory agents. BACKGROUND
Hepatitis C virus (as referred to be ΛHCV ) is the major cause of nonA and nonB type hepatitis. When persons are infected with HCV, 10-20% of the inflected person have progressed in acute hepatitis, and the rest of them in chronic infection state (Alter, H. J. et al . , N. Eng. J. Med. , 321:1494-1500, 1989). It has been reported that one hundred million seven thousands of people are infected with HCV universally, especially in Africa, Japan and Korea, 15-20% of total population is in chronic infection state, and the number of people newly infected with HCV runs into a million annually. The major characteristic which distinguishes the infection with HCV from the infection with HBV (hepatitis B virus), is the point that the possibility of chronic hepatitis is higher than that of HBV. It has been found that the patient suffered from cirrhosis of the liver or liver cancer without infection with HCV, shows a remarkably higher formation • rate of anti-HCV antibody than that of control (Ikeda et al . , Hepatology , 18:47-53, 1993). According to this report, 75% of the infected person with HCV has developed in liver cancer 15 years later. This is very higher than compared with the possibility to be developed in liver cancer is 27% in case of HBV infection, and represents that the HCV infection and the outbreak of cancer are mutually related rather than any other carcinogens .
To cure the chronic hepatitis of HCV effectively, α-interferon has been widely used. However, it has been reported that about 20% of the patient shows only a curative effect of α-interferon when α-interferon is administered into the patient suffering from hepatitis ' (Hoofnagal, J. H., And. Intern . Med. , 39:24-275, 1994). α-interferon is major type of HCV therapeutic agents which is the most widely used in the world, but it has the problem to cure the hepatitis by HCV infection since the curative effect of α-interferon is low for HCV-lb type which has a high transfer rate from hepatitis to liver cirrhosis or liver cancer. In addition, it has been' reported that α-interferon has no effect on HCV having interferon resistance sequence. So it is keenly necessary to develop new therapeutic agents or remedy for hepatitis by HCV infection.
HCV is belong to flavivirus family, and HCV genome is composed of positive single strand RNA having 9.5 kb in size. HCV RNA is polyprotein which is composed of a single ORF (open reading frame) and about 3,000 amino acids, and contains nucleic acid sequences encoding different kinds of about 10 proteins. HCV RNA also contains about 340 nucleotides of 5' untranslated region (UTR) that are important for RNA synthesis and 98 nucleotides of 3' UTR that are a short variable region-polypyrimidine tract. Especially, the 98
■ nucleotides of 3' UTR are a highly conserved sequence in various HCV .
The polyprotein of HCV is composed of NH3-C-E1-E2- p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH in order. That is, a viral nucleocapside protein and structural proteins of Core (C) , El and E2 composed of an envelope, are existed in amino terminal of the polyprotein, and nonstructural proteins of NS2, NS3, NS4A, NS4B and NS5B essential for viral proliferation, are existed in carboxyl terminal in the polyprotein. 9 matured viral proteins are generated by acting host signal peptides and viral protease of NS2-3 and NS3 to the precusor polyproteins and cutting the specific site of precusor polyproteins . Particularly, the host protease cuts between NH-C-El-E2-p7-NS , metailoprotease composed of one-third amino terminal of NS2 and NS3 cuts between NS2-NS3 by autocleavage, and NS3 serin protease cuts the rest of sites . Since NS5B protein of the HCV polyproteins has a conserved GOD motif of RNA-dependent RNA polymerase (RdRp) , it is important for viral proliferation that the minus strand is synthesized from the positive strand, and the virus genome is synthesized by using the minus strand as template. It has been reported that the NS5B protein has no specificity to the RNA genome and performs primer-dependent RNA polymerization or primer-independent RNA polymerization using monopolymeric RNA as template (Behren, S. E. et al . , EMBO J. , 15:12-22, 1996; Luo, G. et al . , J. Virol . , 74:851-863, 2000) . As considered the structure of HCV, it can be easily expected that inhibitory agents to the protease protein or RNA polymerase protein is the most effective for the new type of HCV therapeutic agents or remedy in the same manner of RNA virus like AIDS virus. To develop the inhibitory agents, it has to meet the requirements for development of in vitro assay which rapidly measures the activity of objective materials to be inhibited. To develop the in vitro assay of activity, it has to be also preceded the study about the activity of objective materials to be inhibited. Namely, in vitro study about the activity of HCV viral proteins must be sufficiently accomplished, and for this, the construction of producing system which produces the viral proteins in the active form on a large scale, is needed.
In vitro study about the NS5B protein activity has been performed by using a recombinant NS5B protein. For example, it has been reported that the recombinant NS5B protein is expressed in insect cell using Baculovirus vector system, and the protein has RdRp activity (Behrn, S. E. et al . , EMBO J. , 15:12-22, 1996) In addition, it has been found that the NS5B protein is expressed in E . coli in the form of inclusion body using GST fusion (Yuan, Z. M. et al . , BBRC, 232:231-235, 1997) . Furthermore, it has been also reported that the NS5B protein which is deleted with highly hydrophobic 21 amino acids at the carboxyl terminal to increase the solubility of protein, is fused with GST, and the soluble NS5B-GST fusion protein is expressed by culturing the recombinant E, coli at 30 °C and purified in 1 mg/L of production rate (Yamacita. T. et al. r J. Biol . Chem . f 273:15479-15486, 1998). On the other hand, it has been reported that the NS5B protein in the form of inclusion body is ■ refolded by direct expression of NS5B in E . coli and the active form of NS5B protein is obtained, or little amount of soluble NS5B protein is purified (Al, R. H.,' et al . , Virus Res . , 53:141-149, 1998) .
The existing recombinant NS5B protein expression system has problems that its expression level is very low in case of using E. coli expression system which is widely used for the production of recombinant protein, and the final production yield of the recombinant protein is not much in case of refolding. Therefore, the recombinant NS5B protein expression system using E. col i expression system is not appropriate for in vi tro study of the protein structure analysis or the screening system construction. As described in the above, the NS5B protein which plays an essential role for HCV replication, is the objective material to develop antiviral agents with serine protease. Especially, since HCV is not possible for in vitro cell culture and has no proper animal model, it is of great significance to develop the in vitro assay system or the screening system by using the biochemical property of the NS5B protein. For developing the in vi tro assay system likethis, it is very important to secure the active form of NS5B protein in quantity.
On the other hand, the present inventors have developed that the expression system expresses the protein in the high active form by making up for shortcomings of the existing recombinant protein expression vector, minimizing the protein expression in the inactive form of inclusion body and increasing the solubility. Particularly, the present inventors have reported that the expression vector (pGE-lysN) is constructed by using 154 amino terminal residues of
E . coli lysyl-tRNA synthetase (lysS) , and many proteins are expressed from E . coli in the soluble form by using the expression vector (KR 96-44010; WO PCT/KR97/00186)
At this point, the present inventors have constructed the gene expression system for mass production of HCV NS5B and developed the in vi tro assay system for measuring the protein activity using the fusion protein expressed therefrom.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a gene expression system for mass production of hepatitis C viral RNA-dependent RNA polymeras and an enzyme assay using fusion protein expressed therefrom. It is a further object of this invention to provide the gene expression system which uses N- terminal domain of E . coli lysyl-tRNA synthetase as a fusion partner and produces HCV RNA-dependent RNA polymerase (NS5B) in water-soluble form on a large scale, and the in vitro enzyme assay for measuring the polymerase activity. Further objects and advantages of the present invention will appear hereinafter.
The present invention provides a gene construct which contains a gene for amino terminal of E. coli lysyl-tRNA. polymerase and the whole or part of HCV NS5B This invention also provides a recombinant expression vector which contains the gene construct. In addition, this invention provides a transformant strain which is transformed with the recombinant expression vector. This invention also provides a fusion protein which is expressed by culturing the transformant strain. Finally, the present invention provides an enzyme assay for measuring the activity of RNA-dependent RNA polymerase by using the expressed fusion protein. Further features of the present invention will appear hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a preparing procedure of recombinant expression vector plysNβ-NS5BΔ. FIG.2 shows an enzyme restriction map of plysNβ- NS5BΔ, wherein LysN: amino terminal of E. coli lysyl-tRNA polymerase (154 amino acids) ; GSGSGS: linker peptid composed of glycine and serine; H : histidine; D4K: enterokinase recognition site; NS5BΔ: HCV NS5B of which is removed 20 amino acid residue of carboxyl terminal. FIG.3 shows a SDS-PAGE result of cell debris and supernatant that are obtained by centrifuging the cell lysate of the cultured recombinant strain, wherein Lane 1: protein size marker; Lane 2: HMS174 (plysNβ-NS5BΔ) IPTG induction, total cell lysate; Lane 3: HMS174 (plysNβ-NS5BΔ) IPTG non-induction, total cell lysate; Lane 4: HMS174 (plysNβ-NS5BΔ) IPTG induction, supernatant; Lane 5: purified plysBβ-NS5BΔ. FIG.4 shows a result which measures the polyA- dependent . polyuridylylase activity of LysNβ-NS5BΔ protein, wherein 1st bar: negative control to the RdRp activity; 2nd bar: in case of using oligo dT 12-18 as primer; 3rd bar: in case of using oilgo dT 12-18 as primer and adding rifamycin. FIG.5 shows a result which analyzes the effect of divalent cation g2+ and Mn2+ concentration to the polyA-dependent polyuridylylase activity. FIG.6 shows a result which analyzes the effect of temperature to the polyA-dependent polyuridylylase activity. FIG.7 shows the polyA-dependent polyuridylylase activity of LysNβ-NS5BΔ according to the amount of protein . FIG.8 shows the polyA-dependent polyuridylylase activity of LysNβ-NS5BΔ according to the time course. FIG.9 shows a result of reaction kinetics to the polyA-dependent polyuridylylase activity of LysNβ-NS5BΔ protein, wherein A: Lineweaver-Burk graph to substrate UTP which is obtained from the reaction fixed template/primer into saturated concentration at the fixed value of enzyme concentration; B: a measured value of the early velocity at the various concentration of UTP.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Hereinafter, the present invention is described in detail. In one aspect, the present invention provides a gene construct which contains a gene for amino terminal of E . coli lysyl-tRNA polymerase and the whole or part of HCV NS5B. The gene construct of the present invention is composed of conjugate protein, linker peptide, histidine tagging sequence, recognition site of protein hydrolase, recognition site of restriction enzyme and the whole or part of HCV RNA-dependent RNA-polymerase (NS5B, 1773 base pairs) . The conjugate protein of the present invention uses amino terminal domain of E. coli lysyl-tRNA synthetase (as referred to be λLysN' ) , which contains amino acids from 1 to 154 of amino terminal in the lysyl-tRNA synthetase. The linker peptide of the present invention is GSGSGS represented by the SEQ. ID NO.l following lysN, wherein GSGSGS is oligopeptide of glycine (G) and serine (S). It can be possible to regulate the length of linker peptide more longer. The gene construction' of the present invention contains the tagging sequence at the rear of the linker peptide sequence to make easy for purification and isolation of fusion protein. It is preferable to use 6 to 10 of histidine residues as the tagging sequence, besides can be used polyarginin. The fusion protein containing the tagging sequence can be easily purified using the various affinity column chromatography . In addition, the gene construct of the present invention contains the restriction site recognized by sequence specific protease to easily separate the only foreign protein from the expressed fusion protein. Particularly, the gene construct of the present invention contains the recognition site of enteropeptidase represented by the SEQ. ID MO .2 (DDDDK, D4K) at the rear of histidine tagging sequence. The enteropeptidase cuts the carboxyl terminal of the recognition site. The gene construct of the present invention also contains the recognition site of restriction enzyme for simply cloning of the foreign protein at the rear of the protein restriction site. The recognition site of restriction enzyme in the present invention can be used all kinds of the recognition site of restriction enzyme, particularly, it is preferable to use the recognition site of restriction enzyme composed of Kpnl-BamHI- EcoRV-Sall-Hindlll at the rear of the protein restriction site.
In ' addition, the present invention provides a recombinant expression vector " (plysNβ-NS5B) which contains the gene construct composed of the whole NS5B. To express the fusion protein having more solubility, the present invention also provides a recombinant expression vector (plysNβ-NS5BΔ) which contains the gene construct composed of the deleted NS5BΔ, wherein the NS5BΔ is removed highly hydrophobic 20 amino acids at the carboxyl terminal of HCV NS5B (see FIG .1 and FIG.2) . The recombinant expression vector is constructed by inserting the gene fragment containing the gene construct into the plasmid pGE-lysN (KR 96-44020) . The ι: plasmid pGE-lysN is the recombinant plasmid which carries the cassette composed of T7 promoter-lysN- histidine tag-enterokinase recognition site- multicloning site. The NS5B and its variant are inserted into the modified pGE-lysN to yield plysNβ- NS5B and plysNβ-NS5BΔ respectively.) (see FIG.l).
The recombinant strain is prepared by transforming the recombinant expression vector plysNβ-NS5BΔ of the present invention into E . coli HMS174 (DE3) plysE, and is named as HMS174 (plysNβ-NS5BΔ) . The recombinant strain has been deposited at Korean Culture Center of Microorganism on July 5, 2000 (Accession No.; KCCM 10193) .
The soluble fusion protein LysNβ-NS5BΔ is expressed by culturing the recombinant strain (see FIG.3), and the NS5B protein is isolated by protease treatment from the expressed LysNβ-NS5BΔ.
To measure RNA-dependent RNA polymerase (RdRp) activity of the LysNβ-NS5BΔ fusion protein or NS5B protein, the activity of polyuridylylase has been analyzed by using the mixture containing polyA as template and oilgo dT as primer (see FIG.4) . In case of divalent cation essential for the enzyme activity, it shows an optimal activity at 5 mM Mg2+, and 10 mM Mn2+. However, the optimal activity at Mn2+ only corresponds to 62% of that at Mg2+ (see FIG.5). The optimal temperature is 30°C (see FIG.6), and the amount of enzyme reaction product increases proportional to the amount of enzyme (see FIG.7) and the reaction time (see FIG.8) . In addition, the enzyme does not represent any specificity Ao RNA as primers. As a result of analyzing Michaelis-Menten steady-state kinetics, Km value of the enzyme is 4.5 uM (see FIG.9) .
The present invention can be used for a screening of antiviral agents as the inhibitor of the RNA polymerase by using the in vitro enzyme assay of RNA- dependent RNA polymerase activity which uses the mass- produced fusion protein or NS5B protein.
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples . However, it will be appreciated that those skilled in the art , on consideration of this disclosure , may make modifications and improvements within the spirit and scope of the present invention . Example 1 : Construction of plysBβ-NS5BΔ
DNA fragment containing 5' -H4-DK4-NS5B-BamHI-3' (fragment A) was obtained by polymerase chain reaction (as referred to be ΛPCR' ) using the primer 1 represented by the SEQ. ID NO.3 and the primer 2 represented by the SEQ. ID NO.4 with plasmid pTrcHisA+NS5B as template, which contains NS5B (1773 base pairs) of HCV-3a at the 5'BamHI-3'EcoRI recognition site of pTrcHisA vector. pGE-lysNβ using 154 amino acid residue of amino terminal of E. coli lysyl-tRNA polymerase as a fusion partner, was constructed by inserting GSGSG between the lysN and histidine tagging sequence of pGE-lysN (KR 96-44010) and further inserting 4 to 6 of histidine. DNA fragment (fragment B) containing 5' -Ndel-lysN- GSGSG-H10-D4K-3' was obtained by PCR using the primer 3 represented by the SEQ. ID NO.5 and the primer 4 represented by the SEQ. ID NO.6 with the pGE-lysNβ as template. After the DNA fragment A and B were purified from the agarose gel, fusion PCR amplification was performed by using the primer 3 represented by the SEQ. ID NO.5 and the primer 2 represented by the SEQ. ID NO .4 with the purified fragment A and B as template. After the PCR product was isolated and digested with the restriction enzyme Ndel and BamHI, it was cloned into the recognition site Ndel and BamHI of pGE-lysN. plysNβ-NS5B plasmid was transformed into E. coli HMS174 (DE3) pLysE, and the expression level was measured. As a result, the expression level was low. So, in order to obtain the higher expression yield, the expression vector which carries the C-terminal deleted NS5B was constructed as follows .
To construct the expression vector encoding the 20 carboxyl residues deleted NS5B, PCR amplification was performed using the primer 3 represented by the SEQ. ID NO.3 and the primer 5 represented by the SEQ. ID NO.7 with the recombinant plsmid plysNβ-NS5B as template. After the amplified DNA fragment was digested with Ndel/BamHI restriction enzymes, the expression vector plysNβ-NS5BΔ was constructed by inserting the digested DNA fragment into the Ndel and BamHI restriction site of pGE-lysN. The recombinant strain was prepared by transforming the recombinant plasmid plysNβ-NS5BΔ into E . col i HMS174 (DE3)ρlysE and named as HMS174 (plysNβ- NS5BΔ) . The recombinant strain HMS174 (plysNβ-NS5BΔ) was deposited at Korean Culture Center of Microorganisms on July 5, 2000 (Accession No.; KCCM 10193) .
The construction procedure of the expression vector was shown in the FIG.l, and the restriction map of plysNβ-NS5BΔ in the FIG.2.
Example 2: Expression and purification of HCV NS5B
Single colony of the recombinant strain HMS174 (plysNβ-NS5BΔ) was inoculated into LB medium containing amphicilin 50 ug/ml and chloramphenicol 30 ug/ml, and cultured overnight. The culture solution was . diluted into 1/10 to 1/15 using the same composition of LB medium. After adding 1 mM IPTG when the absorbance of OD6oo at 37°C is 1, the diluted culture solution was incubated at the same temperature for 4 hours . After that, cells were collected by centrifugation, stored in ice box for 30 min, and lysed by sonication. The cell lysate was centrifuged at 12,000 g, 4 °C for 25 min and separated into supernatant and precipitate. First, to check for the existence of the protein expression, the part of cell lysate, supernatant and precipitate was taken respectively, and mixed with 2X SDS buffer solution. After the each reaction mixture was heated at 100°C for 2 min and electrophoresed on 6% SDS-PAGE, the gel was stained with Coomassie blue. As illustrated in the FIG.3, the expressed protein was soluble, cultured at 37 °C, and the expression level was approximately 5 % of the total soluble protein. For separating the NS5B protein, 30% ammonium sulfate was added to the supernatant for precipitating the other proteins. The supernatant obtained by centrifugation of the above mixture, and the precipitate was dialyzed with bl buffer solution composed of 50 mM NaP04 (pH 6.8), 100 mM NaCl, 0.25 M sucrose, 10% glycerol, 10 mM DTT, 1 mM EDTA, 0,1 mM sucrose monolaurate, 0.02% sodium azaide (NaAzaide) and protease inhibitor, and isolated its protein fraction using UNO S6 column (Biorad) . The obtained NS5B protein was dissolved with 50% ammonium sulfate in bl buffer and then passed on gelfiltration column of Superdex 75 column.
Example 3 : Enzyme activity analysis of LysN-NS5BΔ fusion protein
To analyze the activity of RNA-dependent RNA polymerase (RdRp) of LysNβ-NS5BΔ, the amount of n incorporated [ P] UTP was measured by the assay system of polyuridylylase activity using polyA (Pharmacia) as template and oligo dT 12-18 (Pharmacia) as primer. The standard reaction condition for measuring the enzyme activity was composed as following; 1) reacting 20 ul reaction solution at 30°C for 60 min, wherein the reaction solution was composed of 50 mM HEPES (pH 8.0), 25 mM KC1, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 10 uM UTP, 2.0 uCi [32P]UTP, 50 ng/ml actinomycin D (Sigma) 20 ug/ml rifamycin (Sigma), 20 U RNasin (Giboco-BRL) , 0.5 ug/ml purified LysNβ-NS5BΔ, 100 ug/ml polyA and 25 ug/ml oligo dT 12-18, 2) stopping the reaction by adding EDTA into the reaction solution to be 10 mM in final concentration, 3) putting the total reaction solution on DE 81 filter disc (Whatman) and drying in the air, 4) washing the filter disc with 0.5 M Na2HP0 solution for three times, DW for once and 100% ethanol for once and drying in the air, and 5) measuring the radioactivity by using liquid scintillation analyzer (PACKARD) .
In addition, as a negative control to the activity of polyA-dependent polyuridylylase, the reaction solution substituted oligo dT 12-18 with oligo dA 12-18, and as a positive control to the activity of RNA- dependent RNA polymerase, the RdRp activity of E . coli RNA polymerase were used. For measuring the only UTP incorporation derived from RNA-dependent RNA polymerase, actinomycin D as inhibitor of the DNA-dependent RNA polymerase and rifamycin as inhibitor of the E . col RNA polymerase were added to the enzyme reaction solution. In addition, the reaction solution substituted the primers with oligo dA 12-18 was separately prepared from the protein having any other UTP incorporating activity except RdRp. As a result, cpm value corresponding to the reaction without protein was detected in the reaction solution substituted the primers with oligo dA 12-18. On the other hand, cpm value corresponding to the E . coli RNA polymerase activity as the positive control to RdRp activity was- detected in the reaction solution using the primers with oligo dT 12-18. Meanwhile, the activity of E. coli RNA polymerase was almost completely inhibited in the reaction solution, which was added rifamycin and used oligo dT 12-18 as the primer, but there was no change in the activity of LysNβ-NS5BΔ (FIG.4). From these results, it was found that the activity of LysNβ-NS5BΔ was derived from the HCV RdRp activity solely. In addition, Km value to the substrate UTP was measured to examine Michaelis-Menten steady-state kinstics. It was measured in the condition that the amount of enzyme was fixed to 0.5 ug, and the amount of polyA and oligo dT 12-18 to 2 ug and 0.5 ug, respectively, to be saturated its amount to the given amount of enzyme. UTP concentration used 5 points in the range of 0.8 to 30 uM . Since the amount of product in the used reaction condition represented a propotional increase in 10 to
60 min of reaction time, the. initial velocity at the each UTP concentration used the value within 20 min of reaction time. The analyzing result of enzyme activity was represented in the FIG.5 to FIG.9.
INDUSTRIAL APPLICABILITY
The expression system of NS5B protein in the present invention produces the NS5B protein in the active soluble form on a large scale and is used for in vi tro assay system. In addition, the in vi tro assay system of the present invention can be used for: screening inhibitory agents of HCV protein, especially the NS5B protein as antiviral agents, so it may be used to develop therapeutic agents or remedy for hepatitis treatment and prevention by hepatitis C virus.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention
Those skilled in the art will also appreciate that such ?2 equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .
BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM r "I To. Baik-Lin Seong Department of Biotechnology, College of Engineering, Yonsei University RECEIPT IN THE CASE OF ΛN ORIGINAL 134, Shinchon-dong, Seodaemun-gu, issued pursuant, to Rule 7. 1 by the Seoul, 120-749 INTERNATIONAL DEPOSITARY AUTHORITY Republic of Korea identified at the bottom of I his page L. j
Figure imgf000025_0001
nlornational depositary authority is converted into a deposit under the Budapest Treaty, such date on which the microorganism was received by the international depositary authouity. Form B'P/4 Sole page

Claims

What is Claimed is
1. A gene construct composed of conjugate protein, linker peptide, histidine tagging sequence, recognition site of restriction enzyme and the whole or part of RNA-dependent RNA polymerase (NS5B, 1,773 base pairs) of hepatitis C virus (HCV) .
2. The gene construct according to the claim 1, wherein the conjugate protein is 1 "to 154 amino acid domain at the amino terminal of E . coli lysyl-tRNA polymerase.
3. The gene construct according to the claim 1, wherein the part of NS5B is NS5BΔ which is deleted 20 amino acids at the carboxyl terminal.
4. A recombinant expression vector composed of the gene construct of the claim 1, T7 promoter and antibiotics gene as a selective marker.
5. The recombinant expression vector according to the claim 4, which is pl'ysNβ-NS5BΔ having the restriction map of FIG.2.
6. A microorganism transformant which is transformed with the expression vector of the claim 5.
7. The microorganism transformant according to the claim 6 which is HMS174 (plysNβ-NS5BΔ) , wherein the microorganism is E . coli .
8. A fusion protein LysNβ-NS5BΔ having RNA-dependent RNA polymerase of hepatitis C virus which is expressed from the microorganism tramsformant of the claim 7.
9. An enzyme assay for measuring the activity of RNA-dependent RNA polymerase which measures the activity of polyuridylylase by using the reaction misture composed of the fusion protein of the claim 8, primer, inhibitor of DNA-dependent RNA polymerase and UTP.
10. The enzyme assay according to the claim 9, wherein the template is polyA.
11. The enzyme assay according to the claim 9, wherein the primer is oligodeoxythymidine .
PCT/KR2000/000825 2000-07-28 2000-07-28 Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom WO2002010396A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/KR2000/000825 WO2002010396A1 (en) 2000-07-28 2000-07-28 Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom
AU2000261861A AU2000261861A1 (en) 2000-07-28 2000-07-28 Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2000/000825 WO2002010396A1 (en) 2000-07-28 2000-07-28 Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom

Publications (2)

Publication Number Publication Date
WO2002010396A1 WO2002010396A1 (en) 2002-02-07
WO2002010396A9 true WO2002010396A9 (en) 2005-04-14

Family

ID=19198245

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2000/000825 WO2002010396A1 (en) 2000-07-28 2000-07-28 Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom

Country Status (2)

Country Link
AU (1) AU2000261861A1 (en)
WO (1) WO2002010396A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015060235A1 (en) * 2013-10-21 2015-04-30 大鵬薬品工業株式会社 Novel four-ctl epitope-joined peptide

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100203919B1 (en) * 1996-10-04 1999-06-15 신동권 A novel expression vector which produces water soluble protein

Also Published As

Publication number Publication date
AU2000261861A1 (en) 2002-02-13
WO2002010396A1 (en) 2002-02-07

Similar Documents

Publication Publication Date Title
Suzich et al. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes
AU717875B2 (en) HCV NS3 protein fragments having helicase activity and improved solubility
AU2003280239B2 (en) Inhibitor-resistant HCV NS3 protease
US20070172936A1 (en) Purified active HCV NS2/3 protease
Shoji et al. Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli
WO2002010396A9 (en) Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom
KR20020010241A (en) Gene expression system for mass production of hepatitis c viral rna-dependent rna polymerase and enzyme assay using fusion protein expressed therefrom
KR100445604B1 (en) RNA as template for efficient de novo RNA synthesis by HCV NS5B polymerase
KR100241268B1 (en) New active ns4a fused hcv protease and process for preparation thereof
AU2002224688B2 (en) Purified active HCV NS2/3 protease
KR100291102B1 (en) Hcv protease gene and expression thereof in e. coli
KR100921624B1 (en) Novel hepatitis C virus mutants having enhanced replication and infectivity
KR100285585B1 (en) Process for producing nonstructural 5-1.2 protein of hepatitis c virus(hcv)
KR20020070125A (en) Recombinant hepatitis c virus ns5b protein, and preparation process and use thereof
JPH05192160A (en) C type hepatitis virus genome rna, cdna and polypeptide
Luo Expression of an active hepatitis C virus serine proteinase in E. coli
AU2002224688A1 (en) Purified active HCV NS2/3 protease
KR19980069021A (en) Histidine-labeled Korean Non-Structural Protein 3 of Hepatitis C Virus and Its Preparation Method

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 09763567

Country of ref document: US

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

COP Corrected version of pamphlet

Free format text: SEQUENCE LISTING ADDED (3 PAGES); DUE TO A SCANNING ERROR DURING THE TECHNICAL PREPARATIONS FOR INTERNATIONAL PUBLICATION