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Title:
GENE EXPRESSION SYSTEM FOR MASS PRODUCTION OF HEPATITIS C VIRAL RNA-DEPENDENT RNA POLYMERASE AND ENZYME ASSAY USING FUSION PROTEIN EXPRESSED THEREFROM
Document Type and Number:
WIPO Patent Application WO/2002/010396
Kind Code:
A1
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.

Inventors:
MIN MI-KYUNG (KR)
KIM HA-JUNG (KR)
CHUNG SOO-IL (KR)
JIN SEUNG-WON (KR)
CHOI SEONG IL (KR)
SEONG BAIK-LIN (KR)
Application Number:
PCT/KR2000/000825
Publication Date:
February 07, 2002
Filing Date:
July 28, 2000
Export Citation:
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Assignee:
MOGAM BIOTECH RES INST (KR)
HANMI PHARM IND CO LTD (KR)
PROTHEON CO LTD (KR)
MIN MI KYUNG (KR)
KIM HA JUNG (KR)
CHUNG SOO IL (KR)
JIN SEUNG WON (KR)
CHOI SEONG IL (KR)
SEONG BAIK LIN (KR)
International Classes:
C12N15/51; C12N15/70; (IPC1-7): C12N15/51; C12N1/20; C12N15/62; C12N15/70
Domestic Patent References:
WO1998014591A11998-04-09
Other References:
YUAN Z.H. ET AL.: "Expression, purification and partial characterization of RNA polymerase", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 231, no. 1, 1997, pages 231 - 235, XP000877460, DOI: doi:10.1006/bbrc.1997.6249
Attorney, Agent or Firm:
Lee, Won-hee (Sung-ji Heights II 642-16 Yoksam-don, Kangnam-ku Seoul 135-080, KR)
Download PDF:
Claims:
What is Claimed is
1. l.
2. A gene construct composed of conjugate protein, linker peptide, histidine tagging sequence, recognition site of restriction enzyme and the whole or part of RNAdependent RNA polymerase (NS5B, 1,773 base pairs) of hepatitis C virus (HCV).
3. 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 lysyltRNA polymerase.
4. The gene construct according to the claim 1, wherein the part of NS5B is NS5BA which is deleted 20 amino acids at the carboxyl terminal.
5. A recombinant expression vector composed of the gene construct of the claim 1, T7 promoter and antibiotics gene as a selective marker.
6. The recombinant expression vector according to the claim 4, which is plysNßNlS5BX having the restriction map of FIG. 2.
7. A microorganism transformant which is transformed with the expression vector of the claim 5.
8. The microorganism transformant according to the claim 6 which is HMS174 (plysNpNS5BA), wherein the microorganism is E. coli.
9. A fusion protein LysNpNS5BA having RNAdependent RNA polymerase of hepatitis C virus which is expressed from the microorganism tramsformant of the claim 7.
10. An enzyme assay for measuring the activity of RNAdependent RNA polymerase which measures the activity of polyuridylylase by using the reaction misture composed or the fusion protein of the claim 8, primer, inhibitor of DNAdependent RNA polymerase and UTP.
11. The enzyme assay according to the claim 9, wherein the template is polyA. ll. The enzyme assay according to the claim 9, wherein the primer is oligodeoxythymidine.
Description:
GENE 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) RNA-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 vitro 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 vitro 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 HCV') is the

major cause of nonA and nonB type hepatitis. When persons are infected with HCV, 10-20% of the infected 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,

a-interferon has been widely used. However, it has been reported that about 20% of the patient shows only a curative effect of a-interferon when a-interferon is administered into the patient suffering from hepatitis (Hoofnagal, J. H., And. Intem. Med., 39: 24-275,1994). a-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 a-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 a-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), E1 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 NH3-C-El-E2-p7-NS2, metalloprotease 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., <BR> 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 <BR> 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., J.

Biol. Chem., 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. coli expression system is not appropriate for in vitro 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 vitro 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 vitro 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 plysNß-NS5BA.

FIG. 2 shows an enzyme restriction map of plysNp- NS5BA, 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; NS5BA : 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 (plysNp-NSSBA) IPTG induction, total cell lysate; Lane 3: HMS174 (plysNp-NS5BA) IPTG non-induction, total cell lysate ; Lane 4: HMS174 (plysNp-NS5BA) IPTG induction, supernatant ; Lane 5: purified plysBß-NS5BA.

FIG. 4 shows a result which measures the polyA- dependent. polyuridylylase activity of LysNß-NS5BA protein, wherein lst 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 Mg 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 LysNß-NS5BA according to the amount of protein.

FIG. 8 shows the polyA-dependent polyuridylylase activity of LysNß-NS5BA according to the time course.

FIG. 9 shows a result of reaction kinetics to the polyA-dependent polyuridylylase activity of LysNß-NS5BA 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 LysN'), 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. 1 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 NO. 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 KpnI-BamHI- EcoRV-SalI-HindIII at the rear of the protein restriction site.

In'addition, the present invention provides a recombinant expression vector' (plysNß-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 (plysN-NS5BA) which contains the gene construct composed of the deleted NS5BA, wherein the NS5BA 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 plysNp- NS5B and plysNp-NSSBA respectively. ) (see FIG. l).

The recombinant strain is prepared by transforming the recombinant expression vector plysNp-NS5BA of the present invention into E. coli HMS174 (DE3) plysE, and is named as HMS174 (plysNp-NS5BA). 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 (3-NS5BA is expressed by culturing the recombinant strain (see FIG. 3), and the NS5B protein is isolated by protease treatment from the expressed LysNp-NS5BA.

To measure RNA-dependent RNA polymerase (RdRp) activity of the LysNß-NS5BA 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

Mg, 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 30C (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 to 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 plysB-NS5BA DNA fragment containing 5'-H4-DK4-NS5B-BamHI-3' (fragment A) 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-lysNp 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'-NdeI-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. 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 NdeI and BamHI, it was cloned into

the recognition site NdeI and BamHI of pGE-lysN. plysNp-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 plysNp-NS5B as template.

After the amplified DNA fragment was digested with NdeI/BamHI restriction enzymes, the expression vector plysNp-NS5BA was constructed by inserting the digested DNA fragment into the NdeI and BamHI restriction site of pGE-lysN.

The recombinant strain was prepared by transforming the recombinant plasmid plysNp-NS5BA into E. coli HMS174 (DE3) plysE and named as HMS174 (plysNß- NS5BA). The recombinant strain HMS174 (plysN (3-NS5BA) 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. 1, and the restriction map of plysNß-NS5BA in the FIG. 2.

Example 2: Expression and purification of HCV NS5B Single colony of the recombinant strain HMS174 (plysNß-NS5BA) 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 OD600 at 37C 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 100C 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 t of the to al 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-NS5BA fusion protein To analyze the activity of RNA-dependent RNA polymerase (RdRp) of LysNp-NS5BA, the amount of incorporated [322 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 sclution at 30C 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 LysNp-NS5BA, 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 Na2HP04 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. co1= RrlA polyr ; : ras

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 LysNp-NS5BA (FIG. 4). From these results, it was found that the activity of LysNa-NS5BA 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 vitro assay system. In addition, the in vitro 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 : n the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the preser, in---ntion Those skilled in the art will also appreciate e th such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

BUDAPEST TREATY () N THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNAT ! ZONAL FORM r i To. Baik-Lin Seong Department of Biotechnology, College of Engineering, Yonsei University RECEIPT IN THE CASE OF AN 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 this page L J I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR : INTERNATIONAL DEPOSITARY AUTHORITY : Escherichia coli HMS174 (plysN ß-NS5B a) KCCM-10193 n SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by : D a scientific description Mi a proposed taxonomic designation (Mark with a cross where applicable) m. RECEIPT AND ACCEPTANCE This International Depositary Authouity accepts the microorganism identified under I above, which was received by it on Jul. 5. 2000 (date of the original deposit)' ìV. INTERNATIONAL DEPOSITARY AUTHORITY Name : Korean Culture Center of Microorganisms Signature (s) of person (s) having the power to represent the International Depositary Xddress : 361-Z21 Yurim B/D . Authoritv of of autholized--ofEIcial (s) ; Hongje"1-dong, t ; LU Seodaemun-gu .. . flJ : 1 Seodaemun-gu ?)-E SEOUL 120-091 Date : Jul. 11. 2000 ! r-j y - i Republic of Korea r. I __ X i.. ... m ; : 1 Where Rule 6. 4 (d) applies, such date is the date'on which the status of international depositary authority was acquired : where a deposit made outside the Budapest Treaty after the acquisition of the status of i nmrnational depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary auihouily. Furm I3P/4 olo EriLe