Detergent-free hepatitis C protease

Abstract

The protease of Hepatitis C virus (HCV) is purified without detergent, and is useful as a screening tool for HCV antivirals as well as a diagnostic tool for diseases resulting from HCV infection.

Description

This application is a continuation of provisional application Ser. No. 60/027,274, filed Sep. 27, 1996, now abandoned.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is found in 0.5% to 8.0% of blood donors worldwide. Because the infection is chronic in more than 60% of infected persons, the disease is an important public health and economic problem. The management of patients with chronic hepatitis C is complex--the disease is often only mildly symptomatic and slowly progressive, but 20% of patients develop cirrhosis after 20 years of infection and perhaps 10% of those with cirrhosis develop hepatocellular carcinoma. It is also an important indication for liver transplantation. In Europe and Japan the disease is more important numerically than is either hepatitis B or HIV infection. Existing antiviral agents are effective in only a minority of patients, yet good responses can be obtained.

An important target for the treatment of HCV is nonstructural protein 3, a protease encoded by HCV. This NS3 protease associated with human hepatitis C virus is an unstable protein in the absence of high concentrations of detergent. To stabilize the NS3 protease to sufficient quantities for biochemical, kinetic, and biophysical analyses, as well as for the construction of antiviral screening assays, ionic or non-ionic detergents need be incorporated both during purification and analyses. Antiviral leads discovered with detergent treated NS3 protein are not useful. Further, the presence of high quantities of detergents renders significant difficulties in the precise interpretations of biochemical, kinetic, and biophysical analyses. In some cases (e.g., sedimentation, protein crystallization), the presence of detergents preclude biochemical, kinetic, and biophysical analyses.

Prior methods employed detergents and glycerol for purification of NS3. Applicants have discovered a method of purifying NS3 without detergent, with from 5% to about 20% glycerol, preferably 7-12% and with high stability and activity. The resultant enzyme displays a higher catalytic activity than what is known for this protease, that is, 10-500 fold more active than the prior art preparations, depending on the form of the enzyme. Purification according to the methods of the present invention ensures a high stability of the NS3 protease, rendering it amenable to kinetic, biochemical, and biophysical analyses in the absence of detergents. Prior art methods do not afford a stable, detergent free NS3 for enzymologic, biochemical, and biophysical studies.

When properly expressed and prepared from the cloned plasmid in E. coli, the NS3 protease is obtained in milligram quantities in the complete absence of detergent. The resultant enzyme is very soluble and stable for long periods of time (weeks to months at 4.degree. C. and >12 months at -80.degree. C.), and displays high catalytic activity.

An assay with the detergent free HCV NS3 protease is useful as a screening tool for HCV antivirals as well as a diagnostic tool for diseases resulting from HCV infection. The potency range of the HCV antivirals can range from subnanomolar to micromolar concentrations.

BRIEF DESCRIPTION OF THE INVENTION

Detergent free NS3 protease of Hepatitis C virus (HCV) is prepared, and a screening assay for the protein inhibitors is constructed. The detergent free NS3 protease is useful as a screening tool for HCV antivirals, as well as a diagnostic tool for diseases resulting from HCV infection.

DETAILED DESCRIPTION OF THE INVENTION

______________________________________ABBREVIATIONS AND DEFINITIONS______________________________________HCV Hepatitis C VirusIPTG Isopropyl-D(-)thiogalactopyranosideNS3 Nonstructural protein 3 of Hepatitis C virusPMSF Phenylmethylsulfonyl fluorideEDTA ethylendiammino-tetraacetic acidDTT dithiothreitolr.p.m. Revolutions per minutePAGE Polyacrylamide gelSDS Sodium dodecylsulfateNS non-structuralHPLC High Performance Liquid Chromatography______________________________________

In one aspect of the invention a stable, detergent free HCV NS3 protease is claimed.

In another aspect of the invention a screening assay for the detection of compounds that inhibit HCV NS3 protease is claimed.

In still another aspect of the invention, the compounds that inhibit HCV NS3 protease as measured by the screening assay of claim 2 are claimed.

In yet another aspect of the invention, a process for purifying active HCV NS3 protease without detergent is claimed.

There is disclosed stable, detergent free nonstructural protein 3 of Hepatitis C virus, also known as NS3 protease of HCV or NS3. The NS3 protease is useful screening tool for HCV antivirals, as well as a diagnostic tool for diseases resulting from HCV infection.

One utility for HCV NS3 protease is a screening assay for the detection of compounds that inhibit HCV NS3 protease. This assay has a procedure comprising the steps of:

(a) providing a quantity of a compound or compounds to be assayed; (b) incubating said compound or compounds with detergent free HCV NS3 protease in an HCV NS3 protease assay; (c) determining the inhibition of said protease in the HCV NS3 protease substrate cleavage assay.

Also encompassed in the present invention are compounds that substantially inhibit the HCV NS3 protease.

This invention also relates to a process for purifying active HCV NS3 protease without detergent and with from 5% to about 20% glycerol, comprising the steps of:

(a) providing a quantity of cells expressing HCV NS3 protease; (b) disrupting the cells to form a suspension in buffer without detergent; (c) centrifuging the suspension to remove particulate matter; (d) subjecting the supernatant of step (c) to one or more steps of ion exchange chromatography under eluting buffer conditions without detergent; (e) to give active HCV NS3 protease in buffer without detergent.

One embodiment of the process for purifying active HCV NS3 protease without detergent and with from 7% to about 12% glycerol, comprises the steps of:

(a) providing a quantity of cells expressing HCV NS3 protease; (b) disrupting the cells with a microfluidizer to form a suspension in buffer without detergent, said buffer having pH of between about 6.5 and about 7.5; (c) centrifuging the suspension to remove particulate matter, at between about 5000 and about 8000 r.p.m. for about 15 minutes; (d) subjecting the supernatant of step (c) to one or more steps of cation exchange chromatography under eluting buffer conditions in a salt or pH gradient without detergent; (e) to give active HCV NS3 protease in buffer without detergent.

The nonstructural protein 3 of Hepatitis C virus, also known as NS3 protease, can exist in active form as an enzyme or as a complex with the cofactor . It has been discovered by applicants that the complex is about 1000 times more active than the enzyme by itself. The enzyme is itself about 10 times more active than prior art preparations purified with detergent. In the screening assays of the present invention, all active forms are encompassed.

Expression of HCV NS3 Protease in a Recombinant Expression System

It is now a relatively straightforward technology to prepare cells expressing a foreign gene. Such cells act as hosts and include E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells. Expression vectors for many of these host cells have been isolated and characterized, and are used as starting materials in the construction, through conventional recombinant DNA techniques, of vectors having a foreign DNA insert of interest. Any DNA is foreign if it does not naturally derive from the host cells used to express the DNA insert. The foreign DNA insert may be expressed on extrachromosomal plasmids or after integration in whole or in part in the host cell chromosome(s), or may actually exist in the host cell as a combination of more than one molecular form. The choice of host cell and expression vector for the expression of a desired foreign DNA largely depends on availability of the host cell and how fastidious it is, whether the host cell will support the replication of the expression vector, and other factors readily appreciated by those of ordinary skill in the art.

The technology for recombinant procaryotic expression systems is now old and conventional. The typical host cell is E. coli. The technology is illustrated by treatises such as Wu, R (ed) Meth. Enzymol., 68 (1979) and Maniatis, T. et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor 1982.

The foreign DNA insert of interest comprises a DNA sequence coding for HCV NS3 protease (or stable functional mutant thereof) of the present invention, including any synthetic sequence with this coding capacity or any such cloned sequence or combination thereof. For example, HCV peptides coded and expressed by an entirely recombinant DNA sequence is encompassed by this invention.

Vectors useful for constructing eukaryotic expression systems for the production of recombinant HCV comprise the DNA sequence for HCV or variant thereof, operatively linked thereto with appropriate transcriptional activation DNA sequences, such as a promoter and/or operator. Other typical features may include appropriate ribosome binding sites, termination codons, enhancers, terminators, or replicon elements. These additional features can be inserted into the vector at the appropriate site or sites by conventional splicing techniques such as restriction endonuclease digestion and ligation.

Yeast expression systems, which are one variety of recombinant eukaryotic expression systems, generally employ Saccharomyces cerevisiae as the species of choice for expressing recombinant proteins. S. cerevisiae and similar yeasts possess well known promoters useful in the construction of yeast expression systems, including but not limited to GAP491, GAL10, ADH2, and alpha mating factor.

Yeast vectors useful for constructing recombinant yeast expression systems for expressing HCMV include, but are not limited to, shuttle vectors, cosmids, chimeric plasmids, and those having sequences derived from 2-micron circle plasmids.

Insertion of the appropriate DNA sequence coding for HCV, into these vectors will, in principle, result in a useful recombinant yeast expression system for HCV where the modified vector is inserted into the appropriate host cell, by transformation or other means.

One preferred expression system is with baculovirus, under the control of the polyhedrin promoter or the p10 promoter. See, e.g., D.R. O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual W.H. Freeman 1992, for a background description of this expression technology. This system employs the isolation of a recombinant baculovirus carrying the gene of interest. The baculovirus system is especially useful for the simultaneous expression of more than one protein.

Recombinant mammalian expression systems are another means of producing the recombinant HCV for the conjugates of this invention. In general, a host mammalian cell can be any cell that has been efficiently cloned in cell culture. Host mammalian cells useful for the purposes of constructing a recombinant mammalian expression system include, but are not limited to, Vero cells, NIH3T3, GH3, COS, murine C127 or mouse L cells. Mammalian expression vectors can be based on virus vectors, plasmid vectors which may have SV40, BPV or other viral replicons, or vectors without a replicon for animal cells. Detailed discussions on mammalian expression vectors can be found in the treatises of Glover, D.M. (ed.) "DNA Cloning: A Practical Approach," IRL 1985, Vols. I and II.

Recombinant HCV may possess additional and desirable structural modifications not shared with the same organically synthesized peptide, such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation, myristoylation, extension or trimming of either the amino- or carboxy-terminal ends or both. These added features may be chosen or preferred as the case may be, by the appropriate choice of recombinant expression system. On the other hand, recombinant HCV may have its sequence extended by the principles and practice of organic synthesis.

Purification

In the detergent free purification process of the present invention, virtually any source of NS3 protease is suitable, whether recombinant or not. Preferred sources are recombinant, most preferred is an expression system using E. coli.

For expression systems, whether or not recombinant, the NS3 must first be isolated in a soluble fraction. Cells expressing HCV protease are disrupted in buffer to form a suspension in buffer. The disruption is carried out by any of a variety of well known techniques, including but not limited to treatment with a French press, a microfluidizer, sonicator, or self-digestion by inductive expression of lyzozyme. A preferred technique of disruption is with a microfluidizer.

Throughout the purification process without detergent, it is critical to maintain the pH of any buffer between about 6.5 and about 7.5.

The next step involves initial fractionation of the suspension of cellular debris. The suspension is treated to separate the soluble fraction from particulate matter, or such other step is performed that substantially separates soluble from insoluble protein. Appropriate techniques include, but are not limited to, centrifugation at between about 5,000 r.p.m. and about 8,000 r.p.m. for about 15 minutes, filtration, or salt precipitation with e.g. (NH.sub.4).sub.2 SO.sub.4. It is understood that these initial fractionation procedures are well known and are subject to many variations. Appropriate modifications in the initial fractionation of NS3 are well within the skill of the art. The preferred method is centrifugation.

Initial fractionation of the suspension of cellular debris results in a supernatant as well as a precipitate or insoluble pellet. The supernatant is treated further.

The supernatant is then subjected to one or more steps of ion exchange chromatography, and, optionally, gel filtration, to give a substantially pure NS3 in buffer without detergent. Preferred ion exchangers include, but are not limited to, cation exchangers on polystyrene, cation exchangers on dextran, cation exchangers on agarose, cation exchangers on cellulose, or heparin. The cation exchanger is typically a strongly or weakly acidic side chain residue. The ion exchanger is washed in a gradient of salt and/or pH to elute specifically NS3 protease. Preferred eluting conditions are a salt gradient.

Such ion exchange chromatography can be repeated or varied until substantially pure NS3 protease is obtained. Typically two rounds of cation exchange chromatography are employed.

Preferred storage conditions involve having the enzyme in 25 mM HEPES (pH 7.5), 10% glycerol, 10 mM DTT and approximately 300 mM sodium chloride at a concentration of .about.15 uM or above at -80.degree. C.

EXAMPLE 1

Expression of the HCV NS3 Protease

Plasmid DNA encoding amino acids 1027-1206 of the BK strain HCV polypeptide was cloned downstream of the T7-7 vector, in frame with the first ATG of the protein of gene 10 of the T7 phage, to obtain the plasmid pT7-7 (NS3.sub.1027-1206), using methods that are known to the molecular biology practice. See PCT WO 95/22985, published Aug. 31, 1995, incorporated by reference. This plasmid was transfected into E. coli BL21DE3 plysS cells (Novagen) utilizing the heat-shock technique. Cells were grown at 37.degree. C. in LB medium containing 50 ug/ml ampicilin to an optical density of 0.4-0.6 at 600 nm whereupon the temperature was lowered to 25.degree. C. and expression of NS3 was induced with 400 uM IPTG. Cells were allowed to grow further for two hours and then harvested by centrifugation and stored at -80.degree. C. until lysis.

EXAMPLE 2

Purification of the HCV NS3 Protease in the Absence of Detergents

Cells from a 10-L culture were re-suspended in 100 ml of lysis buffer (25 mM sodium phosphate pH 7.5, 1 mM EDTA, 10% glycerol, 5 mM DTT) at 4.degree. C. and treated with 0.02 mg/ml DNase (Type IIS: Bovine Pancreas Sigma) in 20 mM MgCl.sub.2 for 30 min. PMSF (1 mM) was added to the suspension and cells were immediately disrupted by placing them 6 times through a microfluidizer at a pressure of 6 Bar. The lysate was centrifuged at 10,000 rpm for 30 min, and the supernatant was collected and loaded at onto a cation exchange column (Hi-Load SP Sepharose High Performance) pre-equilibrated in 50 mM sodium phosphate pH 6.5, 10% glycerol, 1 mM EDTA, 5 mM DTT, at a flow rate of 2.5 ml/min. The NS3 protease was eluted from the column in a 0-1 M NaCl salt gradient. Fractions were analyzed by SDS-PAGE. Fractions containing the NS3 protease were pooled and first diluted 8-10 fold into a buffer containing 25 mM sodium phosphate(pH 7.5), 10% glycerol, 5 mM DTT buffer and then loaded onto two 4.times.5 ml Heparin columns connected in tandem at a flow rate of 3 ml/min. The enzyme was eluted with a NaCl gradient. Fractions were analyzed by SDS-PAGE and peptide cleavage assay. Enzyme fractions containing >95% pure NS3 protease were pooled and stored at 4.degree. C. in the elution buffer. The yield was 1-2 mg of purified enzyme per liter of E. Coli cell culture. N-terminal sequence analyses were carried out using the Edman degradation method using an Applied Biosystem model 470A gas phase sequencer. The protease concentration was determined by. quantitative amino acid analysis.

EXAMPLE 3

HCV NS3 Substrate Cleavage Assay

The peptides (7-methoxycoumarin-4-acetyl-DEMEECASHLPYK-(.epsilon.-NHCOCH.sub.3) and acetyl-DEMEECASHLPYK-(.epsilon.-NHCOCH.sub.3) mimicking the NS4A/4B cleavage site was purchased from Enzyme Systems Products (Dublin, Calif.) and was >95% pure. A lysine was added to the C-terminus of the acetyl-DEMEECASHLPYK-(.epsilon.-NHCOCH.sub.3) peptide to enable it soluble at high concentrations and a coumarin fluorophore was introduced to the N-terminus of the (7-methoxycoumarin-4-acetyl-DEMEECASHLPYK-(.epsilon.-NHCOCH.sub.3) peptide to enhance detection of the product. The NS4B/5A substrate 7-methoxycoumarin-4-acetyl-EDASTPCSGS-Nph-L (where Nph=para-nitro phenylalanine) was purchased from Bachem Biosciences. The 4A peptide with the sequence of GSVVIVGRIILSGRKK was also synthesized by Enzyme System Products. Peptide cleavage assays were conducted at 25.degree. C. in 100 ul of 50 mM Hepes (pH 7.5) reaction buffer, 10 mM DTT in the presence of varying amounts of glycerol, preferrably 0% to 50% glycerol. The reaction was quenched with 100 ul of 5% phosphoric acid and the mixture was analyzed by reverse phase HPLC on a 4.6/50 mm Vydac C18 column. The cleavage products were separated using a 0.1 % phosphoric acid/acetonitrile gradient and identified by comparison of retention time with authentic peptides representing the reaction products. Cleavage of the NS4A/4B occurred at the expected Cys-Ala scissile bond. UV Absorbance of the products was monitored at 220 nM and fluorescence detection done with with excitation and emission wavelengths set at 328 nm and 393 nm, respectively. The enzyme concentrations used in the assays varied from, but not limiting to, 2 to 1000 nM depending on the reaction conditions desired. For example, the enzyme concentration in the presence of the 4A peptide varied from 2 to 50 nM and 300 to 1000 nM in the absence of the 4A peptide. In the assays with the 4A peptide as the cofactor, the enzyme was preincubated at a temperature of about 0.degree. C. to 10.degree. C. with the 4A peptide for 5 to 10 minutes, followed by 3 to 10 minutes at room temperature at a 10-50 fold greater concentration, before the onset of reaction. For preincubation of enzyme with the 4A peptide, the enzyme was added to the solution already containing the 4A peptide. The concentration of 4A peptide and substrate used ranged from, but not limiting to, 75 nM to 50 .mu.M and 0.1 .mu.M to 250 .mu.M, respectively. All substrates were dissolved in 50 mM HEPES (pH 7.5), 30 mM DTT and 10% glycerol. The reaction was typically allowed to continue for a period of 2.5 to 15 min depending on the initial reaction rate and the sensitive detection of products.

Steady state kinetic parameters (k.sub.cat and K.sub.M) were determined by fitting initial rates (obtained at <5% of total substrate hydrolyzed) verses substrate concentrations to the Michaelis-Menten equation. Initial velocity and steady-state conditions were strictly maintained for all reaction assays performed.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, modifications, deletions or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents.

__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 3(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 631 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vi) ORIGINAL SOURCE:(A) ORGANISM: Hepatitis C. Virus(B) STRAIN: NS3 Serine Protease Domain(C) INDIVIDUAL ISOLATE: BK(vii) IMMEDIATE SOURCE:(A) LIBRARY: described by Tomei et al. in 1993(B) CLONE: cDNA clone pCD (38- 9.4)(viii) POSITION IN GENOME:(B) MAP POSITION: 1-180(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AlaProIleThrAlaTyrSerGlnGlnThrArgGlyLeuLeuGlyCys151015IleIleThrSerLeuThrGlyArgAspLysAsnGlnValGluGlyGlu202530ValGlnValValSerThrAlaThrGlnSerPheLeuAlaThrCysVal354045AsnGlyValCysTrpThrValTyrHisGlyAlaGlySerLysThrLeu505560AlaGlyProLysGlyProIleThrGlnMetTyrThrAsnValAspGln65707580AspLeuValGlyTrpGlnAlaProProGlyAlaArgSerLeuThrPro859095CysThrCysGlySerSerAspLeuTyrLeuValThrArgHisAlaAsp100105110ValIleProValArgArgArgGlyAspSerArgGlySerLeuLeuSer115120125ProArgProValSerTyrLeuLysGlySerSerGlyGlyProLeuLeu130135140CysProSerGlyHisAlaValGlyIlePheArgAlaAlaValCysThr145150155160ArgGlyValAlaLysAlaValAspPheValProValGluSerMetGlu165170175ThrThrMetArgSerProValPheThrAspAsnSerSerProProAla180185190ValProGlnSerPheGlnValAlaHisLeuHisAlaProThrGlySer195200205GlyLysSerThrLysValProAlaAlaTyrAlaAlaGlnGlyTyrLys210215220ValLeuValLeuAsnProSerValAlaAlaThrLeuGlyPheGlyAla225230235240TyrMetSerLysAlaHisGlyIleAspProAsnIleArgThrGlyVal245250255ArgThrIleThrThrGlyAlaProValThrTyrSerThrTyrGlyLys260265270PheLeuAlaAspGlyGlyCysSerGlyGlyAlaTyrAspIleIleIle275280285CysAspGluCysHisSerThrAspSerThrThrIleLeuGlyIleGly290295300ThrValLeuAspGlnAlaGluThrAlaGlyAlaArgLeuValValLeu305310315320AlaThrAlaThrProProGlySerValThrValProHisProAsnIle325330335GluGluValAlaLeuSerAsnThrGlyGluIleProPheTyrGlyLys340345350AlaIleProIleGluAlaIleArgGlyGlyArgHisLeuIlePheCys355360365HisSerLysLysLysCysAspGluLeuAlaAlaLysLeuSerGlyLeu370375380GlyIleAsnAlaValAlaTyrTyrArgGlyLeuAspValSerValIle385390395400ProThrIleGlyAspValValValValAlaThrAspAlaLeuMetThr405410415GlyTyrThrGlyAspPheAspSerValIleAspCysAsnThrCysVal420425430ThrGlnThrValAspPheSerLeuAspProThrPheThrIleGluThr435440445ThrThrValProGlnAspAlaValSerArgSerGlnArgArgGlyArg450455460ThrGlyArgGlyArgArgGlyIleTyrArgPheValThrProGlyGlu465470475480ArgProSerGlyMetPheAspSerSerValLeuCysGluCysTyrAsp485490495AlaGlyCysAlaTrpTyrGluLeuThrProAlaGluThrSerValArg500505510LeuArgAlaTyrLeuAsnThrProGlyLeuProValCysGlnAspHis515520525LeuGluPheTrpGluSerValPheThrGlyLeuThrHisIleAspAla530535540HisPheLeuSerGlnThrLysGlnAlaGlyAspAsnPheProTyrLeu545550555560ValAlaTyrGlnAlaThrValCysAlaArgAlaGlnAlaProProPro565570575SerTrpAspGlnMetTrpLysCysLeuIleArgLeuLysProThrLeu580585590HisGlyProThrProLeuLeuTyrArgLeuGlyAlaValGlnAsnGlu595600605ValThrLeuThrHisProIleThrLysTyrIleMetAlaCysMetSer610615620AlaAspLeuGluValValThr625630(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(A) LIBRARY: cDNA clone (See Seq. ID No:1)(B) CLONE: NS4A Protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:SerThrTrpValLeuValGlyGlyValLeuAlaAlaLeuAlaAlaTyr151015CysLeuThrThrGlySerValValIleValGlyArgIleIleLeuSer202530GlyArgProAlaIleValProAspArgGluLeuLeuTyrGlnGluPhe354045AspGluMetGluGluCys50(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(v) FRAGMENT TYPE: internal(vii) IMMEDIATE SOURCE:(A) LIBRARY: Cofactor of NS3 serine protease(B) CLONE: Solid phase peptide synthesis(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GlySerValValIleValGlyArgIleIleLeuSerGlyArgProAla151015IleValProAspArgGluValLeuTyrGlnGluPheAspGluMetGlu202530GluAsx__________________________________________________________________________

Claims

1. Stable, detergent free, and substantially pure Hepatitis C virus NS3 protease.

2. A process for purifying active HCV NS3 protease without detergent, comprising the steps of:

(a) providing a quantity of cells expressing HCV NS3 protease;

(b) disrupting the cells to form a suspension in buffer without detergent;

(c) centrifuging the suspension to remove particulate matter;

(d) subjecting the supernatant of step (c) to one or more steps of ion exchange chromatography under eluting buffer conditions without detergent;

(e) to give active HCV NS3 protease in buffer without detergent.

3. A process for purifying active HCV NS3 protease without detergent, comprising the steps of:

(a) providing a quantity of cells expressing HCV NS3 protease;

(b) disrupting the cells with a microfluidizer to form a suspension in buffer without detergent, said buffer having pH of between about 6.5 and about 7.5;

(c) centrifuging the suspension to remove particulate matter, at between about 5000 and about 8000 r.p.m. for about 15 minutes;

(d) subjecting the supernatant of step (c) to one or more steps of cation exchange chromatography under eluting buffer conditions in a salt or pH gradient without detergent;

(e) to give active HCV NS3 protease in buffer without detergent.