CA2265512A1 - Detergent-free hepatitis c protease - Google Patents

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

?CA 02265512 1999-03-15WO 98113482 PCTIUS97/17029-1-TITLE OF THE INVENTIONDETERGENT-FREE HEPATITIS C PROTEASEBACKGROUND OF THE INVENTIONHepatitis C virus (HCV) infection is found in 0.5% to 8.0%of blood donors worldwide. Because the infection is chronic in morethan 60% of infected persons, the disease is an important public healthand economic problem. The management of patients with chronichepatitis C is complex——the disease is often only mildly symptomatic andslowly progressive, but 20% of patients develop cirrhosis after 20 yearsof infection and perhaps 10%. of those with cirrhosis develophepatocellular carcinoma. It is also an important indication for livertransplantation. In Europe and Japan the disease is more importantnumerically than is either hepatitis B or HIV infection. Existingantiviral agents are effective in only a minority of patients, yet goodresponses can be obtained.An important target for the treatment of HCV isnonstructural protein 3, a protease encoded by HCV. This NS3 proteaseassociated with human hepatitis C virus is an unstable protein in theabsence of high concentrations of detergent. To stabilize the NS3protease to sufficient quantities for biochemical, kinetic. and biophysicalanalyses, as well as for the construction of antiviral screening assays,ionic or non—ionic detergents need be incorporated both duringpurification and analyses. Antiviral leads discovered with detergenttreated NS3 protein are not useful. Further, the presence of highquantities of detergents renders significant difficulties in the preciseinterpretations of biochemical, kinetic, and biophysical analyses. Insome cases (e.g., sedimentation, protein crystallization), the presence ofdetergents preclude biochemical, kinetic, and biophysical analyses.Prior methods employed detergents and glycerol forpurification of NS3. Applicants have discovered a method of purifyingNS3 without detergent, with from 5% to about 20% glycerol,preferably 7-12% and with high stability and activity. The resultantenzyme displays a higher catalytic activity than what is known for this?CA 02265512 1999-03-15W0 98l13482 PCT/US97/17029-2-protease, that is, 10-500 fold more active than the prior artpreparations, depending on the form of the enzyme. Purificationaccording to the methods of the present invention ensures a highstability 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 forenzymologic, biochemical, and biophysical studies.When properly expressed and prepared from the clonedplasmid in E. coli, the NS3 protease is obtained in milligram quantitiesin the complete absence of detergent. The resultant enzyme is verysoluble and stable for long periods of time (weeks to months at 4°C and>12 months at —80°C), and displays high catalytic activity.An assay with the detergent free HCV NS3 protease isuseful as a screening tool for HCV antivirals as well as a diagnostic toolfor diseases resulting from HCV infection. The potency range of theHCV antivirals can range from subnanomolar to micromolarconcentrations.BRIEF DESCRIPTION OF THE INVENTIONDetergent free NS3 protease of Hepatitis C virus (HCV) isprepared, and a screening assay for the protein inhibitors is constructed.The detergent free NS3 protease is useful as a screening tool for HCVantivirals, as well as a diagnostic tool for diseases resulting from HCVinfection.DETAIIJED DESCRIPTION OF THE INVENTIONABBREVIATIONS AND DEFINITIONSHCV Hepatitis C VirusIPTG lsopropyl—D(—)thiogalactopyranosideNS3 Nonstructural protein 3 of Hepatitis C virus?CA 02265512 1999-03-15W0 98/ 13482 i PCT/US97/ 17029-3-PMSF Phenylmethylsulfonyl ?uorideEDTA ethylendiammino-tetraacetic acidDTT dithiothreitolr.p.m. Revolutions per minutePAGE Polyacrylamide gelSDS Sodium dodecylsulfateNS non—structuralHPLC High Performance Liquid ChromatographyIn one aspect of the invention a stable, detergent free HCV CNS3 protease is claimed.In another aspect of the invention a screening assay for the detection ofcompounds that inhibit HCV NS3 protease is claimed.In still another aspect of the invention, the compounds thatinhibit HCV NS3 protease as measured by the screening assay of Claim2 are claimed.In yet another aspect of the invention, a process forpurifying active HCV NS3 protease without detergent is claimed.There is disclosed stable, detergent free nonstructuralprotein 3 of Hepatitis C virus , also known as NS3 protease of HCV orNS3. The NS3 protease is useful screening tool for HCV antivirals, aswell as a diagnostic tool for diseases resulting from HCV infection.One utility for HCV NS3 protease is a screening assay forthe detection of compounds that inhibit HCV NS3 protease. This assayhas a procedure comprising the steps of:?CA 02265512 1999-03-15W0 93/13482 i PCT/US97/17029-4-(a) providing a quantity of a compound or compounds tobe assayed;(b) incubating said compound or compounds withdetergent free HCV NS3 protease in an HCV NS3protease assay;(0) determining the inhibition of said protease in theHCV NS3 protease substrate cleavage assay.Also encompassed in the present invention are compoundsthat substantially inhibit the HCV N S3 protease.This invention also relates to a process for purifying activeHCV NS3 protease without detergent and with from 5% to about 20%glycerol, comprising the steps of:(a) providing a quantity of cells expressing HCV NS3protease;(b) disrupting the cells to form a suspension in bufferwithout detergent;(c) centrifuging the suspension to remove particulatematter;((1) subjecting the supernatant of step (c) to one or moresteps of ion exchange chromatography under elutingbuffer conditions without detergent;(e) to give active HCV NS3 protease in buffer withoutdetergent.One embodiment of the process for purifying active HCVNS3 protease without detergent and with from 7% to about 12%glycerol, comprises the steps of:(a) providing a quantity of cells expressing HCV NS3protease;?CA 02265512 1999-03-15W0 98/ 13482 PCT/U S97/ 17029-5-(b) disrupting the cells with a microfluidizer to form asuspension in buffer without detergent, said bufferhaving pH of between about 6.5 and about 7.5;(c) centrifuging the suspension to remove particulatematter, at between about 5000 and about 8000 r.p.m.for about 15 minutes;(d) subjecting the supernatant of step (c) to one or moresteps of cation exchange chromatography undereluting buffer conditions in a salt or pH gradientwithout detergent;(e) to give active HCV NS3 protease in buffer withoutdetergent.The nonstructural protein 3 of Hepatitis C virus, alsoknown as NS3 protease, can exist in active form as an enzyme or as acomplex with the cofactor . It has been discovered by applicants that thecomplex is about 1000 times more active than the enzyme by itself. Theenzyme is itself about 10 times more active than prior art preparationspurified with detergent. In the screening assays of the present invention,all active forms are encompassed.Expression of HCV NS3 Protease in a Recombinant Eimression SystemIt is now a relatively straightforward technology to preparecells expressing a foreign gene. Such cells act as hosts and includeE. coli, B. subtilis, yeasts, fungi, plant cells or animal cells. Expressionvectors for many of these host cells have been isolated andcharacterized, and are used as starting materials in the construction,through conventional recombinant DNA techniques, of vectors having aforeign DNA insert of interest. Any DNA is foreign if it does notnaturally derive from the host cells used to express the DNA insert.The foreign DNA insert may be expressed on extrachromosomalplasmids or after integration in whole or in part in the host cellchromosome(s), or may actually exist in the host cell as a combinationof more than one molecular form. The choice of host cell and?CA 02265512 1999-03-15W0 98/ 13482 PCT/US97/ 17029-5-expression vector for the expression of a desired foreign DNA largelydepends on availability of the host cell and how fastidious it is, whetherthe host cell will support the replication of the expression vector, andother factors readily appreciated by those of ordinary skill in the art.The technology for recombinant procaryotic expressionsystems is now old and conventional. The typical host cell is E. coli.The technology is illustrated by treatises such as Wu, R (ed) Meth.Enzym0l., 68 (1979) and Maniatis, T. et al., Molecular Cloning.’ ALaboratory Manual Cold Spring Harbor 1982.The foreign DNA insert of interest comprises a DNAsequence coding for HCV NS3 protease (or stable functional mutantthereof) of the present invention, including any synthetic sequence withthis coding capacity or any such cloned sequence or combinationthereof. For example, HCV peptides coded and expressed by an entirelyrecombinant DNA sequence is encompassed by this invention.Vectors useful for constructing eukaryotic expressionsystems for the production of recombinant HCV comprise the DNAsequence for HCV or variant thereof, operatively linked thereto withappropriate transcriptional activation DNA sequences, such as apromoter and/or operator. Other typical features may includeappropriate ribosome binding sites, termination codons, enhancers,tenninators, or replicon elements. These additional features can beinserted into the vector at the appropriate site or sites by conventionalsplicing techniques such as restriction endonuclease digestion andligation.Yeast expression systems, which are one variety ofrecombinant eukaryotic expression systems, generally employSaccharomyces cerevisiae as the species of choice for expressingrecombinant proteins. 5. cerevisiac and similar yeasts possess wellknown promoters useful in the construction of yeast expression systems,including but not limited to G_/32491, Q_PLL_. 10, _A_l?l_2, and alpha matingfactor.Yeast vectors useful for constructing recombinant yeastexpression systems for expressing HCMV include, but are not limited to,?CA 02265512 1999-03-15W0 98/13482 PCT/US97l17029-7-shuttle vectors, cosmids, chimeric plasmids, and those having sequencesderived from 2-micron circle plasmids.Insertion of the appropriate DNA sequence coding forHCV, into these vectors will, in principle, result in a useful recombinantyeast expression system for HCV where the modified vector is insertedinto the appropriate host cell, by transformation or other means.One preferred expression system is with baculovirus, underthe control of the polyhedrin promoter or the p10 promoter. See, e.g.,D.R. O'Reilly et al., Baculovirus Expression Vectors: A LaboratoryManual W.H. Freeman 1992, for a background description of thisexpression technology. This system employs the isolation of arecombinant baculovirus carrying the gene of interest. The baculovirussystem is especially useful for the simultaneous expression of more thanone protein.Recombinant mammalian expression systems are anothermeans of producing the recombinant HCV for the conjugates of thisinvention. In general, a host mammalian cell can be any cell that hasbeen efficiently cloned in cell culture. Host mammalian cells useful forthe purposes of constructing a recombinant mammalian expressionsystem include, but are not limited to, Vero cells, NIH3T3, GH3, COS,murine C127 or mouse L cells. Mammalian expression vectors can bebased on virus vectors, plasmid vectors which may have SV40, BPV orother viral replicons, or vectors without a replicon for animal cells.Detailed discussions on mammalian expression vectors can be found inthe treatises of Glover, D.M. (ed.) "DNA Cloning: A PracticalApproach," IRL 1985, Vols. I and II.Recombinant HCV may possess additional and desirablestructural modifications not shared with the same organicallysynthesized peptide, such as adenylation, carboxylation, glycosylation,hydroxylation, methylation, phosphorylation, myristoylation, extensionor trimming of either the amino— or carboxy—terminal ends or both.These added features may be chosen or preferred as the case may be, bythe appropriate choice of recombinant expression system. On the other?CA 02265512 1999-03-15WO 98/13482 PCT/US97/17029-3-hand, recombinant HCV may have its sequence extended by theprinciples and practice of organic synthesis.PurificationIn the detergent free purification process of the presentinvention, virtually any source of NS3 protease is suitable, whetherrecombinant or not. Preferred sources are recombinant, most preferredis an expression system using E.c0li.For expression systems, whether or not recombinant, theN S3 must first be isolated in a soluble fraction. Cells expressing HCVprotease are disrupted in buffer to form a suspension in buffer. Thedisruption is carried out by any of a variety of well known techniques,including but not limited to treatment with a French press, amicrofluidizer, sonicator, or self-digestion by inductive expression oflyzozyme. A preferred technique of disruption is with a microfluidizer.Throughout the purification process without detergent, it iscritical to maintain the pH of any buffer between about 6.5 and about7.5.The next step involves initial fractionation of the suspensionof cellular debris. The suspension is treated to separate the solublefraction from particulate matter, or such other step is performed thatsubstantially separates soluble from insoluble protein. Appropriatetechniques include, but are not limited to, centrifugation at betweenabout 5,000 r.p.m. and about 8,000 r.p.m. for about 15 minutes,filtration, or salt precipitation with e.g. (NH4)2SO4. It is understoodthat these initial fractionation procedures are well known and are subjectto many variations. Appropriate modifications in the initialfractionation of NS3 are well within the skill of the art. The preferredmethod is centrifugation.lnitial fractionation of the suspension of cellular debrisresults in a supernatant as well as a precipitate or insoluble pellet. Thesupernatant is treated further.?CA 02265512 1999-03-15WO 98/13482 . PCT/US97/17029-9-The supernatant is then subjected to one or more steps ofion exchange chromatography, and, optionally, gel filtration, to give asubstantially pure NS3 in buffer without detergent. Preferred ionexchangers include, but are not limited to, cation exchangers onpolystyrene, cation exchangers on dextran, cation exchangers onagarose, cation exchangers on cellulose, or heparin. The cationexchanger is typically a strongly or weakly acidic side chain residue.The ion exchanger is washed in a gradient of salt and/or pH to elutespecifically NS3 protease. Preferred eluting conditions are a saltgradient.Such ion exchange chromatography can be repeated orvaried until substantially pure NS3 protease is obtained. Typically tworounds of cation exchange chromatography are employed.Preferred storage conditions involve having the enzyme in25mM HEPES (pH 7.5), 10% glycerol, l0mM DTT and approximately300mM sodium chloride at a concentration of ~15uM or above at -80°C.EXAMPLE 1Expression of the HCV NS3 ProteasePlasmid DNA encoding amino acids 1027-1206 of the BKstrain HCV polypeptide was cloned downstream of the T7-7 vector, inframe with the first ATG of the protein of gene 10 of the T7 phage, toobtain the plasmid pT7-7 (NS31027-1206), using methods that areknown to the molecular biology practice. See PCT WO 95/22985,published August 31, 1995, incorporated by reference. This plasmidwas transfected into E. coli BL21DE3 plysS cells (Novagen) utilizingthe heat-shock technique. Cells were grown at 37°C in LB mediumcontaining 50 ug/ml ampicilin to an optical density of 0.4-0.6 at 600 nmwhereupon the temperature was lowered to 25°C and expression of NS3was induced with 400 uM IPTG. Cells were allowed to grow furtherfor two hours and then harvested by centrifugation and stored at —80°Cuntil lysis.?CA 02265512 1999-03-15WO 98/13482 PCT/U S97/ 17029_ 10 _EXAMPLE 2Purification of the HCV NS3 Protease in the Absence of DetergentsCells from a 10-L culture were re—suspended in 100 ml oflysis buffer (25 mM sodium phosphate pH 7.5, 1 mM EDTA, 10%glycerol, 5 mM DTT) at 4°C and treated with 0.02 mg/ml DNase (TypeIIS: Bovine Pancreas Sigma) in 20 mM MgCl2 for 30min. PMSF (1mM) was added to the suspension and cells were immediately disruptedby 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 thesupernatant was collected and loaded at onto a cation exchange column(Hi-Load SP Sepharose High Performance) pre—equilibrated in 50 mMsodium phosphate pH 6.5, 10% glycerol, 1 mM EDTA, 5 mM DTT, at aflow rate of 2.5 ml/min. The NS3 protease was eluted from the columnin a 0-1 M NaCl salt gradient. Fractions were analyzed by SDS—PAGE.Fractions containing the NS3 protease were pooled and first diluted 8-10fold into a buffer containing 25 mM sodium phosphate( pH 7.5), 10%glycerol, 5 mM DTT buffer and then loaded onto two 4x5 ml Heparincolumns connected in tandem at a flow rate of 3 ml/min. The enzymewas eluted with a N aCl gradient. Fractions were analyzed by SDS-PAGE and peptide cleavage assay. Enzyme fractions containing > 95%pure NS3 protease were pooled and stored at 4°C in the elution buffer.The yield was 1-2 mg of purified enzyme per liter of E. Cali cellculture. N-terminal sequence analyses were carried out using theEdman degradation method using an Applied Biosystem model 470A gasphase sequencer. The protease concentration was determined byquantitative amino acid analysis.?CA 02265512 1999-03-15W0 98/ 13482 PCT/US97/ 17029- 11 _EXAMPLE 3HCV NS3 Substrate Cleavaae AssavThe peptides (7—methoxycoumarin-4-acetyl-DEMEECASHLPYK—(e-NHCOCH3) and acetyl—DEMEECASHLPYK-(8-NHCOCH3) mimicking the NS4A/4B cleavage site was purchasedfrom Enzyme Systems Products (Dublin, CA) and was > 95% pure. Alysine was added to the C-terrninus of the acetyl—DEMEECASHLPYK-(8-NHCOCH3) peptide to enable it soluble at high concentrations and acoumarin ?uorophore was introduced to the N-terrninus of the (7-methoxycoumarin—4-acetyl-DEMEECASHLPYK—(£-NHCOCH3) peptideto enhance detection of the product. The NS4B/5A substrate 7-methoxycoumarin—4—acety1—EDASTPCSGS—Nph-L (where Nph = para-nitro phenylalanine) was purchased from Bachem Biosciences. The 4Apeptide with the sequence of GSVVIVGRHLSGRKK was alsosynthesized by Enzyme System Products. Peptide cleavage assays wereconducted at 25°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 100111 of 5% phosphoric acid and the mixture was analyzed by reverse phaseHPLC on a 4.6/50 mm Vydac C18 column. The cleavage products wereseparated using a 0.1% phosphoric acid/acetonitrile gradient andidentified by comparison of retention time with authentic peptidesrepresenting the reaction products. Cleavage of the NS4A/4B occurredat the expected Cys-Ala scissile bond. UV Absorbance of the productswas monitored at 220 nM and fluorescence detection done with withexcitation 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 conditionsdesired. For example, the enzyme concentration in the presence of the4A peptide varied from 2 to 50 nM and 300 to 1000 nM in the absenceof the 4A peptide. In the assays with the 4A peptide as the cofactor, theenzyme was preincubated at a temperature of about 0°C to 10°C with the4A peptide for 5 to 10 minutes, followed by 3 to 10 minutes at room?CA 02265512 1999-03-15W0 98/ 13482 PCT/US97/17029-12-temperature at a 10-50 fold greater concentration, before the onset ofreaction. For preincubation of enzyme with the 4A peptide, the enzymewas added to the solution already containing the 4A peptide. Theconcentration of 4A peptide and substrate used ranged from, but notlimiting to, 75 nM to 50uM and 0.luM to 250uM, respectively. Allsubstrates were dissolved in 50 mM HEPES (pH 7.5), 30 mM DTT and10% glycerol. The reaction was typically allowed to continue for aperiod of 2.5 to 15 min depending on the initial reaction rate and thesensitive detection of products.Steady state kinetic parameters (kcat and KM) weredetermined by fitting initial rates (obtained at <5 % of total substratehydrolyzed) verses substrate concentrations to the Michaelis-Mentenequation. Initial velocity and steady—state conditions were strictlymaintained for all reaction assays performed.While the foregoing specification teaches the principles ofthe present invention, with examples provided for the purpose ofillustration, it will be understood that the practice of the inventionencompasses all of the usual variations, adaptations, modifications,deletions or additions of procedures and protocols described herein, ascome within the scope of the following claims and its equivalents.?CA 02265512 1999-03-15WO 98/13482 PCT/US97/17029- 13 _SEQUENCE LISTING(1) GENERAL INFORMATION:(i) APPLICANT: Sardana, Vinod VBlue, Jeffrey T(ii) TITLE OF INVENTION: DETERGENT—FREE HEPATITIS C PROTEASE(iii) NUMBER OF SEQUENCES: 3(iv) CORRESPONDENCE ADDRESS:(A) ADDRESSEE: MERCK & CO., INC.(B) STREET: P.O. Box 2000, l26 E. Lincoln Ave.(C) CITY: Rahway(D) STATE: NJ(E) COUNTRY: US(F) ZIP: 07065-0907(V) COMPUTER READABLE FORM:) MEDIUM TYPE: Floppy disk( ) COMPUTER: IBM PC compatible( ) OPERATING SYSTEM: PC—DOS/MS—DOS( ) SOFTWARE: Patentln Release #l.0, Version #l.30(vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE:(C) CLASSIFICATION:(viii) ATTORNEY/AGENT INFORMATION:(A) NAME: Ayler, Sylvia A(B) REGISTRATION NUMBER: 36,436(C) REFERENCE/DOCKET NUMBER: l969lPV(ix) TELECOMMUNICATION INFORMATION:(A) TELEPHONE: 908-594-4909(B) TELEFAX: 908-594-4720(2) INFORMATION FOR SEQ ID NO:l:(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?CA 02265512 1999-03-15W0 98/ 13482 PCT/US97/17029_ [4 _(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:l:Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys1 5 10 15Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly Glu20 25 30Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val35 40 45Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu50 55 60Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln65 70 75 80Asp Leu Val Gly Trp Gln Ala Pro Pro Gly Ala Arg Ser Leu Thr Pro85 90 95Cys Thr Cys Gly Sex Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp100 105 110Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser115 120 125Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu130 135 140Cys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys Thr145 150 155 160Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser Met Glu165 170 175Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Ala180 185 190Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser195 200 205?CA 02265512 1999-03-15W0 98/ 13482 9 PCT/US97/ 17029Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys210 215 220Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala225 230 235 240Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val245 250 255Arg Thr Ile Thr Thr Gly Ala Pro Val Thr Tyr Ser Thr Tyr Gly Lys260 265 270Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile275 280 - 285Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly290 295 300Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu305 310 315 320Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile325 330 335Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys340 345 350Ala Ile Pro Ile Glu Ala Ile Arg Gly Gly Arg His Leu Ile Phe Cys355 360 365His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Leu370 375 380Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile385 390 395 400Pro Thr Ile Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr405 410 415Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val420 425 430Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr435 440 445Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg450 455 460Thr Gly Arg Gly Arg Arg Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu465 470 475 480Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp485 490 495Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg500 505 510 ?CA 02265512 1999-03-15WO 98/13482 PCT/US97/17029-16-Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His515 S20 S25Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala530 535 540His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu545 550 555 560Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro565 S70 S75Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu580 585 590His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu595 600 605Val Thr Leu Thr His Pro Ile Thr Lys Tyr Ile Met Ala Cys Met Ser610 615 620Ala Asp Leu Glu Val Val Thr625 630(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) IMEDIATE SOURCE:(A) LIBRARY: CDNA clone (See Seq. ID No:l)(B) CLONE: NS4A Protein(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyrl 5 10 15Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser20 25 30?CA 02265512 1999-03-15W0 98/13482 PCT/US97/ 17029-17-Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe35 40 45Asp Glu Met Glu Glu Cys50(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 N33 serine protease(B) CLONE: Solid phase peptide synthesis(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Pro Ala1 5 10 15Ile Val Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe Asp Glu Met Glu20 25 30Glu Asx

Claims (5)

WHAT IS CLAIMED IS:

1. Stable, detergent free and substantially pure Hepatitis C virus NS 3 protease.

2. A screening assay for the detection of compounds that inhibit HCV NS3 protease, said assay having 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 the HCV NS3 protease of Claim 1 in an HCV NS3 protease assay;
(c) determining the inhibition of said protease in the HCV NS3 protease assay.

3. The compounds that inhibit HCV NS3 protease as measured by the screening assay of Claim 2.

4. 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.

5. 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.