IN-VITRO CLEAVAGE OF HEPATITIS-C VIRUS POLYPROTEIN SUBSTRATES BY PURIFIED RECOMBINANT NS3 PROTEASE

被引：29

作者：

DSOUZA, EDA ^{[1
]}

GRACE, K ^{[1
]}

SANGAR, DV ^{[1
]}

ROWLANDS, DJ ^{[1
]}

CLARKE, BE ^{[1
]}

机构：

[1] WELLCOME RES LABS,DIV BIOL,BECKENHAM BR3 3BS,KENT,ENGLAND

来源：

JOURNAL OF GENERAL VIROLOGY | 1995年 / 76卷

关键词：

D O I：

10.1099/0022-1317-76-7-1729

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using the assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

引用

页码：1729 / 1736

页数：8

共 17 条

[1] NONSTRUCTURAL PROTEIN-3 OF THE HEPATITIS-C VIRUS ENCODES A SERINE-TYPE PROTEINASE REQUIRED FOR CLEAVAGE AT THE NS3/4 AND NS4/5 JUNCTIONS [J].

BARTENSCHLAGER, R ;

AHLBORNLAAKE, L ;

MOUS, J ;

JACOBSEN, H .

JOURNAL OF VIROLOGY, 1993, 67 (07) :3835-3844

[2] KINETIC AND STRUCTURAL-ANALYSES OF HEPATITIS-C VIRUS POLYPROTEIN PROCESSING [J].

BARTENSCHLAGER, R ;

AHLBORNLAAKE, L ;

MOUS, J ;

JACOBSEN, H .

JOURNAL OF VIROLOGY, 1994, 68 (08) :5045-5055

[3] ISOLATION OF A CDNA CLONE DERIVED FROM A BLOOD-BORNE NON-A, NON-B VIRAL-HEPATITIS GENOME [J].

CHOO, QL ;

KUO, G ;

WEINER, AJ ;

OVERBY, LR ;

BRADLEY, DW ;

HOUGHTON, M .

SCIENCE, 1989, 244 (4902) :359-362

[4] GENETIC ORGANIZATION AND DIVERSITY OF THE HEPATITIS-C VIRUS [J].

CHOO, QL ;

RICHMAN, KH ;

HAN, JH ;

BERGER, K ;

LEE, C ;

DONG, C ;

GALLEGOS, C ;

COIT, D ;

MEDINASELBY, A ;

BARR, PJ ;

WEINER, AJ ;

BRADLEY, DW ;

KUO, G ;

HOUGHTON, M .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (06) :2451-2455

[5] ANALYSIS OF NS3-MEDIATED PROCESSING OF THE HEPATITIS-C VIRUS NONSTRUCTURAL REGION IN-VITRO [J].

DSOUZA, EDA ;

OSULLIVAN, E ;

AMPHLETT, EM ;

ROWLANDS, DJ ;

SANGAR, DV ;

CLARKE, BE .

JOURNAL OF GENERAL VIROLOGY, 1994, 75 :3469-3476

[6] THE HEPATITIS-C VIRUS ENCODES A SERINE PROTEASE INVOLVED IN PROCESSING OF THE PUTATIVE NONSTRUCTURAL PROTEINS FROM THE VIRAL POLYPROTEIN PRECURSOR [J].

ECKART, MR ;

SELBY, M ;

MASIARZ, F ;

LEE, C ;

BERGER, K ;

CRAWFORD, K ;

KUO, C ;

KUO, G ;

HOUGHTON, M ;

CHOO, QL .

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1993, 192 (02) :399-406

[7] BOTH NS3 AND NS4A ARE REQUIRED FOR PROTEOLYTIC PROCESSING OF HEPATITIS-C VIRUS NONSTRUCTURAL PROTEINS [J].

FAILLA, C ;

TOMEI, L ;

DEFRANCESCO, R .

JOURNAL OF VIROLOGY, 1994, 68 (06) :3753-3760

[8] CHARACTERIZATION OF THE HEPATITIS-C VIRUS-ENCODED SERINE PROTEINASE - DETERMINATION OF PROTEINASE-DEPENDENT POLYPROTEIN CLEAVAGE SITES [J].

GRAKOUI, A ;

MCCOURT, DW ;

WYCHOWSKI, C ;

FEINSTONE, SM ;

RICE, CM .

JOURNAL OF VIROLOGY, 1993, 67 (05) :2832-2843

[9] EXPRESSION AND IDENTIFICATION OF HEPATITIS C VIRUS POLYPROTEIN CLEAVAGE PRODUCTS [J].

GRAKOUI, A ;

WYCHOWSKI, C ;

LIN, C ;

FEINSTONE, SM ;

RICE, CM .

JOURNAL OF VIROLOGY, 1993, 67 (03) :1385-1395

[10] 2 DISTINCT PROTEINASE ACTIVITIES REQUIRED FOR THE PROCESSING OF A PUTATIVE NONSTRUCTURAL PRECURSOR PROTEIN OF HEPATITIS-C VIRUS [J].

HIJIKATA, M ;

MIZUSHIMA, H ;

AKAGI, T ;

MORI, S ;

KAKIUCHI, N ;

KATO, N ;

TANAKA, T ;

KIMURA, K ;

SHIMOTOHNO, K .

JOURNAL OF VIROLOGY, 1993, 67 (08) :4665-4675

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