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Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in escherichia coli
Author(s) -
Shoji Ikuo,
Suzuki Tetsuro,
Chieda Shinya,
Sato Mitsuru,
Harada Takashi,
Chiba Tsutomu,
Matsuura Yoshiharu,
Miyamura Tatsuo
Publication year - 1995
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840220606
Subject(s) - ns3 , serine , proteinase 3 , biochemistry , affinity chromatography , serine proteinase inhibitors , recombinant dna , ns2 3 protease , serine protease , microbiology and biotechnology , enzyme , biology , chemistry , protease , antibody , gene , immunology , autoantibody
The serine proteinase of hepatitis C virus (HCV) nonstructural protein NS3 was efficiently expressed in an active form as a fused protein with oligohistidine in Escherichia coli . The recombinant fusion protein was purified to near homogeneity by affinity chromatography on a metal chelation column. Trans ‐cleavage activity of this protein was investigated by using the substrate NS5 protein expressed in insect cells. The purified serine proteinase trans ‐cleaved the partially purified NS5 protein. In contrast, the NS3 proteins with mutations at the proposed catalytic site, Ser 1165 or His 1083 , lost the trans ‐cleavage activity. Analysis of the authentic enzyme and variants with site‐directed mutations provides a useful tool for understanding the structure‐function relationship of the NS3 serine proteinase. We then developed an in vivo trans ‐cleavage assay system by coexpression of the NS3 proteinase and the NS5 substrate in E coli , and examined the effect of known inhibitors of serine proteinase. Inhibition of its proteolytic activity by N‐p ‐tosyl‐ L ‐lysine chloromethyl ketone (TLCK) was observed, but only at high concentrations. The in vitro and in vivo trans ‐cleavage assays for NS3 serine proteinase will facilitate efficient testing for inhibitors of the replication of HCV and specific treatment for hepatitis C. (Hepatology 1995; 22:1648‐1655).