title: Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli.
abstract: The serine proteinase of hepatitis C virus (HCV) non-structural 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, Ser1165 or His1083, 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.
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D016174:Hepacivirus / Q000201:enzymology*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D006639:Histidine; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D014107:Tosyllysine Chloromethyl Ketone; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase