PROTEOLYTIC ACTIVITY OF NS3 SERINE PROTEINASE OF HEPATITIS-C VIRUS EFFICIENTLY EXPRESSED IN ESCHERICHIA-COLI

The serine proteinase of hepatitis C virus (HCV) nonstructural protein NS3 was efficiently expressed in an active form as a fused protein wi th oligohistidine in Escherichia coil. The recombinant fusion protein was purified to near homogeneity by affinity chromatography on a metal chelation column, Trans-cleavage activity of this protein was investi gated by using the substrate NS5 protein expressed in insect cells. Th e 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 activi ty. Analysis of the authentic enzyme and variants with site-directed m utations provides a useful tool for understanding the structure-functi on 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 coil, and examined the effect of known inh ibitors of serine proteinase. Inhibition of its proteolytic activity b y N-p-tosyl-L-lysine chloromethyl ketone (TLCK) was observed, but only at high concentrations, The in vitro and in vivo trans-cleavage assay s for NS3 serine proteinase will facilitate efficient testing for inhi bitors of the replication of HCV and specific treatment for hepatitis C.