AIM:To establish a simple and convenient assay in vitroforthe Hepatitis C virus NS3 serine protease based on therecombinant protease and substrate,and to evaluate itsfeasibility in screening the enzyme inhibitors.METHODS:Based on the crystallographic structure ofhepatitis C virus(HCV)serine protease,a novel single-chainserine protease was designed,in which the central sequenceof cofactor NS4A was linked to the N-terminus of NS3 serineprotease domain via a flexible linker GSGS.The fusion genewas obtained by two-step PCR that was carried out withthree primers and then cloned into the prokaryotic expressionvector pQE30,and the recombinant clone was verified byDNA sequencing.The single-chain recombinant protease wasexpressed when the E.coliwas induced with IPTG and theexpression conditions were optimized to produce largeamount of soluble protease.The recombinant substrateNS5ab that covers the cleavage point NS5A/B was alsoexpressed in E.coli.Both of the protease and substrate werepurified by using Ni-NTA agarose metal affinity resin,thenthey were mixed together in a specific buffer,and the mixturewas analyzed by SDS-PAGE.The cleavage system was usedto evaluate some compounds for their inhibitory activity onserine protease.RESULTS:The single-chain recombinant protease was over-expressed as soluble protein when the E.coliwas inducedat a low dosage of IPTG(0.2 mM)and cultured at a lowtemperature(15 ℃).The protease was purified by usingNi-NTA agarose metal affinity resin(the purity is over 95 %).The recombinant subs-b-ate NS5ab was expressed in an insolubleform and could refold successfully after purification and dialysis.A simple and convenient assay in vitro was established,inwhich the purified single-chain serine protease could cleavethe recombinant substrate NS5ab into two fragments that werevisualized by SDS-PAGE.PMSF had an effect on inhibiting activityof serine protease,while EDTA had not.CONCLUSION:A simple and convenient assay in vitro forhepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease.This assaycan be used in screening of enzyme inhibitors. AIM: To establish a simple and convenient assay in vitroforthe Hepatitis C virus NS3 serine protease based on therecombinant protease and substrate, and to evaluate its sensitivity in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protein domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with primers cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease wasexpressed when the E. coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. recombinant substrate NS5ab that covers the cleavage point NS5A / B was alsoexpressed in E.coli.Both of the protease and substrate werepurified by using Ni-NTA agarose metal affinity resin, thenthey were mixed together in a specific buffer, and the mixturewas analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity onserine protease .RESULTS: The single-chain recombinant protease was over-expressed as soluble protein when the E. coli was induced to a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 ° C.). The protease was purified by using the Ni-NTA agarose metal affinity resin 95%). The recombinant subs-b-ate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, inwhich the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not. CONCLUSION: A simple and convenient assay in vitro for hepatitis C virus NS3 se rine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assaycan be used in screening of enzyme inhibitors.