Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease.
OBJECTIVE
To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility 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 protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease.


RESULTS
The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which 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.


CONCLUSIONS
A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.
Du
Gui-Xin
GX
Department of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Fengtai, Beijing 100071, China. dugx@hotmail.com
Hou
Li-Hua
LH
Guan
Rong-Bin
RB
Tong
Yi-Gang
YG
Wang
Hai-Tao
HT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004279:DNA, Viral; D004492:Edetic Acid; D010664:Phenylmethylsulfonyl Fluoride; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases