title: Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.
abstract: Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).
authors.affiliation: Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Taremi
authors.forename: B
authors.initials: B
authors.lastname: Beyer
authors.forename: M
authors.initials: M
authors.lastname: Maher
authors.forename: N
authors.initials: N
authors.lastname: Yao
authors.forename: W
authors.initials: W
authors.lastname: Prosise
authors.forename: P C
authors.initials: PC
authors.lastname: Weber
authors.forename: B A
authors.initials: BA
authors.lastname: Malcolm
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005960:Glucosides / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D005960:Glucosides; D005990:Glycerol; D010455:Peptides; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C018619:octyl-beta-D-glucoside