Construction, expression, and characterization of a novel fully activated recombinant single‐chain hepatitis C virus protease

• Published in: Protein science 1998-10, Vol.7 (10), p.2143-2149
• Main Authors: Taremi, S. Shane, Beyer, Brian, Maher, Maureen, Yao, Nanhua, Prosise, Winifred, Weber, Patricia C., Malcolm, Bruce A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Enzyme Activation - drug effects; Escherichia coli - genetics; Glucosides - pharmacology; Glycerol - pharmacology; Hepacivirus - enzymology; hepatitis C virus; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Molecular Sequence Data; NS3; NS4A; Osmolar Concentration; overexpression; Peptides - metabolism; protease; recombinant; Recombinant Fusion Proteins - chemistry; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Viral Proteins - chemistry
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560071011

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 3ndash181) 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 kcat of 20.0 ± 2.0 μM and 9.6 ± 2.0 min−1, respectively; parameters identical to those of the authentic NS31_631/NS4A1_54 protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392‐3401).