Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli

Abstract

The serine protease domain of HCV comprising amino acids 1027–1218 (ΔNS3) was expressed in E. coli with a His tag at its N-terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (∼3 mg/l of cultured cells). The ΔNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with k cat/ K m=160×10 −3 min −1 μM −1 in the presence of NS4A peptide. Our ΔNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The ΔNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203–2506 amino acids) that was synthesized by in vitro transcription and translation system.