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

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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.