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

FEBS Letters

Shimotohno, Kunitada¹  Kakiuchi, Nobuko¹  Vishnuvardhan, Daesety²  Kumar, P.K.R²  Urvil, Petri T²  Nishikawa, Satoshi² 

[1] Virology Division, National Cancer Center Research Institute, Tsukiji, Tokyo 104, Japan;National Institute of Bioscience and Human Technology, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan

关键词: Hepatitis C virus;    Serine protease;    Non-structural protein 3;   

DOI  :  10.1016/S0014-5793(96)01532-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown