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Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli
Author(s) -
Vishnuvardhan Daesety,
Kakiuchi Nobuko,
Urvil Petri T,
Shimotohno Kunitada,
Kumar P.K.R,
Nishikawa Satoshi
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(96)01532-3
Subject(s) - ns3 , protease , serine protease , recombinant dna , amino acid , ns5a , microbiology and biotechnology , peptide , chemistry , ns2 3 protease , biochemistry , biology , hepatitis c virus , enzyme , virology , virus , gene , hepacivirus
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.