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 x 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.
Vishnuvardhan, D., Kakiuchi, N., Urvil, P. T., Shimotohno, K., Kumar, P. K. R., & Nishikawa, S. (1997). Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli. FEBS Letters, 402(2–3), 209–212. https://doi.org/10.1016/S0014-5793(96)01532-3