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- Title
Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions.
- Authors
Uchiyama, Yasutoshi; Huang, Ying; Kanamori, Hiroshi; Uchida, Masao; Doi, Takefumi; Takamizawa, Akihisa; Hamakubo, Takao; Kodama, Tatsuhiko
- Abstract
The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer. The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 °C and a pH of 7.5. The addition of 2.5 mM Mn2+ increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg2+. At higher concentrations of Mn2+, GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted. This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp.
- Subjects
BIOLOGICAL assay; THERAPEUTIC use of proteins; HEPATITIS C virus
- Publication
Hepatology Research, 2002, Vol 23, Issue 2, p90
- ISSN
1386-6346
- Publication type
Article
- DOI
10.1016/S1386-6346(01)00167-X