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J. Biochem, 2004, Vol. 135, No. 2 245-252
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Expression and Purification of a Hepatitis C Virus NS3/4A Complex, and Characterization of Its Helicase Activity with the Scintillation Proximity Assay System

Kiyoshi Kyono*,1, Masahiko Miyashiro2 and Ikuhiko Taguchi2

1 Medicinal Chemistry Research Laboratories and 2 Discovery & Pharmacology Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505

The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) Anal. Biochem. 257, 120–126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 protease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn2+, and that the optimal pH was 6.0–6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.

* To whom correspondence should be addressed. Tel: +81-6-6300-2574, Fax: +81-6-6300-2593, E-mail: k-kyono{at}tanabe.co.jp


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