Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system.

Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Research Abstract Details 

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Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Abstract Text:

Kiyoshi Kyono,Masahiko Miyashiro,Ikuhiko Taguchi,

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 Mn(2+), 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.

Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Publishing Authors By Initials

K Kyono,M Miyashiro,I Taguchi,

For similar proteins: viral proteins: viral nonstructural proteins research abstracts see: proteins: viral proteins: viral nonstructural proteins research

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Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 135

Page Numbers: 245-52

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Feb

YEAR: 2004

Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Keywords Mesh Terms:

KEYWORDS: Viral Nonstructural Proteins

MESH TERMS: metabolism

Chemical & Substance for Abstract: Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system. Information

Substance Name: Endopeptidases

Registry Number: EC 3.4.-

Grant and Affiliation Information for Expression and purification of a hepatitis C virus NS3/4A complex, and characterization of its helicase activity with the Scintillation Proximity Assay system.

AFFILIATION: Medicinal Chemistry Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505. k-kyono@tanabe.co.jp

Country: Japan

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MEDLINETA: J Biochem

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