Persistent infection with hepatitis C virus (HCV) is a common cause of chronic liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1). HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products (reviewed in 2).
NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (3). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 4).
The HCV NS3/4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (5-7). It has been shown that the central region (amino acids 21–30) of the 54-residue NS4A protein is essential and sufficient for the enhancement of proteolytic activity of the NS3 serine protease (8, 9). In recent phase I trials, a 2–3-log reduction of HCV viral load was observed after a 2-day treatment with a serine protease inhibitor, which provided the first proof-of-concept evidence that HCV NS3/4A protease inhibitors could be a new therapeutic option for hepatitis C patients (10).
Recombinant HCV-NS3-3 was expressed in a E.coli system and purified by an affinity column in combination with FPLC chromatography Research Use Only. HCV-NS3-3 can be applied in in vitro assay development and screening of protease inhibitors. The purified recombinant protein is greater than 95% homogeneous and contains no detectable DNase and RNase activity. 1 unit equals 1 nanogram of purified protein 0.5 mg/ml (in 1X dilution buffer A) Buffer A: Tris-Cl, Glycerol, KCl, DTT, EDTA.
References: 1. Alter, M. J. 1997. Epidemiology of hepatitis C. Hepatology 26:62S-65S. 2. Blight, K. J., et al., (1998) Antiviral Ther. 3, Suppl. 3, 71-81 3. Kolykhalov, A. et al., (2000) J. Virol. 74, 2046-2051 4. De Francesco, R., et al., (2003) Antiviral Res. 58, 1-16 5. Grakoui, A., et al., (1993) J. Virol. 67, 2832-2843 6. Hijikata, M., et al., (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10773-10777 7. Tomei, L., et al., (1993) J. Virol. 67, 4017-4026 8. Bartenschlager, et al., (1995) J. Virol. 69, 7519-7528 9. Lin, C., and Rice, C. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7622-7626 10. Lamarre, D., et al., (2003) Nature 426, 186-189
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