EXPRESSION, PURIFICATION, AND PARTIAL CHARACTERIZATION OF HCV RNA-POLYMERASE

The product of the NS5B gene of Hepatitis C Virus (HCV) has been expre ssed in Escherichia Coli both as a fusion protein with glutathione-S-t ransferase (GST) of molecular weight 91KDa and at high level as a sing le protein of molecular weight 65KDa. The protein was sequestered with in inclusion bodies and a variety of procedures designed to minimize i nclusion body formation proved unsuccessful. The method finally adopte d involved the purification of inclusion bodies followed by the solubi lization, purification, and refolding of the expressed protein. A good recovery and protein purity of the order of 80-90% were achieved. The purified protein was shown to possess RNA polymerase activity in an a ssay using polyA/oligoU as template. The enzymatic activity is rifampi cin resistant, poly A dependent, and requires Mg++. The availability o f purified HCV RNA polymerase will allow the study of viral replicatio n and constitute the basis for testing new anti-viral drugs. (C) 1997 Academic Press.