PURIFICATION AND CHARACTERIZATION OF THE NS3 SERINE-PROTEASE DOMAIN OF HEPATITIS-C VIRUS EXPRESSED IN SACCHAROMYCES-CEREVISIAE

cDNA encoding the putative core of the hepatitis C virus NS3 serine pr otease domain (residues 1-181 of NS3; NS3(181)) was expressed as an N- terminally (His)(6)-tagged fusion protein in Saccharomyces cerevisiae. NS3(181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purificat ion of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181) protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated su bstrates, and subsequently a previously unobserved cleavage site withi n the NS5A region was identified. The inhibitory effect of known prote ase inhibitors was also examined. It is hoped that availability of thi s method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.