title: Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.
abstract: cDNA encoding the putative core of the hepatitis C virus NS3 serine protease 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 purification 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 substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.
authors.affiliation: Vertex Pharmaceuticals Inc., Cambridge, MA 02139-4242, USA.
authors.forename: W
authors.initials: W
authors.lastname: Markland
authors.forename: R A
authors.initials: RA
authors.lastname: Petrillo
authors.forename: M
authors.initials: M
authors.lastname: Fitzgibbon
authors.forename: T
authors.initials: T
authors.lastname: Fox
authors.forename: R
authors.initials: R
authors.lastname: McCarrick
authors.forename: T
authors.initials: T
authors.lastname: McQuaid
authors.forename: J R
authors.initials: JR
authors.lastname: Fulghum
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: M A
authors.initials: MA
authors.lastname: Fleming
authors.forename: J A
authors.initials: JA
authors.lastname: Thomson
authors.forename: S P
authors.initials: SP
authors.lastname: Chambers
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D020365:RNA Helicases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases