title: Expression and characterization of recombinant hepatitis A virus 3C proteinase.
abstract: The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy expression vector in Escherichia coli under control of the tac promoter. The resulting plasmid construction produced 3C proteinase as a soluble and active enzyme constituting approximately 10% of total cellular proteins. The enzyme was purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was developed, and synthetic peptides derived from the predicted cleavage sites of the HAV polyprotein were tested for proteolysis of the enzyme. The peptide representing the 2B/2C cleavage site was cleaved most efficiently with a Km and kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed mutagenesis was then used to identify the cysteine at position 172 as the active site nucleophile. Finally, the purified enzyme showed the expected endoproteinase activity on the P1 precursor protein generated by in vitro transcription/translation.
authors.affiliation: Chiron Corporation, Emeryville, California 94608.
authors.forename: B A
authors.initials: BA
authors.lastname: Malcolm
authors.forename: S M
authors.initials: SM
authors.lastname: Chin
authors.forename: D A
authors.initials: DA
authors.lastname: Jewell
authors.forename: J R
authors.initials: JR
authors.lastname: Stratton-Thomas
authors.forename: K B
authors.initials: KB
authors.lastname: Thudium
authors.forename: R
authors.initials: R
authors.lastname: Ralston
authors.forename: S
authors.initials: S
authors.lastname: Rosenberg
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D006507:Hepatovirus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D014764:Viral Proteins
chemical_list: D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases