title: Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli.
abstract: A cDNA encoding the viral protease from the 3C region of human rhinovirus type 14 was expressed in Escherichia coli through the use of a periplasmic secretion vector. The recombinant protease contained an eight amino acid N-terminal extension that enabled its detection by a specific antibody. It was expressed at a level of approximately 1 mg/L of E. coli culture. Biological activity of the protease was assessed in vitro by using a chemically synthesized peptide consisting of a consensus picornavirus protease cleavage site, Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu. The peptide was cleaved by the recombinant protease at the Gln-Gly bond, generating the product peptides Arg-Ala-Glu-Leu-Gln and Gly-Pro-Tyr-Asp-Glu, which could be separated from the substrate peptide by reversed-phase HPLC. An in vitro assay for the rhinovirus 3C protease was developed by observing the rate of disappearance of the substrate peak from chromatograms of the supernatants of digestion mixtures.
authors.affiliation: Department of Molecular Biology, Immunex Corporation, Seattle, Washington 98101.
authors.forename: R T
authors.initials: RT
authors.lastname: Libby
authors.forename: D
authors.initials: D
authors.lastname: Cosman
authors.forename: M K
authors.initials: MK
authors.lastname: Cooney
authors.forename: J E
authors.initials: JE
authors.lastname: Merriam
authors.forename: C J
authors.initials: CJ
authors.lastname: March
authors.forename: T P
authors.initials: TP
authors.lastname: Hopp
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: D010447:Peptide Hydrolases