title: Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.
abstract: Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+ Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY-p-nitroanilide was found to be cleaved by the 2A protease with a k(cat)/Km ratio of approximately 335 M(-1)s(-1), which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. qmwang@lilly.com
authors.forename: Q M
authors.initials: QM
authors.lastname: Wang
authors.forename: R B
authors.initials: RB
authors.lastname: Johnson
authors.forename: G A
authors.initials: GA
authors.lastname: Cox
authors.forename: E C
authors.initials: EC
authors.lastname: Villarreal
authors.forename: L M
authors.initials: LM
authors.lastname: Churgay
authors.forename: J E
authors.initials: JE
authors.lastname: Hale
mesh_terms: D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D017510:Protein Folding; D012229:Rhinovirus / Q000201:enzymology*; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus