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  • Title: Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.
    Author: Wang QM, Johnson RB, Cox GA, Villarreal EC, Churgay LM, Hale JE.
    Journal: J Virol; 1998 Feb; 72(2):1683-7. PubMed ID: 9445078.
    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.
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