Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli |
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Authors: | QM Wang RB Johnson GA Cox EC Villarreal LM Churgay JE Hale |
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Affiliation: | Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. qmwang@lilly.com |
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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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