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J Virol, February 1998, p. 1683-1687, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in Escherichia coli

Q. May Wang,* Robert B. Johnson, Gregory A. Cox, Elcira C. Villarreal, Lisa M. Churgay, and John E. Hale

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Received 5 August 1997/Accepted 4 November 1997

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 kcat/Km ratio of ~335 M-1s-1, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

* Corresponding author. Mailing address: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285. Phone: (317) 277-6975. Fax: (317) 276-1743. E-mail: qmwang{at}lilly.com.




This article has been cited by other articles:

  • Wang, Q. M., Johnson, R. B., Jungheim, L. N., Cohen, J. D., Villarreal, E. C. (1998). Dual Inhibition of Human Rhinovirus 2A and 3C Proteases by Homophthalimides. Antimicrob. Agents Chemother. 42: 916-920 [Abstract] [Full Text]  


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