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Expression, Purification, and Activity of Recombinant MHV-A59 3CLpro

  • A. C. Sims
  • X. T. Lu
  • M. R. Denison
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 440)

Abstract

The 3C-like proteinase (3CLpro) of MHV-A59 is predicted to mediate the majority of proteolytic processing events within the gene 1 polyprotein. We have overexpressed 3CLpro in E. coli as a fusion protein with maltose binding protein (MBP). The MBP-3CLpro fusion protein was purified from contaminating E. coli proteins by amylose column chromatography, and r3CLpro was cleaved from the fusion protein by factor Xa. Recombinant 3CLpro (r3CLpro) was able to cleave a polypeptide substrate containing mutated inactive 3CLpro and portions of the flanking domains. R3CLpro cleaved substrate completely within 5 minutes and the activity of r3CLpro was sensitive to inhibition by serine and cysteine proteinase inhibitors; however, it was not inhibited by EDTA, suggesting that metal ions were not critical for 3CLpro activity.

Keywords

Cleavage Site Maltose Binding Protein Cysteine Proteinase Inhibitor Mouse Hepatitis Virus Carboxy Terminal Domain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • A. C. Sims
    • 1
    • 2
  • X. T. Lu
    • 2
    • 3
  • M. R. Denison
    • 1
    • 2
    • 3
  1. 1.Department of Microbiology and ImmunologyVanderbilt University Medical CenterNashvilleUSA
  2. 2.Elizabeth B. Lamb Center for Pediatric ResearchVanderbilt University Medical CenterNashvilleUSA
  3. 3.Department of PediatricsVanderbilt University Medical CenterNashvilleUSA

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