Expression, purification, and activity of recombinant MHV-A59 3CLpro.
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.
Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
A C
AC
Sims
X T
XT
Lu
M R
MR
Denison
D000818:Animals; D003001:Cloning, Molecular; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D006517:Murine hepatitis virus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases