Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli.
The 3C region of human rhinovirus 14 was expressed in Escherichia coli. The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity. Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules. Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site. This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease. The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly.
Cordingley
M G
MG
Department of Virus and Cell Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Register
R B
RB
Callahan
P L
PL
Garsky
V M
VM
Colonno
R J
RJ
D000086042:3C Viral Proteases; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000235:genetics; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015345:Oligonucleotide Probes; D010455:Peptides; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics*
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D010455:Peptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases