Activity of two-chain recombinant human cytomegalovirus protease.
The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.
Holwerda
B C
BC
Searle Research and Development, St. Louis, Missouri 63198.
Wittwer
A J
AJ
Duffin
K L
KL
Smith
C
C
Toth
M V
MV
Carr
L S
LS
Wiegand
R C
RC
Bryant
M L
ML
D000595:Amino Acid Sequence; D001483:Base Sequence; D003587:Cytomegalovirus / Q000201:enzymology*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D006639:Histidine / Q000235:genetics; D006801:Humans; D007531:Isoflurophate / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011994:Recombinant Proteins; D014764:Viral Proteins; D007531:Isoflurophate; D006639:Histidine; D010450:Endopeptidases; C087225:human cytomegalovirus protease UL80