ACTIVITY OF 2-CHAIN RECOMBINANT HUMAN CYTOMEGALOVIRUS PROTEASE

The human cytomegalovirus U(L)80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine t ag engineered at the amino terminus. Cleavage of the 30-kDa protease a t an internal site, VEA/A(144), resulted in the recovery of 16- plus 1 4-kDa two chain protease. The amino-terminal 16-kDa chain and the carb oxyl-terminal 14-kDa chain remained associated as an active enzyme tha t was modified specifically at Ser(132) on the 16-kDa chain by [H-3]di isopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-cha in enzyme that could be similarly modified at Ser(132) by [3H]diisopro pyl fluorophosphate. Both one- and two-chain enzymes cleaved recombina nt assembly protein at the maturation site, VNA/S, and a peptide, GVVN ASARL, mimicking this site. Internal processing does not inactivate th e protease but forms a two-chain enzyme that retains activity.