Activity of two-chain recombinant human cytomegalovirus protease.
1994; Elsevier BV; Volume: 269; Issue: 41 Linguagem: Inglês
10.1016/s0021-9258(18)47332-2
ISSN1083-351X
AutoresBarry C. Holwerda, Arthur J. Wittwer, Kevin L. Duffin, Christine E. Smith, Máté Tóth, Linda Carr, Roger C. Wiegand, Martin L. Bryant,
Tópico(s)Biochemical and Molecular Research
ResumoThe human cytomegalovirus U,SO protease was expressed in Escherichia coli and purified by metalchelate chromatography using a histidine tag engineered at the amino terminus.Cleavage of the 30-kDa protease at an internal site, 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 Seri3' on the 16-kDa chain by [3H]diisopropyl fluorophosphate.Disruption of the cleavage site by mutation from VENA to AEAIA facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser13' by r3H1diisopropyl fluorophosphate.Both one-and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNAIS, and a peptide, GVVNASARL, mimicking this site.Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.Herpesviruses encode a unique protease that is necessary for viral replication (1-9).A combination of studies employing class-specific inhibitors (2), site-directed mutagenesis (81, and covalent modification (9) have shown these proteases possess a conserved serine as the active site nucleophile.However, they show little or no primary structure identity to other well characterized serine proteases.Herpesvirus proteases are expressed in virus-infected cells as a fusion with a carboxyl-terminal capsid assembly protein (1, 10).The protease autocatalytically cleaves after alanine at specific L X N S or VXA/S sites (where X is any amino acid and/ indicates the scissile bond) found at a consensus maturation site near the carboxyl terminus of the assembly protein and a release site between the protease and the assembly protein (1, 7).The human cytomegalovirus (HCMV)' and simian cytomegalovirus ( S C M V ) proteases each undergo an additional cleavage within the protease domain at a conserved I-site (8,11,12).HCMV protease (11,12,16) and herpes simplex virus type-1 (HSV-1) protease (9,13-15) have been expressed in Escherichia coli to allow further study of these novel enzymes.The recombinant proteases maintain autocatalytic processing activity of
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