Activity of two-chain recombinant human cytomegalovirus protease
Autor: | M V Toth, Arthur J. Wittwer, R.C. Wiegand, Christine E. Smith, L S Carr, K L Duffin, M L Bryant, Barry C. Holwerda |
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Rok vydání: | 1994 |
Předmět: |
chemistry.chemical_classification
Protease medicine.medical_treatment Peptide Cell Biology medicine.disease_cause Biochemistry Molecular biology law.invention Enzyme chemistry law medicine Recombinant DNA Diisopropyl fluorophosphate Molecular Biology Escherichia coli Histidine medicine.drug Assemblin |
Zdroj: | Journal of Biological Chemistry. 269:25911-25915 |
ISSN: | 0021-9258 |
DOI: | 10.1016/s0021-9258(18)47332-2 |
Popis: | 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. |
Databáze: | OpenAIRE |
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