PMID: 9008301Nov 1, 1996Paper

Initial characterization of autoprocessing and active-center mutants of CMV proteinase

Journal of Protein Chemistry
S W SnyderT H Holzman

Abstract

Human cytomegalovirus (CMV) encodes a unique serine proteinase that is required in the maturation of the viral capsid. The CMV proteinase can undergo autocatalytic activation and is subject to proteolytic self-inactivation. Mutant enzyme forms were prepared to eliminate the initial autoprocessing site and thus form an active single-chain protein for structure-function studies. Two mutants of CMV proteinase were cloned and expressed in Escherichia coli. The A143V mutant was a conservative substitution at the first internal cleavage site. The S132A mutant modified one of the triad of residues responsible for catalytic activity. Through the use of computer-controlled high-cell-density fermentations the mutant proteins were expressed in E. coli at approximately 170 mg/L as both soluble (approximately 40% of total) and inclusion-body forms (approximately 60% of total). The soluble enzyme was purified by standard methods; inclusion-body protein was isolated by standard methods after refolding and solubilization in guanidine or urea. Sedimentation equilibrium and sedimentation velocity analyses reveal that the enzyme undergoes concentration-dependent aggregation. It exhibits a monomer <==> dimer equilibrium (Kd = 1 microM) at low conc...Continue Reading

References

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