作者: P J Burck , D H Berg , T P Luk , L M Sassmannshausen , M Wakulchik
DOI: 10.1128/JVI.68.5.2937-2946.1994
关键词:
摘要: Abstract The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation its C terminus, is an essential step virion maturation. The proteinase responsible for this cleavage amino-terminal half protein encoded by UL80a open reading fame. We have obtained high expression levels 256-amino-acid HCMV proteinase, assemblin, Escherichia coli. In addition to 28-kDa a 15-kDa comprising first 143 amino acids and 13-kDa last 113 were present. Both purified two-step chromatographic procedure utilizing anion exchange urea dithiothreitol size exclusion NaSCN dithiothreitol. Activation required denaturation as well complete reduction all five cysteine residues molecule. Removal dialysis with retention reducing agent yielded active proteinase. Addition glycerol 50% enhanced activity. cleaved peptides RGVVNASSRLAK SYVKASVSPE, which are mimics maturational (M)- release (R)-site sequences, respectively, UL80a-encoded protein. site was at same Ala-Ser scissile bond observed Km value (M-site mimic) similar that SYVKASVSPE (R-site mimic), but turnover (kcat) M-site peptide mimic substrate six eight times faster. homologs herpes simplex virus type 1 M- R-site sequences UL26-encoded also although rates slower than those substrates. inhibited Zn2+ alkylating agents, only very inhibitor concentrations. subjected activation conditions had no enzymatic activity against