FIG. 4.
Purification of SARS-CoV PLpro1541-1855 and in vitro assays for enzymatic activity. (A) Expression and purification of wild-type SARS-CoV PLpro. Samples (20 μg) from each stage of the purification were analyzed on an SDS-10% polyacrylamide gel and stained with Coomassie brilliant blue. Lane 1, soluble, crude cell lysate from E. coli BL21(DE3) cells expressing pET11a-SARS CoV-PLpro; lane 2, resuspended pellet of the 40% ammonium sulfate cut of the lysate; lane 3, pooled peak fractions from the phenyl-Sepharose column; lane 4, pooled peak fractions after elution from the Q Sepharose and Mono Q columns. (B) Kinetic assay of peptide hydrolysis and deubiquitination. The peptide hydrolysis (▪) and deubiquitinating (▴) activity of wild-type SARS-CoV PLpro were tested at different concentrations of substrate in 50 mM HEPES, pH 7.5, at 25°C. Peptide hydrolysis reactions contained 1 μM enzyme, whereas deubiquitination assays contained 60 nM enzyme. PLproD1826A was also assayed for proteolytic activity against the fluorescent peptide substrate (□) under the same conditions listed for the wild-type enzyme. The data were fit to the equation v/[E]Total = kapp [S] by linear regression and then plotted as log [peptide] (peptide concentration) in order to see the differences in rates with the various substrates. (C) SARS-CoV PLpro cleavage of diubiquitin. Wild-type and alanine substitution mutants of SARS-CoV PLpro were tested for their ability to process diubiquitin in vitro. Purified proteins were incubated at 37°C with diubiquitin in a reaction buffer containing bovine serum albumin (BSA) for the times indicated. Cleavage products were analyzed by electrophoresis on a 10 to 20% gradient acrylamide gel and stained with Coomassie blue. The components in each reaction and the incubation time are indicated above each lane. ub, ubiquitin; di-ub, diubiquitin.