Figure 4.

Expression and catalytic activity of plastid E2. A, Expression of plastid E2. Mature plastid E2 was expressed in E. coli. Isopropylthio-β-galactoside induced the expression of plastid E2 (GST::Plastid E2). Plastid E2 was eluted from a glutathione-Sepharose affinity column in four fractions (Elution). The purified protein was then digested with the PreScission protease. Uncut and cut samples were separated by SDS-PAGE in triplicate, followed by staining with Coomassie Brilliant Blue (Stain), or transfer to nitrocellulose and immunolabeling with plastid E2 antibody (E2 Ab) or GST monoclonal antibody (GST MAb). The products of digestion are indicated. B, Activity of purified recombinant plastid E2. Controls were no enzyme and enzyme plus oxidized dihydrolipoamide (DHL). Catalytic activity of recombinant GST::plastid E2 and cleaved enzyme was measured at the protein concentrations indicated in the presence of reduced dihydrolipoamide. Error bars indicate the sd of triplicate assays; absence of error bars indicates negligible sd.