Peptide Oxidation Assay with Recombinant CtDsbB

The peptide oxidation assay was performed as reported in [36 (link)] except that crude membranes containing recombinant CtDsbB were used instead of oxidised glutathione to sustain CtDsbA activity. Briefly the assay was performed in a 384-well plate (Perkin Elmer, USA). A solution of 50 mM MES, 50 mM NaCl, 2 mM EDTA, pH 5.5, 8 μM of the DsbB and either 80 nM (EcDsbA) or 640 nM (CtDsbA or CtDsbA-SSS) were added to the wells in a total volume of 25 μL. Adding 25 μL peptide to a final concentration of 10 μM started the reaction. Change in fluorescence was monitored at excitation 340 nm and emission 615 nm, with a delay of 100 μs and read time of 100 μs, using a Synergy H1 Multimode plate reader (BioTek, USA). Plotted data shows mean and SD for two biological replicates.