Supplementary Figure 4: Western blot of total membrane fractions from NR1154 cells transformed with pEXT21 encoding either wild-type (WT) or mutant MurJTA.
From: Crystal structure of the MOP flippase MurJ in an inward-facing conformation
MurJTA proteins carrying a C-terminal FLAG tag were expressed by the addition of 0.1 mM IPTG in the presence of arabinose. All of the mutants expressed with the exception of mutants D235A and N374A (image is representative of n = 3, technical replicates). There is no correlation between expression levels and complementation outcomes as long as the proteins are expressed: the lowest expression level was observed for the N231A mutant, which clearly complements MurJEC, while the highest expression level was observed in R24A, which clearly fails to complement. MurJTA (55 kD) migrates at an apparent molecular weight of 37 kD on a 10% SDS-PAGE gel.
