FIGURE 8.

Co-purification demonstrates interactions between WbbM, WbbN, and WbbO. Co-purification of pairwise combinations of the synthesis components of d-galactan I O-PS biosynthesis was examined in lysates from E. coli containing two plasmids expressing combinations of biosynthesis components. The protein of interest was bound to TALON metal affinity resin via a polyhistidine tag on one of the partner proteins (His₆-WbbN), or to GST affinity resin via a glutathione tag (GST-WbbO). Washes were completed before buffer containing imidazole (300 mm) or glutathionine (10 mm) was added to elute the appropriately tagged protein and any interacting proteins from the resin. S, lysate added to the resin; FT, flow through; W1, wash one (10 resin volumes); W2, wash two (10 resin volumes); E1, elution one (1 resin volume); E2, elution two (1 resin volume). A, samples of 5 μl of each fraction were separated by SDS-PAGE and visualized with Simply Blue stain. Corresponding Western blots for each partner pair are shown below the corresponding gels. B, negative controls verifying that purification of each partner protein is dependent on its interaction with His- or GST-tagged protein.