FIG. 3.

Complex formation between His-HasA and HasR and its mutants. In all cases, about 20 μg of purified His-HasA (holo) was mixed with 1.6 ml of ZW3-14-solubilized membranes from strain C600ΔhemA harboring the various recombinant plasmids grown for 3 h in the presence of arabinose (20 μg/ml). After 1 h of incubation at 4°C, Ni-nitrilotriacetic acid agarose beads (50 μl; QIAGEN) were added, and the incubation continued for 1 h. The beads were pelleted, washed three times with a solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM imidazole, and 0.02% ZW3-14, and finally eluted with the same buffer containing 0.5 M imidazole before being loaded onto a gel. The top shows the stained gel of the various bound fractions, whereas the bottom represents the immunodetection of HasR proteins in solubilized fractions. C indicates empty vector.