FIGURE 2.

SDS-PAGE analysis of overexpression and purification of HiLpxH. A, cell-free extract (CFE) of VC_t10 and HiH_t10 prepared as described under “Experimental Procedures.” The 27-kDa band that is present in the HiH_t10 lane and not in the VC_t10 lane corresponds to overexpressed HiLpxH. B, cytosolic (Cyt) and membrane (Mem) fractions of HiH_t10 cell-free extracts incubated in the absence or presence of Triton X-100 (TX-100). The activity of each fraction, as determined by autoradiographic assay, is listed below the lanes as a percentage of total LpxH activity observed in the corresponding cell-free extract. C, analysis of fractions from HiLpxH purification outlined under “Experimental Procedures.” Fractions from the Ni-NTA purification include the Load, denoting the solubilized cell-free extract from HiH10_t that was loaded onto the column, FT representing the flow-through from the column, W1 signifying the 50 mm imidazole wash, and Elute indicating the tagged HiLpxH obtained from the 400 mm imidazole wash. The CU elute lane shows protein that flowed through the second clean-up Ni-NTA column after TEV protease digestion. The purified HiLpxH fractions depict the final sample of purified HiLpxH that was used in autoradiographic assays and EPR experiments. The labels 5 μg and 10 μg indicate how much protein was loaded in each lane.