FIGURE 2.

Cellular localization, purification, and characterization of the periplasmic domain of LptC. A and B, His₆-LptC(24–191) was expressed in E. coli CWG905. Fractions were separated by SDS-PAGE, and proteins were visualized using either SimplyBlue SafeStain (A) or Western immunoblotting with anti-His₅ monoclonal antibody (B). The His₆-LptC(24–191) construct migrates as a 21-kDa band (indicated by the arrow on the right), consistent with the predicted value of 21,300 Da. Lane 1 contains whole cell lysate from induced E. coli CWG905 cells. Lanes 2 and 4 represent the soluble fractions after centrifugation at 15,000 and 100,000 × g, respectively. Lanes 3 and 5 represent the membrane fractions after centrifugation at 12,000 and 100,000 × g, respectively. The purification of His₆-LptC(24–191) by Ni²⁺-NTA affinity chromatography is shown on the right side of A and B. FT, flow-through with unbound proteins; W, wash; E, elution with imidazole.