Figure 5.

Purification of FtsE‐His and determination of ATP binding. (A) SDS‐PAGE: lane 1, Ni‐NTA‐column affinity‐purified FtsE‐His, which was further purified by size‐exclusion chromatogrphy; lanes 2 and 3, fractions from size‐exclusion chromatography of affinity‐purified FtsE‐His. Additional bands were not visible in these lanes elsewhere in the gels (data not shown) A section of the gel lacking samples was removed in the image between lanes 1 and 2. (B). Size‐exclusion chromatogram (Superdex 200 30/100 in 20 mmol/L Tris pH 8.0, 100 mmol/L NaCl; flow rate = 0.5 mL per min; A₂₈₀ detection) of purified FtsE‐His. The elution time of 33 min corresponds to an elution volume of 16.5 mL. Minimal additional peaks were detected elsewhere in chromatograms (data not shown). FtsE‐His overexpression and purification were carried out as described in Materials and Methods. (C) TNP‐ATP fluorescence binding assay of purified FtsE‐His. The assay and titration procedure are described in Materials and Methods. The K_d determined by the concentration of TNP‐ATP at half saturation of fluorescence intensity is indicated. Data points were averaged from two measurements.