FIGURE 6.

ApoCcmE forms a ternary complex with CcmI and apocytochrome c₂. A, co-purification of FLAG-CcmI and Strep-apocytochrome c₂ with His-apoCcmE using Ni²⁺-Sepharose resin is shown. B, co-purification of FLAG-CcmI and His-apoCcmE with Strep-apocytochrome c₂ using Strep-Tactin-Sepharose resin is shown. C, shown is an elution profile (absorbance at 280 nm as a function of the volume) of a mixture containing His-apoCcmE, FLAG-CcmI, and Strep-apocytochrome c₂ from the Sephacryl S200 column in buffer containing 0.01% DDM. Estimation of the molecular masses (kDa) was done according to the calibration of the column using bovine serum albumin (67 kDa), carbonic anhydrase (39 kDa), and horse heart cytochrome c (12.4 kDa). Elution volume of the different standards and their respective molecular masses are depicted on the top of the chromatogram. The area of the chromatogram where FLAG-CcmI, His-apoCcmE, and Strep-apocytochrome c₂ are co-eluted as a ternary complex is highlighted with diagonal lines. D, SDS-PAGE profiles of selected fractions are shown. Fractions 35–40 (left panel) and 65–70 (right panel) contain mainly aggregates of apoCcmE and apocytochrome c₂, respectively, and fractions 48, 52, and 55 contain the quasi-stoichiometric ternary complex composed of FLAG-CcmI, His-apoCcmE, and Strep-apocytochrome c₂, as indicated on the right. Note that proteins with higher molecular masses (e. i., CcmI) stained more intensely with Coomassie Blue as compared with smaller proteins (i.e. apoCcmE and apocytochrome c₂).