Reconstitution of Cgs Protein in Liposomes

E. coli polar lipids at a concentration of 10 mg ml⁻¹ in 25 mM HEPES pH 7.5, 100 mM NaCl were extruded through a 100 nm filter to generate liposomes. Purified Cgs at a concentration of 2 mg ml⁻¹ was added to liposomes destabilized by 0.3% DDM at a 1:2’000 protein:lipid molar ration. Detergent was then removed by two rounds of fresh Bio-Beads. Resulting proteoliposomes were loaded onto a Sephadex G50 column equilibrated with buffer 1 (25 mM Tris pH 7.6, 100 mM NaCl). Concentration of protein in the eluted fractions was measured using the Bradford method (protein assay dye purchased form Bio-Rad). Fractions containing the proteoliposomes were combined and adjusted to 0.2 mg ml⁻¹ protein concentration. The incorporation of Cgs into proteoliposomes was confirmed by SDS-PAGE.