*5.3. Purification and Reconstitution of P-gp*

A C-terminal dodecyl-histidine version of human P-gp was expressed in *Trichoplusia ni* (High-Five) insect cells using recombinant baculovirus as previously described [19]. Following expression, crude membranes were prepared using differential ultra-centrifugation and stored at −80 ◦C. P-gp was extracted from the High-Five crude membranes using the detergent dodecyl-β-D-maltoside (DDM) and purified by metal affinity chromatography on Ni-NTA His-Bind resin. Chromatography buffers contained 0.1% (*w*/*v*) DDM and were supplemented with 0.1% (*w*/*v*) of a lipid mixture comprising a 4:1 ratio of *E. coli* total lipid extract and cholesterol. This enabled rapid reconstitution into vesicles by detergent adsorption using SM2 BioBeads.
