Membrane protein reconstitution in liposomes is an invaluable technique to study numerous properties of membrane proteins in vitro. Kinetics, substrate specificity, and protein-protein interaction of membrane proteins can be investigated once they are embedded in the liposome bilayer. Both protocols described here aim to investigate the efflux pump MexA-MexB-OprM from Pseudomonas aeruginosa, a proteins complex embedded in both membranes of this Gram-negative bacteria. This tri-partite system, which by-passes the periplams, is involved in antibiotic resistance. First, we describe a protocol to study MexB, the actual transporter, and second we propose an alternate protocol where the tripartite system is investigated as a whole.
Keywords: Detergent; Fluorescence; Membrane protein purification and reconstitution; Proteoliposomes; Substrate transport.