Reconstitution of tripartite efflux pumps MexAB-OprM and MacAB-TolC in biomimetic systems.

Tripartite efflux pumps are central to multidrug resistance in Gram-negative bacteria, actively extruding antibiotics across the cell envelope. They operate as tripartite complexes spanning the inner and outer membranes, connected by periplasmic adaptors. Here, we describe the in vitro reconstitution of two representative systems into biomimetic environments: the RND-type pump MexAB-OprM from Pseudomonas aeruginosa and the ABC-type pump MacAB-TolC from Escherichia coli. We provide detailed protocols for heterologous expression and purification of individual subunits, followed by their stepwise incorporation into proteoliposomes, nanodiscs, or amphipols. The protocols are adaptable to other Gram-negative multidrug efflux systems and provide a robust platform for dissecting structure-function relationships.