Large complexes: Cloning strategy, production, and purification

Abstract

Membrane proteins can assemble and form complexes in the cell envelope. In Gram-negative bacteria, a number of multiprotein complexes, including secretion systems, efflux pumps, molecular motors, and pilus assembly machines, comprise proteins from the inner and outer membranes. Besides the structures of isolated soluble domains, only a few atomic structures of these assembled molecular machines have been elucidated. To better understand the function and to solve the structure of protein complexes, it is thus necessary to design dedicated production and purification processes. Here we present cloning procedures to overproduce membrane proteins into Escherichia coli cells and describe the cloning and purification strategy for the Type VI secretion TssJLM membrane complex.