Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes

Abstract

Protein translocation is one of the examples of a complex membrane-bound biological process that has been functionally reconstituted into proteoliposomes. This process relies on several membrane proteins that need to be coreconstituted to yield a functional system. This chapter describes methods to express, detergent solubilize, purify, and functionally reconstitute preprotein translocation in proteoliposomes using Escherichia coli translocase components. A coreconstitution protocol and a fluorescence assay to monitor preprotein translocation in vitro are described. The proteins can be solubilized from the membrane and purified to homogeneity (>95%) by anion exchange and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. A multitude of methods are described for the reconstitution of membrane proteins into proteoliposomes. The methods of hydrophobic absorption or rapid dilution for detergent removal are the most significant methods for the functional reconstitution. The functional-membrane integration of newly synthesized inner membrane proteins, using the purified translocase and associated components, remains a challenge for future research. Other challenges lie in the reconstitution of the complete assembly of multisubunit membrane proteins.