Preprotein Translocase of Escherichia coli: Solubilization, Purification, and Reconstitution of the Integral Membrane Subunits SecY/E

This chapter describes the procedures to solubilize and purify a functional secY/E protein that, upon reconstitution, supports an authentic translocation reaction of precursor proteins. The secY/E protein is purified from octylglucoside-extracted membranes by the ability of the reconstituted enzyme to stimulate the ATP-hydrolyzing activity of the purified secA protein in the presence of proOmpA. The chapter describes the reconstitution procedure and the various assays required to determine the activity of the reconstituted secY/E protein. Protocols used for the isolation and solubilization of E. coli inner membranes and the purification of the secY/E protein are described. The purified secY/E protein contains three major polypeptide species. These are identified by immunoblots with antisera to secY and secE and by N-terminal sequence analysis. The largest polypeptide of the purified protein reacts with antibodies to the secY N-terminus and migrates on SDS-PAGE with an apparent molecular mass of 29 kDa. The use of glycerol and phospholipids with octylglucoside has a dramatic effect on the solubility of the secY protein