Chapter 6 Preprotein Translocase of Escherichia coli: Solubilization, Purification, and Reconstitution of the Integral Membrane Subunits SecY/E

Publisher Summary This chapter describes the procedures to solubilize and purify a functional sec Y/E protein that, upon reconstitution, supports an authentic translocation reaction of precursor proteins. The sec Y/E protein is purified from octylglucoside-extracted membranes by the ability of the reconstituted enzyme to stimulate the ATP-hydrolyzing activity of the purified sec A protein in the presence of proOmpA. The chapter describes the reconstitution procedure and the various assays required to determine the activity of the reconstituted sec Y/E protein. Protocols used for the isolation and solubilization of E. coli inner membranes and the purification of the sec Y/E protein are described. The purified sec Y/E protein contains three major polypeptide species. These are identified by immunoblots with antisera to sec Y and sec E and by N -terminal sequence analysis. The largest polypeptide of the purified protein reacts with antibodies to the sec Y 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 sec Y protein.