20] Use of cell-free systems to determine P-glycoprotein transmembrane topology

Abstract

This chapter describes the application of a cell-free expression system combined with cDNA engineering to dissect the detailed topological folding of P-Glycoprotein (Pgp). The methods described in the chapter help to determine the topogenic sequences in Pgp and identify other factors that control the Pgp topology. This method can also be used to determine the topological folding of other simple membrane proteins. There are two well characterized and frequently used cell-free expression systems, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE), both supplemented with microsomal membranes (RM) from dog pancreatic endoplasmic reticulum (ER). The use of these systems determines the topology that is based on the belief that most transmembrane proteins in eukaryotic cells acquire their final membrane topologies during or immediately after synthesis on the rough ER.