EXPRESSION AND PURIFICATION OF THE SACCHAROMYCES-CEREVISIAE ALPHA-FACTOR RECEPTOR (STE2P), A 7-TRANSMEMBRANE-SEGMENT G-PROTEIN-COUPLED RECEPTOR

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor recept or (the STE2 gene product) tagged at its C-terminal end with an epitop e (FLAG) and a His(6) tract. When expressed in yeast from this plasmid , Ste2p was produced at a level at least S-fold higher than that repor ted previously for any other 7-transmembrane-segment receptor expresse d in the same cells. For purification, isolated cell membranes contain ing the overexpressed receptor were solubilized with detergent under s pecific conditions and subjected to immobilized metal affinity chromat ography. Yields as high as 1 mg of nearly homogeneous (95%) receptor w ere routinely obtained even from relatively small scale preparations ( 60 g of frozen cell paste). The purified receptor was :reconstituted i nto artificial phospholipid vesicles. Radioligand binding studies demo nstrated that the purified receptor, in the reconstituted vesicles, bo und its tridecapeptide ligand (alpha-factor) with a K-D (155 nM) consi stent with the affinity expected for this receptor in the absence of i ts associated G protein. Efficient restoration of ligand binding activ ity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physica l studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.