부산대학교 도서관

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiaeα-factor receptor (the STE2gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (α-factor) with a KD(155 nm) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity 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 physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.