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 receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. 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 (alpha-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.
David
N E
NE
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.
Gee
M
M
Andersen
B
B
Naider
F
F
Thorner
J
J
Stevens
R C
RC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D019204:GTP-Binding Proteins / Q000378:metabolism; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D011869:Radioligand Assay; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors
D004274:DNA, Recombinant; D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor; D019204:GTP-Binding Proteins