Purification of functional human P-glycoprotein expressed in Saccharomyces cerevisiae.
A system for expression and facile purification of the human P-glycoprotein (Pgp) from the yeast Saccharomyces cerevisiae is described. The wild-type human mdr1 cDNA was cloned into a high copy number yeast expression vector under the control of the constitutive promoter of the yeast plasma membrane H+-ATPase. Western blots of membranes from the stable transformants confirmed that the Pgp is expressed in yeast cells in amounts approximately 0.4% of the total yeast membrane protein. Density gradient sedimentation analysis of the yeast membranes indicated that the expressed Pgp is localized in the plasma membrane. Yeast cells transformed with the Pgp expression plasmid acquire increased resistance to valinomycin, suggesting that the expressed Pgp is properly folded and functional. The expressed Pgp can be solubilized from the yeast membranes with lysophosphatidylcholine, and when tagged with ten histidines at its C-terminus, can be readily purified to about 90% homogeneity by Ni2+ affinity chromatography. About 50 microg of the Pgp can be purified from 20 mg of crude yeast membranes. The purified human Pgp exhibits a verapamil-stimulated ATPase activity and the maximal activity is 2.5 +/- 0.5 micromol/min per mg of Pgp, suggesting that the purified Pgp from yeast is highly functional. The Pgp expressed in yeast has the same electrophoretic mobility (ca. 130 kDa) as the Pgp produced in Sf9 insect cells and is unaffected by N-glycosidase treatment, suggesting that it is not glycosylated. Because of the relative ease of growing yeast in massive quantities this expression system appears to be excellent for producing this membrane transporter at levels sufficient for further biochemical and biophysical studies, and for site-directed mutagenesis studies as well.
Mao
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Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Scarborough
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GA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D006801:Humans; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018076:DNA, Complementary