Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: High-yield expression and purification of human P-glycoprotein

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance t he heterologous expression of ATP-binding cassette transporters in Saccharo myces cerevisiae, Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0.4-0.7 mu mol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated, By use of the PMA1 promoter, P-gp could be expresse d at 3% of total membrane protein. The expression level could be further en hanced to 8% when cells were grown in the presence of 10% glycerol as a che mical chaperone, Similarly, glycerol enhanced protein levels of P-gp expres sed under control of the GAL1 promoter. Glycerol was demonstrated to enhanc e posttranslational stability of P-gp, Polyhistidine-tagged P-gp was purifi ed by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K-M(MgAT P),V-max, and drug activation were dependent on the lipid composition of pr oteoliposomes and pH of the assay and were similar to P-gp purified from ma mmalian sources. In conclusion, we developed a system for cost-effective, h igh-yield, heterologous expression of functional P-gp useful in producing l arge quantities of normal and mutant P-gp forms for structural and mechanis tic studies. (C) 2000 Academic Press.