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

Robert A. Figler, Hiroshi Omote, Robert K. Nakamoto, Marwan K. Al-Shawi

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2μ 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 μmol/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 expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified 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)/(MgATP), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P- gp forms for structural and mechanistic studies. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)34-46
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume376
Issue number1
DOIs
Publication statusPublished - Apr 1 2000
Externally publishedYes

Keywords

  • ABC transporters
  • Chemical chaperones
  • Glycerol
  • Heterologous expression
  • Membrane proteins
  • P-glycoprotein
  • Saccharomyces cerevisiae
  • Yeast

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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