Use of Chemical Chaperones in the Yeast Saccharomyces Cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-glycoprotein

Arch Biochem Biophys. 2000 Apr 1;376(1):34-46. doi: 10.1006/abbi.2000.1712.

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 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 micromol/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(MgATP)(M), 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / isolation & purification
  • Base Sequence
  • Cell Membrane / metabolism
  • DNA Primers / genetics
  • Gene Expression / drug effects
  • Genetic Vectors
  • Glycerol / pharmacology
  • Humans
  • In Vitro Techniques
  • Ionophores / pharmacology
  • Kinetics
  • Monensin / pharmacology
  • Mutation
  • Plasmids / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • DNA Primers
  • Ionophores
  • Recombinant Proteins
  • Monensin
  • Glycerol