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 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.
Figler
R A
RA
Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908-0736, USA.
Omote
H
H
Nakamoto
R K
RK
Al-Shawi
M K
MK
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008985:Monensin / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers; D007476:Ionophores; D011994:Recombinant Proteins; D008985:Monensin; D005990:Glycerol