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

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

Auteurs : Robert A. Figler ; Hiroshi Omote ; Robert K. Nakamoto ; Marwan K. Al-Shawi

Source :

Archives of Biochemistry and Biophysics [ 0003-9861 ] ; 2000.

RBID : ISTEX:2D3D9D818C527204D94CC9FF1885D78DEDAD4702

English descriptors

Abstract

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 KMgATPM, Vmax, 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.

Url:

https://api.istex.fr/ark:/67375/6H6-4T8G8FFT-G/fulltext.pdf

DOI: 10.1006/abbi.2000.1712

Affiliations:

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<author><name sortKey="Nakamoto, Robert K" sort="Nakamoto, Robert K" uniqKey="Nakamoto R" first="Robert K." last="Nakamoto">Robert K. Nakamoto</name>
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<term>Atpase activities</term>
<term>Atpase activity</term>
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<term>Basal activity</term>
<term>Basal atpase activity</term>
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<term>Blot analysis</term>
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<term>Chemical chaperone</term>
<term>Chemical chaperone glycerol</term>
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<term>Lipid vesicles</term>
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<term>Open reading frame</term>
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<term>Promoter region</term>
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<term>Transport substrates</term>
<term>Transporter</term>
<term>Tris base</term>
<term>Unpublished results</term>
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<front><div type="abstract" xml:lang="en">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 KMgATPM, Vmax, 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.</div>
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<name sortKey="Omote, Hiroshi" sort="Omote, Hiroshi" uniqKey="Omote H" first="Hiroshi" last="Omote">Hiroshi Omote</name>
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Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein

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

Source :

English descriptors

Abstract

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Links to Exploration step

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