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Recombinant Synthesis of Human ABCG2 Expressed in the Yeast Saccharomyces cerevisiae: an Experimental Methodological Study

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Abstract

Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS–PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.

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Abbreviations

MDR:

Multidrug resistance

ABC:

ATP-binding cassette

BCRP:

Breast cancer resistance protein

ABCP:

Placental ABC protein

MXR:

Mitoxantrone resistance protein

MRP:

Multidrug resistance associated protein

MM:

Minimal media

PMSF:

Phenylmethansulfonyl fluoride

DFP:

Diisopropyl fluorophosphate

PVDF:

Polyvinylidene fluoride

HRP:

Horseradish peroxidase

Ni-NTA:

Ni2+-nitrilotriacetate

CHAPSO:

3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate

DM:

n-decyl-β-D-maltopyranoside

DDM:

n-dodecyl-β-D-maltopyranoside

OG:

n-octyl-β-D-glucopyranoside

FC-12:

Fos-choline 12

FC-13:

Fos-choline 13

FC-14:

Fos-choline 14

FC-15:

Fos-choline 15

FC-16:

Fos-choline 16

PMA1:

Plasma membrane H+-ATPase

PDR:

Pleiotropic drug resistance

PGK:

Phosphoglycerate kinase

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Acknowledgments

We would like to thank Dr. Jens Meyer for his helpful discussions and suggestions during this work. We also thank Dieter Baumert for excellent technical assistance and Dr. Ilza Pajeva for thoroughly revising the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GRK804).

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany

    Anna Jacobs, Svenja Wieschrath & Michael Wiese

  2. Department of Chemistry and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA

    Dana Emmert & Christine A. Hrycyna

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  1. Anna Jacobs
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  2. Dana Emmert
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  3. Svenja Wieschrath
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  5. Michael Wiese
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Correspondence to Michael Wiese.

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Jacobs, A., Emmert, D., Wieschrath, S. et al. Recombinant Synthesis of Human ABCG2 Expressed in the Yeast Saccharomyces cerevisiae: an Experimental Methodological Study. Protein J 30, 201–211 (2011). https://doi.org/10.1007/s10930-011-9321-5

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