ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.
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Research Article|
June 26 2014
Detergent-free purification of ABC (ATP-binding-cassette) transporters
Sonali Gulati;
Sonali Gulati
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
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Mohammed Jamshad;
Mohammed Jamshad
†School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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Timothy J. Knowles;
Timothy J. Knowles
‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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Kerrie A. Morrison;
Kerrie A. Morrison
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
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Rebecca Downing;
Rebecca Downing
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
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Natasha Cant;
Natasha Cant
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
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Richard Collins;
Richard Collins
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
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Jan B. Koenderink;
Jan B. Koenderink
∥Department of Pharmacology and Toxicology 149, Radboud University Medical Centre, P.O. Box 9101, 65 HB Nijmegen, The Netherlands
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Robert C. Ford;
Robert C. Ford
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
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Michael Overduin;
Michael Overduin
‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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Ian D. Kerr;
Ian D. Kerr
¶School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.
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Timothy R. Dafforn;
Timothy R. Dafforn
†School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
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Alice J. Rothnie
Alice J. Rothnie
1
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
1To whom correspondence should be addressed (email a.rothnie@aston.ac.uk).
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Publisher: Portland Press Ltd
Received:
November 13 2013
Revision Received:
April 07 2014
Accepted:
April 23 2014
Accepted Manuscript online:
April 23 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 461 (2): 269–278.
Article history
Received:
November 13 2013
Revision Received:
April 07 2014
Accepted:
April 23 2014
Accepted Manuscript online:
April 23 2014
Citation
Sonali Gulati, Mohammed Jamshad, Timothy J. Knowles, Kerrie A. Morrison, Rebecca Downing, Natasha Cant, Richard Collins, Jan B. Koenderink, Robert C. Ford, Michael Overduin, Ian D. Kerr, Timothy R. Dafforn, Alice J. Rothnie; Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochem J 15 July 2014; 461 (2): 269–278. doi: https://doi.org/10.1042/BJ20131477
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