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Detergent-free purification of ABC (ATP-binding-cassette) transporters

Gulati, Sonali; Jamshad, Mohammed; Knowles, Timothy J.; Morrison, Kerrie A.; Downing, Rebecca; Cant, Natasha; Collins, Richard; Koenderink, Jan B.; Ford, Robert C.; Overduin, Michael; Kerr, Ian D.; Dafforn, Timothy R.; Rothnie, Alice J.

Authors

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 KERR ian.kerr@nottingham.ac.uk
Associate Professor

Timothy R. Dafforn

Alice J. Rothnie



Abstract

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 awide range of possibilities for the future study of their structure and function. © The Authors Journal compilation © 2014 Biochemical Society.

Citation

Gulati, S., Jamshad, M., Knowles, T. J., Morrison, K. A., Downing, R., Cant, N., …Rothnie, A. J. (2014). Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochemical Journal, 461(2), 269-278. https://doi.org/10.1042/BJ20131477

Journal Article Type Article
Acceptance Date Apr 23, 2014
Online Publication Date Jun 26, 2014
Publication Date Jul 15, 2014
Deposit Date Feb 12, 2020
Journal Biochemical Journal
Print ISSN 0264-6021
Electronic ISSN 1470-8728
Publisher Portland Press
Peer Reviewed Peer Reviewed
Volume 461
Issue 2
Pages 269-278
DOI https://doi.org/10.1042/BJ20131477
Keywords Cell Biology; Biochemistry; Molecular Biology
Public URL https://nottingham-repository.worktribe.com/output/3088496