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

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

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91 Citations (Scopus)

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 utilises detergents to solubilise 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. We present here the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead using a styrene maleic acid co-polymer (SMA). SMA inserts in a bilayer and assembles into discrete particles, essentially solubilising the membrane into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). We show that this polymer can extract several eukaryotic ABC transporters; P-glycoprotein (ABCB1), MRP1 (ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (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 to those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared to detergent solubilisation. This 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.
Original languageEnglish
Pages (from-to)269-278
Number of pages10
JournalBiochemical Journal
Volume461
Issue number2
DOIs
Publication statusPublished - 15 Jul 2014

Keywords

  • Detergents
  • membrane protein
  • nanodisc
  • polymer
  • Purification
  • solubilisation

Cite this

Gulati, S., Jamshad, M., Knowles, T. J., Morrison, K., Downing, R., Cant, N., Collins, R., Koenderink, J. B., Ford, R. C., Overduin, M., Kerr, I. D., Dafforn, T. R., & 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