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.
Bibliographical noteThis research was originally published in Biochemical journal. Gulati, S., Jamshad, M., Knowles, T. J., Morrison, K. A., Downing, R., Cant, N., Collins, R., Koenderink, J. B., Ford, R. C., Overduin, M., Kerr, I. D., Dafforn, T. R., & Rothnie, A. J., Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochemical journal. 2014; 421: 269-278. © the Biochemical Society.
A.J.R. was the recipient of a Royal Society Research Grant [grant number RG110156], an ARCHA (Aston Research Centre for Health Aging) pump-priming grant and a Biochemical Society Guildford Bench Fund. T.R.D. and M.O. also received support from the Biotechnology and Biological Sciences Research Council [grant numbers BB/J017310/1, BB/I020349/1, BB/G010412/1, BB/J010812/1 and FoF/295].
- membrane protein