Detergent-free purification of ABC (ATP-binding-cassette) transporters.
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.
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
Sonali
S
Gulati
†School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Mohammed
M
Jamshad
‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Timothy J
TJ
Knowles
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
Kerrie A
KA
Morrison
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
Rebecca
R
Downing
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
Natasha
N
Cant
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
Richard
R
Collins
∥Department of Pharmacology and Toxicology 149, Radboud University Medical Centre, P.O. Box 9101, 65 HB Nijmegen, The Netherlands.
Jan B
JB
Koenderink
§Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.
Robert C
RC
Ford
‡School of Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Michael
M
Overduin
¶School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.
Ian D
ID
Kerr
†School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Timothy R
TR
Dafforn
*School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
Alice J
AJ
Rothnie
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000302:isolation & purification*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008298:Maleates / Q000737:chemistry*; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000302:isolation & purification*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011137:Polystyrenes / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008024:Ligands; D008298:Maleates; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D011137:Polystyrenes; D011994:Recombinant Proteins; C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; C421109:multidrug resistance-associated protein 1