Abstract
Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.
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References
Gulati S, Jamshad M, Knowles TJ, Morrison KA, Downing R, Cant N, Collins R, Koenderink JB, Ford RC, Overduin M, Kerr ID, Dafforn TR, Rothnie AJ (2014) Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochem J 461:269–278
Knowles TJ, Finka R, Smith C, Lin YP, Dafforn T, Overduin M (2009) Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc 131:7484–7485
Jamshad M, Charlton J, Lin Y P, Routledge S J, Bawa Z, Knowles T J, Overduin M, Dekker N, Dafforn T R, Bill R M, Poyner D R, Wheatley M (2015) G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. Biosci Rep 35(2). pii: e00188
Swainsbury DJ, Scheidelaar S, van Grondelle R, Killian JA, Jones MR (2014) Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability. Angew Chem Int Ed Engl 53:11803–11807
Dorr JM, Koorengevel MC, Schafer M, Prokofyev AV, Scheidelaar S, van der Cruijsen EA, Dafforn TR, Baldus M, Killian JA (2014) Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs. Proc Natl Acad Sci U S A 111:18607–18612
Paulin S, Jamshad M, Dafforn TR, Garcia-Lara J, Foster SJ, Galley NF, Roper DI, Rosado H, Taylor PW (2014) Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a. Nanotechnology 25
Postis V, Rawson S, Mitchell JK, Lee SC, Parslow RA, Dafforn TR, Baldwin SA, Muench SP (2015) The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy. Biochim Biophys Acta 1848:496–501
Long AR, O’Brien CC, Malhotra K, Schwall CT, Albert AD, Watts A, Alder NN (2013) A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs. BMC Biotechnol 13:41
Orwick-Rydmark M, Lovett JE, Graziadei A, Lindholm L, Hicks MR, Watts A (2012) Detergent-free incorporation of a seven-transmembrane receptor protein into nanosized bilayer Lipodisq particles for functional and biophysical studies. Nano Lett 12:4687–4692
Jamshad M, Grimard V, Idini I, Knowles TJ, Dowle MR, Schofield N, Sridhar P, Lin YP, Finka R, Wheatley M, Thomas ORT, Palmer RE, Overduin M, Govaerts C, Ruysschaert JM, Edler KJ, Dafforn TR (2015) Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins. Nano Res 8:774–789
Sahu ID, McCarrick RM, Troxel KR, Zhang R, Smith HJ, Dunagan MM, Swartz MS, Rajan PV, Kroncke BM, Sanders CR, Lorigan GA (2013) DEER EPR measurements for membrane protein structures via bifunctional spin labels and lipodisq nanoparticles. Biochemistry 52:6627–6632
Scheidelaar S, Koorengevel MC, Pardo JD, Meeldijk JD, Breukink E, Killian JA (2015) Molecular model for the solubilization of membranes into nanodisks by styrene maleic Acid copolymers. Biophys J 108:279–290
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Rothnie, A.J. (2016). Detergent-Free Membrane Protein Purification. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 1432. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3637-3_16
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DOI: https://doi.org/10.1007/978-1-4939-3637-3_16
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3635-9
Online ISBN: 978-1-4939-3637-3
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