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A method for detergent-free isolation of membrane proteins in their local lipid environment


Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1–2 h); investigate SMALP protein purity by SDS–PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

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Figure 1: Styrene maleic acid (SMA) co-polymer production and SMALP formation from lipids.
Figure 2: Styrene maleic acid (SMA) co-polymer preparation.
Figure 3: Forming SMALP from membrane preparations using SMA.
Figure 4: Purification and biophysical characterization of SMALP protein.


  1. Filmore, D. It's a GPCR world. Mod. Drug Discov. 7, 24–28 (2004).

    CAS  Google Scholar 

  2. Arinaminpathy, Y., Khurana, E., Engelman, D.M. & Gerstein, M.B. Computational analysis of membrane proteins: the largest class of drug targets. Drug Discov. Today 14, 1130–1135 (2009).

    CAS  Article  Google Scholar 

  3. Seddon, A.M., Curnow, P. & Booth, P.J. Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophys. Acta 1666, 105–117 (2004).

    CAS  Article  Google Scholar 

  4. le Maire, M., Champeil, P. & Moller, J.V. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508, 86–111 (2000).

    CAS  Article  Google Scholar 

  5. Gohon, Y. & Popot, J.-L. Membrane protein–surfactant complexes. Curr. Opin. Colloid Interface Sci. 8, 15–22 (2003).

    CAS  Article  Google Scholar 

  6. Lin, S.-H. & Guidotti, G. Purification of membrane proteins. Methods Enzymol. 463, 619–629 (2009).

    CAS  Article  Google Scholar 

  7. Damianoglou, A. et al. The synergistic action of melittin and phospholipase A2 with lipid membranes: development of linear dichroism for membrane-insertion kinetics. Protein Pept. Lett. 17, 1351–1362 (2010).

    CAS  Article  Google Scholar 

  8. Charalambous, K., Miller, D., Curnow, P. & Booth, P.J. Lipid bilayer composition influences small multidrug transporters. BMC Biochem. 9, 31 (2008).

    Article  Google Scholar 

  9. Wiener, M.C. & White, S.H. Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of X-ray and neutron diffraction data. III. Complete structure. Biophys. J. 61, 434–447 (1992).

    CAS  Article  Google Scholar 

  10. Simons, K. & Sampaio, J.L. Membrane organization and lipid rafts. Cold Spring Harb. Perspect. Biol. 3, a004697 (2011).

    Article  Google Scholar 

  11. Kellosalo, J., Kajander, T., Honkanen, R. & Goldman, A. Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases. Mol. Membr. Biol. 30, 64–74 (2013).

    Article  Google Scholar 

  12. Breyton, C., Tribet, C., Olive, J., Dubacq, J.P. & Popot, J.L. Dimer to monomer conversion of the cytochrome b6 f complex. Causes and consequences. J. Biol. Chem. 272, 21892–21900 (1997).

    CAS  Article  Google Scholar 

  13. Esmann, M. Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study. Biochim. Biophys. Acta 857, 38–47 (1986).

    CAS  Article  Google Scholar 

  14. Popot, J.-L. Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. Annu. Rev. Biochem. 79, 737–775 (2010).

    CAS  Article  Google Scholar 

  15. Zoonens, M. & Popot, J.-L. Amphipols for each season. J. Membr. Biol. 247, 759–796 (2014).

    CAS  Article  Google Scholar 

  16. Debnath, D.K., Basaiawmoit, R.V., Nielsen, K.L. & Otzen, D.E. The role of membrane properties in Mistic folding and dimerisation. Protein Eng. Des. Sel. 24, 89–97 (2011).

    CAS  Article  Google Scholar 

  17. Denisov, I.G., Grinkova, Y.V., Lazarides, A.A. & Sligar, S.G. Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).

    CAS  Article  Google Scholar 

  18. Luthra, A., Gregory, M., Grinkova, Y.V., Denisov, I.G. & Sligar, S.G. Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes. Methods Mol. Biol. 987, 115–127 (2013).

    CAS  Article  Google Scholar 

  19. Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S.G. & Duong, F. Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J. 26, 1995–2004 (2007).

    CAS  Article  Google Scholar 

  20. Leitz, A.J., Bayburt, T.H., Barnakov, A.N., Springer, B.A. & Sligar, S.G. Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling nanodisc technology. Biotechniques 40 601–2, 604, 606, passim (2006).

  21. Knowles, T.J. et al. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J. Am. Chem. Soc. 131, 7484–7485 (2009).

    CAS  Article  Google Scholar 

  22. Jamshad, M. et al. G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. Biosci. Rep. 35 (2015).

  23. Tonge, S.R. & Tighe, B.J. Responsive hydrophobically associating polymers: a review of structure and properties. Adv. Drug Deliv. Rev. 53, 109–122 (2001).

    CAS  Article  Google Scholar 

  24. Tonge, S., Stephen, T., Vincent, R. & Tighe, B.J. Dynamic surface activity of biological fluids and ophthalmic solutions. Cornea 19, S133 (2000).

    Article  Google Scholar 

  25. Jamshad, M. et al. Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins. Nano Res. 8, 774–789 (2014).

    Article  Google Scholar 

  26. Scheidelaar, S. et al. Molecular model for the solubilization of membranes into nanodisks by styrene maleic acid copolymers. Biophys. J. 108, 279–290 (2015).

    CAS  Article  Google Scholar 

  27. Postis, V. et al. The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy. Biochim. Biophys. Acta 1848, 496–501 (2015).

    CAS  Article  Google Scholar 

  28. Gulati, S. et al. Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochem. J 461, 269–278 (2014).

    CAS  Article  Google Scholar 

  29. Dörr, J.M. et al. Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs. Proc. Natl. Acad. Sci. USA 111, 18607–18612 (2014).

    Article  Google Scholar 

  30. Paulin, S. et al. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a. Nanotechnology 25, 285101 (2014).

    Article  Google Scholar 

  31. Orwick-Rydmark, M. et al. Detergent-free incorporation of a seven-transmembrane receptor protein into nanosized bilayer Lipodisq particles for functional and biophysical studies. Nano Lett. 12, 4687–4692 (2012).

    CAS  Article  Google Scholar 

  32. Long, A.R. et al. A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs. BMC Biotechnol. (2013).

  33. Gomori, G., Colowick, S.P. & Kaplan, N.O. in Methods in Enzymology 138–148 (Academic Press, 1955).

    Book  Google Scholar 

  34. Lin, Y. Over-expression and Biophysical Characterisation of Membrane Proteins Solubilised in a Styrene Maleic Acid Polymer (2011).

  35. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    CAS  Article  Google Scholar 

  36. Fotiadis, D., Harder, D. & Fotiadis, D. Preparation of detergent-solubilized membranes from Escherichia coli. Protoc. Exchange (2012).

  37. Panaretou, B. & Piper, P. in Yeast Protocols (ed. Evans, I.H.) 117–121 (Humana Press).

  38. Jamshad, M. et al. G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. Biosci. Rep. 35, 1–10 (2015).

    CAS  Article  Google Scholar 

  39. Scott, R.E. Plasma membrane vesiculation: a new technique for isolation of plasma membranes. Science 194, 743–745 (1976).

    CAS  Article  Google Scholar 

  40. Del Piccolo, N., Placone, J., He, L., Agudelo, S.C. & Hristova, K. Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions. Anal. Chem. 84, 8650–8655 (2012).

    CAS  Article  Google Scholar 

  41. Cohen, S., Ushiro, H., Stoscheck, C. & Chinkers, M. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J. Biol. Chem. 257, 1523–1531 (1982).

    CAS  PubMed  Google Scholar 

  42. Gulati, S. et al. Detergent free purification of ABC transporters. Biochem. J 44, 1–24 (2014).

    Google Scholar 

  43. Nordén, B., Rodger, A. & Dafforn, T. Linear Dichroism and Circular Dichroism: A Textbook on Polarized-light Spectroscopy (Royal Society of Chemistry, 2010).

  44. Burgess, S.A., Walker, M.L., Sakakibara, H., Oiwa, K. & Knight, P.J. The structure of dynein-c by negative stain electron microscopy. J. Struct. Biol. 146, 205–216 (2004).

    CAS  Article  Google Scholar 

  45. Booth, D.S., Avila-Sakar, A. & Cheng, Y. Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J. Vis. Exp. 58, 3227 (2011).

    Google Scholar 

  46. Grassucci, R.A., Taylor, D.J. & Frank, J. Preparation of macromolecular complexes for cryo-electron microscopy. Nat. Protoc. 2, 3239–3246 (2007).

    CAS  Article  Google Scholar 

  47. Grassucci, R.A., Taylor, D. & Frank, J. Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat. Protoc. 3, 330–339 (2008).

    CAS  Article  Google Scholar 

  48. Scheres, S.H.W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  49. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    CAS  Article  Google Scholar 

  50. Rico, A.I., Krupka, M. & Vicente, M. In the beginning, Escherichia coli assembled the proto-ring: an initial phase of division. J. Biol. Chem. 288, 20830–20836 (2013).

    CAS  Article  Google Scholar 

  51. Hale, C.A., Rhee, A.C. & De Boer, P.A.J. ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains. J. Bacteriol. 182, 5153–5166 (2000).

    CAS  Article  Google Scholar 

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This work was supported by the Biotechnology and Biosciences Research Council (BBSRC; grants BB/J017310/1, BB/H016309/1, BB/I005579/1, BB/I020349/1, BB/K004441/1 and BB/M018261/1 to T.R.D.) and the Wellcome Trust (ref. 091322/7/10/Z to V.L.G.P.). S.P.M. is supported by an MRC Career Development Award (G100567). We dedicate this work to the late Professor Stephen A. Baldwin.

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S.C.L., V.L.G.P., T.J.K., R.A.P., M.J., A.G., S.P.M., M.O. and T.R.D. designed the research. T.J.K., T.R.D. and M.O. devised the SMALP method. S.C.L., V.L.G.P., Y.L., M.J., R.A.P., T.J.K. and P.S. optimized the detailed protocol. S.C.L., V.L.G.P., T.J.K., A.G., R.A.P., S.P.M. and T.R.D. wrote the paper.

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Correspondence to Timothy R Dafforn.

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Lee, S., Knowles, T., Postis, V. et al. A method for detergent-free isolation of membrane proteins in their local lipid environment. Nat Protoc 11, 1149–1162 (2016).

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