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

Nature Protocols volume 11, pages 1149–1162 (2016)Cite this article

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Abstract

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.

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Acknowledgements

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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Author notes

  1. Tim J Knowles & Pooja Sridhar

    Present address: Present address: Department of Biosciences, University of Birmingham, Birmingham, UK.,

  2. Sarah C Lee, Tim J Knowles and Vincent L G Postis: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biosciences, University of Birmingham, Birmingham, UK

    Sarah C Lee, Mohammed Jamshad, Rosemary A Parslow, Yu-pin Lin, Michael Overduin & Timothy R Dafforn

  2. School of Cancer Sciences, University of Birmingham, Birmingham, UK

    Tim J Knowles & Pooja Sridhar

  3. School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK

    Vincent L G Postis, Adrian Goldman & Stephen P Muench

  4. Biomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett University, Leeds, UK

    Vincent L G Postis

  5. Department of Biosciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland

    Adrian Goldman

  6. Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada

    Michael Overduin

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Contributions

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). https://doi.org/10.1038/nprot.2016.070

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