Lee, SC, Knowles, TJ, Postis, VLG orcid.org/0000-0001-9251-4567 et al. (8 more authors) (2016) A method for detergent-free isolation of membrane proteins in their local lipid environment. Nature Protocols, 11 (7). pp. 1149-1162. ISSN 1754-2189
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2016, Author(s). This is an author produced version of a paper published in Nature Protocols. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | Styrene Maleic Acid, SMA, SMALPs, Membrane proteins, Detergent free, Protein |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 091322/Z/10/Z MRC G1000567/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 11 Oct 2016 12:44 |
Last Modified: | 05 Dec 2016 20:42 |
Published Version: | http://dx.doi.org/10.1038/nprot.2016.070 |
Status: | Published |
Publisher: | Nature Publishing Group |
Identification Number: | 10.1038/nprot.2016.070 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:100008 |