Journal
NATURE PROTOCOLS
Volume 11, Issue 7, Pages 1149-1162Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.070
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Funding
- Biotechnology and Biosciences Research Council (BBSRC) [BB/J017310/1, BB/H016309/1, BB/I005579/1, BB/I020349/1, BB/K004441/1, BB/M018261/1]
- Wellcome Trust [091322/7/10/Z]
- MRC Career Development Award [G100567]
- BBSRC [BB/H024697/1, BB/M021610/1, BB/I020349/1, BB/K004441/1, BB/J010812/1, BB/I005579/1, BB/M018261/1, BB/H016309/1, BB/L015056/1, BB/J017310/1] Funding Source: UKRI
- MRC [G1000567] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/L015056/1, BB/H024697/1, BB/M021610/1, BB/J010812/1, BB/M018261/1, BB/I020349/1, BB/I005579/1, BB/J017310/1] Funding Source: researchfish
- Medical Research Council [G1000567] Funding Source: researchfish
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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 (similar to 2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.
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