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

Sarah C. Lee, Tim J. Knowles, Vincent L G Postis, Mohammed Jamshad, Rosemary A Parslow, Yu-pin Lin, Adrian Goldman, Pooja Sridhar, Michael Overduin, Stephen P. Muench, Timothy R Dafforn (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

193 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)1149-1162
Number of pages14
JournalNature Protocols
Volume11
DOIs
Publication statusPublished - 2 Jun 2016

Fingerprint

Dive into the research topics of 'A method for detergent-free isolation of membrane proteins in their local lipid environment.'. Together they form a unique fingerprint.

Cite this