title: A method for detergent-free isolation of membrane proteins in their local lipid environment.
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.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Lee
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, UK.|Present address: Department of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Knowles
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.|Biomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett University, Leeds, UK.
authors.forename: Vincent L G
authors.initials: VL
authors.lastname: Postis
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Jamshad
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Parslow
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Yu-Pin
authors.initials: YP
authors.lastname: Lin
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.|Department of Biosciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland.
authors.forename: Adrian
authors.identifier: 0000-0001-8032-9700
authors.initials: A
authors.lastname: Goldman
authors.affiliation: School of Cancer Sciences, University of Birmingham, Birmingham, UK.|Present address: Department of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Pooja
authors.initials: P
authors.lastname: Sridhar
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.|Department of Biochemistry, Faculty of Medicine &Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Overduin
authors.affiliation: School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Stephen P
authors.identifier: 0000-0001-6869-4414
authors.initials: SP
authors.lastname: Muench
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Dafforn
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000302:isolation & purification*; D008298:Maleates / Q000737:chemistry*; D008563:Membrane Lipids / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008958:Models, Molecular; D011137:Polystyrenes / Q000737:chemistry*; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D008298:Maleates; D008563:Membrane Lipids; D008565:Membrane Proteins; D011137:Polystyrenes; C024074:styrene-maleic acid polymer