title: Practical aspects in expression and purification of membrane proteins for structural analysis.
abstract: A surge of membrane protein structures in the last few years can be attributed to advances in technologies starting at the level of genomes, to highly efficient expression systems, stabilizing conformational flexibility, automation of crystallization and data collection for screening large numbers of crystals and the microfocus beam lines at synchrotrons. The substantial medical importance of many membrane proteins provides a strong incentive to understand them at the molecular level. It is becoming obvious that the major bottleneck in many of the membrane projects is obtaining sufficient amount of stable functional proteins in a detergent micelle for structural studies. Naturally, large effort has been spent on optimizing and advancing multiple expression systems and purification strategies that have started to yield sufficient protein and structures. We describe in this chapter protocols to refold membrane proteins from inclusion bodies, purification from inner membranes of Escherichia coli and from mammalian cell lines.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Kutti R
authors.initials: KR
authors.lastname: Vinothkumar
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.forename: Joerg
authors.initials: J
authors.lastname: Standfuss
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D018272:Porins / Q000235:genetics / Q000302:isolation & purification; D012243:Rhodopsin / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; C114347:OmpG protein, E coli; D018272:Porins; D012243:Rhodopsin; D010450:Endopeptidases