title: Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF).
abstract: A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).
authors.affiliation: Pharmaceutical Sciences Division, King's College London, Franklin Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.
authors.forename: Irene
authors.initials: I
authors.lastname: Neophytou
authors.forename: Richard
authors.initials: R
authors.lastname: Harvey
authors.forename: Jayne
authors.initials: J
authors.lastname: Lawrence
authors.forename: Phil
authors.initials: P
authors.lastname: Marsh
authors.forename: Barry
authors.initials: B
authors.lastname: Panaretou
authors.forename: David
authors.initials: D
authors.lastname: Barlow
mesh_terms: D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D062506:Claudin-4; D003001:Cloning, Molecular; D004386:Duodenum / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism*; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D062793:Occludin; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*