Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF).
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).
Neophytou
Irene
I
Pharmaceutical Sciences Division, King's College London, Franklin Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.
Harvey
Richard
R
Lawrence
Jayne
J
Marsh
Phil
P
Panaretou
Barry
B
Barlow
David
D
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*