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).
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, Itoh M, Yonemura S, Furuse M, Tsukita S (1996) Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues. J Cell Biol 133:43–47
Ashen MD, O’Rourke B, Kluge KA, Johns DC, Tomaselli GF (1997) Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line. Am J Physiol 268:506–511
Bibi E, Gros P, Kaback HR (1993) Functional expression of mouse mdr1 in Escherichia coli. Proc Natl Acad Sci U S A 90:9209–9213
Doyle DA, Cabral JM, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, Mackinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77
Drew DE, Heijne G, Nordlund P, de Gier JW (2001) Green fluorescent protein as an indicator to monitor membrane protein over-expression in Escherichia coli. FEBS Lett 507:220–224
Erni B, Zanolari B (1986) Glucose-permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc. J Biol Chem 261:16398–16403
Fiermonte G, Palmieri L, Todisco S, Agrimi G, Palmieri F, Walker JE (2002) Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J Biol Chem 272:19289–19294
Fu D, Libson A, Miercke LJW, Weitzman C, Nollert P, Krucinski J, Stroud RM (2000) Structure of a glycerol conducting channel and the basis for its selectivity. Science 290:481–486
Guan C, Li P, Riggs PD, Inouye H (1988) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67:21–30
Hofmann K, Stoffel W (1993) Tmbase—A database of membrane spanning protein segments. Biol Chem Hoppe-Seyler 374:166
Jung Y, Kwak J, Lee Y (2001) High-level production of heme-containing holoproteins in Escherichia coli. Appl Microbiol Biotechnol 55:187–191
Katahira J, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N (1997) Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol 136:1239–1247
Kobe B, Center RJ, Kemp BE, Poumbourios P (1999) Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. Proc Natl Acad Sci U S A 96:4319–4324
Laage R, Langosch D (2001) Strategies for prokaryotic expression of eukaryotic membrane proteins. Traffic 2:99–104
Liu Y, Manna A, li R, Martin WE, Murphy RC, Cheung AL, Zhang G (2001) Crystal structure of the SarR protein from Staphylococcus aureus. Proc Natl Acad Sci U S A 98:6877–6882
Luirink J, Heijne G, Houben E, de Gier JW (2005) Biogenesis of inner membrane proteins in Escherichia coli. Annu Rev Microbiol 59:329–355
Manley DM, McComb ME, Perreault H, Donald LJ, Duckworth HW, O’Neil JD (2000) Secondary structure and oligomerization of the E. coli glycerol facilitator. Biochem 39:12303–12311
McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C, Barlow D, Bomford A, Peters TJ, Raja KB, Shirali S, Hediger MA, Farzaneh F, Simpson RJ (2001) An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291:1755–1759
Menke JG, Borkowski JA, Bierilo KK, MacNeil T, Derrick AW, Schneck KA, Ransom RW, Strader CD, Linemeyer DL, Hess JF (1994) Expression cloning of a human B1 bradykinin receptor. J Biol Chem 269:21583–21586
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298
Nakamuta M, Takayanagi R, Sakai Y, Sakamoto S, Hagiwara H, Mizuno T, Saito Y, Hirose S, Yamamoto M, Nawata H (1991) Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor. Biochem Biophys Res Commun 177:34–39
Niemann HH, Schmoldt HU, Wentzel A, Kolmar H, Heinz DW (2006) Barnase fusion as a tool to determine the crystal structure of the small disulfide rich protein McoEeTI. J Mol Biol 356:1–8
Quick M, Wright EM (2002) Employing Escherichia coli to functionally express, purify, and characterize a human transporter. Proc Natl Acad Sci U S A 99:8597–8601
Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S (2005) NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307:1317–1321
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Smith DB, Johnson KS (1988) Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40
Tate CG (2001) Over-expression of mammalian integral membrane proteins for structural studies. FEBS Lett 504:94–98
Tjaden J, Schwoppe C, Mohlmann T, Quick P, Neuhaus H (1998) Expression of a plastidic ATP/ADP transporter gene in Escherichia coli leads to a functional adenine nucleotide transport system in the bacterial cytoplasmic membrane. J Biol Chem 273:9630–9636
Venter H, Ashcroft AE, Keen JN, Henderson PJ, Herbert RB (2002) Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose–H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of ring-2-13C histidine and 15NH(3). Biochem J 363:243–252
Wilson ML, Macnab RM (1988) Overproduction of the MotA protein of Escherichia coli and estimation of its wild-type level. J Bacteriol 170:588–597
This work was supported by an award from the Heptagon Fund. Special thanks go to Prof. Robert Hider and Drs Tom Seddon, Sophie Khanna, Ian Hill, Jon Oakhill and Colin Dolphin.
About this article
Cite this article
Neophytou, I., Harvey, R., Lawrence, J. et al. Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF). Appl Microbiol Biotechnol 77, 375–381 (2007). https://doi.org/10.1007/s00253-007-1174-7
- Recombinant protein
- Membrane protein
- Gene expression
- Protein engineering
- Escherichia coli