did anybody try E.coli cell expression for GPCR?

I am trying E. coli system to express human whole GPCR, haven't got positive result. Anybody succeeded? Thanks.

Two examples of human GPCRs expressed in E Coli.

Tian C, Breyer RM, Kim HJ, Karra MD, Friedman DB, Karpay A, Sanders CR.
Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor.
J Am Chem Soc. 2005 Jun 8;127(22):8010-1


Baneres JL, Mesnier D, Martin A, Joubert L, Dumuis A, Bockaert J.
Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy.
J Biol Chem. 2005 May 27;280(21):20253-60. Epub 2005 Mar 17.

One of the better characterized GPCRs expressed in E coli is NTS1 from Grisshammer

http://intramural.niddk.nih.gov/research/faculty.asp?People_ID=1555

It actively binds ligand and presubambly reasonably well folded.

Then there's the mistic stuff:

NMR Structure of Mistic, a Membrane-Integrating Protein for Membrane Protein Expression
Tarmo P. Roosild, Jason Greenwald, Mark Vega, Samantha Castronovo, Roland Riek,* Senyon Choe*
Science, 2005:
Vol. 307. no. 5713, pp. 1317 - 1321

They show GPCR class B expression in E.coli.

TonyW said:

One of the better characterized GPCRs expressed in E coli is NTS1 from Grisshammer http://intramural.niddk.nih.gov/research/faculty.asp?People_ID=1555 It actively binds ligand and presubambly reasonably well folded.

This review may probably help you:

Heterologous expression of G-protein-coupled receptors:
comparison of expression systems from the standpoint
of large-scale production and purification
V. Sarramegna, F.Talmont, P. Demange and A. Milon

CMLS, Cell. Mol. Life Sci. 60 (2003) 15291546

In the paper below, published in the Journal of Molecular Pharmacology, January 2001, the method of expression in E.coli has been desribed:

http://molpharm.aspetjournals.org/cgi/content/abstract/59/1/30

SCH-202676: An Allosteric Modulator of Both Agonist and Antagonist Binding to G Protein-Coupled Receptors Ahmad B. Fawzi, Douglas Macdonald,1 Lawrence L. Benbow, April Smith-Torhan, Hongtao Zhang, Blair C. Weig, Ginny Ho, Deen Tulshian, Maurine E. Linder, and Michael P. Graziano

Methods

Expression of Human 2-Adrenergic Receptor in DH5 Bacterial Cells. The 2-adrenergic receptor was expressed in Escherichia coli by a modification of methods described previously by Marullo et al. (1988) and Freissmuth et al. (1991). The human 2-adrenergic receptor (cDNA obtained from Dr. R. Lefkowitz, GenBank accession number 4501968) was amplified using polymerase chain reaction primers designed to incorporate an EcoRI site at the 5'-end, and SalI at the 3'-end (upper primer: 5'-CTTGAATTCGGGCAACCCGGGAACGG-3', lower primer: 5'-TCTGTCGACTTACAGCAGTGAGTCATT-3', respectively). After digestion with the corresponding restriction enzymes, the polymerase chain reaction product was ligated into a pFLAG-1 vector (Eastman Kodak Co., Rochester, NY). The nucleotide sequence of the pFLAG-1 2-adrenergic receptor cDNA was verified using the dRhodamine Terminator Cycle Sequencing Reaction system (PE Biosystems, Foster City, CA) and analyzed on an ABI PRISM 377 automated DNA sequencer (PE/ABI, Foster City, CA). Transformation of the purified plasmid into E. coli strain DH5 was performed using the standard commercial protocol provided with the DH5 competent cells (Life Technologies, Gaithersburg, MD). The pFLAG/2-adrenergic receptor-positive DH5 transformants were cultured at 37°C in ampicillin-containing (100 g/ml) Luria broth culture to an optical density of 500 (= 600 nm) at which point 0.5 mM isopropylthio--D-galactoside was added. After additional incubation for 2.5 h at 23°C, membranes were isolated as described previously (Stanasila et al., 1999). Membrane pellets were resuspended in 1 ml of cold 50 mM Tris-HCl, pH 7.4, containing 10% glycerol and 1% BSA. Aliquots were frozen in liquid nitrogen and stored at 80°C. Protein determinations were made before the BSA addition using the micro bicinchoninic acid assay (BCA; Pierce, Rockford, IL). Competition binding of [125I]iodocyanopindolol to 50 g of pFLAG-1/2-adrenergic receptor DH5 membranes in 500 l of buffer containing 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA, and protease inhibitors (Complete+, EDTA; Boehringer Mannheim) was performed as described before (Perkin Elmer Life Sciences, Norwalk, CT).