Expression of a rat neurotensin receptor in Escherichia coli.
With the goal of obtaining sufficient quantities of seven-helix G-protein-coupled receptors for structural analysis, we have studied the functional expression of a rat neurotensin receptor cDNA in Escherichia coli with and without a signal sequence and as a fusion with the gene coding for maltose-binding protein. The addition of an N-terminal signal peptide resulted in increased expression levels. In vitro translation at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neurotensin receptor cDNA fused to the 3' end of the gene encoding maltose-binding protein resulted in a 40-fold increase in neurotensin-binding sites. Binding of [3H]neurotensin to intact bacteria or E. coli membranes was saturable, with a dissociation constant, KD, of 0.23 nM (Bmax. = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). The binding properties of all recombinant receptors presented in this study were similar and corresponded to those of the high-affinity binding sites in rat brain. For immunological detection and future purification of neurotensin receptor, a C-terminal pentahistidine/c-myc tail was introduced. Western-blot analysis revealed the association of neurotensin receptor with E. coli membranes.
Grisshammer
R
R
Cambridge Centre for Protein Engineering/MRC Centre, U.K.
Duckworth
R
R
Henderson
R
R
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D018076:DNA, Complementary; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007930:Leucine / Q000235:genetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D014176:Protein Biosynthesis; D051381:Rats; D018028:Receptors, Neurotensin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D007930:Leucine; D003545:Cysteine