EXPRESSION OF A RAT NEUROTENSIN RECEPTOR IN ESCHERICHIA-COLI

With the goal of obtaining sufficient quantities of seven-helix G-prot ein-coupled receptors for structural analysis, we have studied the fun ctional expression of a rat neurotensin receptor cDNA in Escherichia c oli with and without a signal sequence and as a fusion with the gene c oding for maltose-binding protein. The addition of an N-terminal signa l peptide resulted in increased expression levels. In vitro translatio n at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neur otensin 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 [H-3]neurotensin to intact bacteria or E. coli memb ranes was saturable, with a dissociation constant, K(D, of 0.23 nM (B( max) = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). T he binding properties of all recombinant receptors presented in this s tudy were similar and corresponded to those of the high-affinity bindi ng sites in rat brain. For immunological detection and future purifica tion of neurotensin receptor, a C-terminal pentahistidine/c-myc tail w as introduced. Western-blot analysis revealed the association of neuro tensin receptor with E. coli membranes.