Characterization of an antibody Fv fragment that binds to the human, but not to the rat neurotensin receptor NTS-1.
The cDNAs coding for the heavy and light chain variable domains of an antibody, recognizing the human G-protein-coupled receptor for neurotensin, NTS-1, were obtained from a hybridoma cell line, B-N6. The Fv B-N6 fragment was expressed in Escherichia coli and purified. To characterize the properties of the antibody fragment, human and rat high-affinity neurotensin receptors were expressed in E. coli in functional form, linked at their N-termini to the maltose-binding protein. Fv B-N6 was found to compete for [3H]neurotensin binding to the human neurotensin receptor, but not to the rat neurotensin receptor, with IC50 values of 1.6 microM (membrane-bound receptor) and 1.9 microM (detergent-solubilized, purified receptor). The formation of a relatively stable complex of Fv B-N6 with purified human neurotensin receptor fusion protein was also demonstrated by gel filtration experiments. The Fv B-N6 fragment will be used to isolate a high-affinity binder to the human neurotensin receptor as a valuable tool for cocrystallization and receptor structure determination.
Grisshammer
Reinhard
R
MRC Laboratory of Molecular Biology, Cambridge, UK. rkgriss@helix.nih.gov
Grunwald
Thomas
T
Sohal
Awinder K
AK
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D018076:DNA, Complementary; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D051381:Rats; D018028:Receptors, Neurotensin / Q000276:immunology*; D013045:Species Specificity; D014316:Tritium
D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor; D014316:Tritium