Characterization of an antibody Fv fragment that binds to the human, but not to the rat neurotensin receptor NTS-1

Protein Expression and Purification
R GrisshammerAwinder K Sohal

Abstract

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.

References

Mar 11, 1986·Nucleic Acids Research·P H SchreierA Bothwell
Dec 1, 1973·Analytical Biochemistry·W Schaffner, C Weissmann
Aug 1, 1995·Quarterly Reviews of Biophysics·R Grisshammer, C G Tate
Nov 1, 1993·Mayo Clinic Proceedings·M WatsonE Richelson
Oct 15, 1993·The Biochemical Journal·R GrisshammerR Henderson
Jun 21, 1996·The Journal of Biological Chemistry·Y P PangE Richelson
Aug 1, 1996·The Biochemical Journal·J Tucker, R Grisshammer
Nov 5, 1997·Protein Expression and Purification·R Grisshammer, J Tucker
Jun 1, 2000·Biochemical Society Transactions·R GrisshammerA K Sohal
Aug 5, 2000·Science·K PalczewskiM Miyano

❮ Previous
Next ❯

Citations

Sep 10, 2003·Proceedings of the National Academy of Sciences of the United States of America·Sorin LucaMarc Baldus
Aug 8, 2007·Biomolecular Engineering·Yan YangZhiren Zhang
Mar 26, 2005·Protein Engineering, Design & Selection : PEDS·David OttAndreas Plückthun

❮ Previous
Next ❯

Related Concepts

Related Feeds

Antibody Specificity

Antibodies produced by B cells are highly specific for antigen as a result of random gene recombination and somatic hypermutation and affinity maturation. As the main effector of the humoral immune system, antibodies can neutralize foreign cells. Find the latest research on antibody specificity here.