EXPRESSION OF THE EXTRACELLULAR DOMAIN OF THE HUMAN HEAT-STABLE ENTEROTOXIN RECEPTOR IN ESCHERICHIA-COLI AND GENERATION OF NEUTRALIZING ANTIBODIES

The entire extracellular domain of the human heat-stable enterotoxin ( ST) receptor as well as a truncated N-terminal domain were cloned as g lutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cyto sol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No bindi ng was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the en tire extracellular domain fusion protein as well as the truncated extr acellular domain fusion protein, and the antibodies were purified by a ffinity chromatography. Addition of the purified antibodies to T84 cel ls inhibited ST binding and abolished ST-mediated cGMP production, ind icating that critical epitopes involved in ligand interaction are pres ent in the N-terminal fragment of the receptor, Purified antibodies re cognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated rece ptor in T84 cells. These studies are the first report of the expressio n, purification, and characterization of any member of the guanylyl cy clase family of receptors in E. coli and show that binding of the toxi n to the extracellular domain of the receptor is possible in the absen ce of any posttranslational modifications such as glycosylation. The r ecombinant fusion proteins as well as the antibodies that we have gene rated could serve as useful tools in the identification of critical re sidues of the extracellular domain involved in ligand interaction. (C) 1996 Academic Press, Inc.