Recombinant extracellular domain of rabbit growth hormone receptor (rbGHR-ECD): Preparation and use for comparing binding capacity and biological activity of somatogenic hormones

The cDNA of the extracellular domain of rabbit growth hormone receptor (rbG HR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The ba cterially expressed rbPRLR-ECD protein, contained within the refractile-bod y pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Seph arose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 28-kDa fraction was eluted in 0.15 M NaCl, yielding 50 mg/2.5 l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Gel filtration was used to determine the stoichiometry of rbGHR-ECD 's interaction with human (h), ovine (o), chicken (ch) and common carp (cc) GHs and with bovine (b) and caprine (c) placental lactogens (PLs). The for mation of 2:1 complexes was indicated in all cases. Binding experiments usi ng radiolabelled oGH as a ligand revealed it to be the most effective compe titor, followed by bPL, cPL, hGH chGH and ccGH, with respective IC50 values of 0.27, 0.94, 1.55, 2.13, 41.9 and 51.2 nM. Rabbit GHR-ECD inhibited the bPL-inducible proliferation of FDC-P1 cells stably transfected with rbGHR a nd Nb, cells possessing rat PRLR. The biological activity of oGH, hGH, cPL, bPL, chGH and ccGH was tested in the FDC-P1 cells stably transfected with rbGHR and yielded the respective E C50 values (in nM) of 0.024, 0.023, 0.021, 0.24, 4.71 and 0.49. These resul ts indicate remarkable discrepancies between the binding capacities and bio logical activities: the possible reasons for these findings are discussed.