Recombinant Extracellular Domain of Rabbit Growth Hormone Receptor (rbGHR-ECD): Preparation and use for Comparing Binding Capacity and Biological Activity of Somatogenic Hormones

Abstract

The cDNA of the extracellular domain of rabbit growth hormone receptor (rbGHR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed rbPRLR-ECD protein, contained within the refractile-body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose 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 formation of 2:1 complexes was indicated in all cases. Binding experiments using radiolabelled oGH as a ligand revealed it to be the most effective competitor, 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 and Nb2 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 EC50 values (in nM) of 0.024, 0.023, 0.021, 0.24, 4.71 and 0.49. These results indicate remarkable discrepancies between the binding capacities and biological activities: the possible reasons for these findings are discussed.

Crystallization and preliminary X-ray diffraction analysis of the unliganded human growth hormone receptor

“…The cells were incubated for an additional 3 h at 293 K, harvested, PMSF was added to a ®nal concentration of 0.2 mM and the paste was stored frozen at 203 K overnight. In a modi®cation of the method of Sakal et al (2000), the inclusionbody pellet obtained from 2 l bacterial culture by French Press lysis and washing was solubilized in 400 ml 4.5 M urea buffered with 40 mM Tris base pH 10.4. l-Cysteine was added to 0.1 mM and the clear solution was stirred at 277 K for 48 h. This solution was then dialyzed for 48 h against six changes of 10 mM Tris±HCl pH 8.1 at 277 K. The solution was subsequently loaded at 120 ml h À1 onto a Q-Sepharose column (1.6 Â 20 cm) pre-equilibrated with 10 mM Tris±HCl pH 8.1 at 277 K. Elution was carried out with a continuous NaCl gradient (0±0.4 M) in the same buffer. Fractions eluting between 0.05 and 0.25 M NaCl were analyzed by 12.5% SDS±PAGE in the presence or absence of reducing agents and fractions judged to be greater than 95% purity were pooled.…”