Preparation and characterization of recombinant prolactin receptor extracellular domain from rat

Complementary (c)DNA of the extracellular domain of rat prolactin receptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A, and expressed in Escherichia coli following induction with isopropyl-b-D-thiogalactopyranoside. The expressed rPRLR-ECD protein, contained within the refractile body pellet was solubilized in 4.5 M urea, refolded and purified on a Q-Sepharose column by stepwise elution with NaCl. Only approximately 10% of the expressed protein refolded as a monomeric fraction, yielding 5-6 mg/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. Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa. Binding experiments indicated high affinity for bovine placental lactogen (bPL) and human growth hormone (hGH) as compared to ovine (o) or rat PRLs. Gel filtration was used to determine the stoichiometry of rPRLR-ECD's interaction with these hormones. At a 5 microM initial concentration of the hormones, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hGH and oPRL whereas only 1:1 complex was formed with rPRL. Dilution (25-fold) of these complexes did not affect the stoichiometry with bPL, whereas with hGH a clear tendency towards dissociation of the initial 2:1 complex to 1:1 complex was observed. This tendency was even stronger in the case of oPRL. Although all four hormones exhibited nearly identical activities in the Nb2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic activity generally reflected their affinity for the respective hormones. In view of these and former results, we suggest that unlike in the GH:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily predicts lack of biological activity.