Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): Subcloning, preparation, and characterization

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression v ector pMON to enable its expression in Escherichia coli after induction wit h nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained w ithin 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 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneo us, as shown by SDS-PAGE in the presence or absence of reducing agent and b y chromatography on a Superdex column. Binding experiments using [I-125]ovi ne placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nhl. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Onl y oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes , with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or tr PRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its i nability to compete with [I-125]opL or with trPRL because of its low affini ty toward trPRLR-ECD. The present results agree with previous findings indi cating, as in mammals, that homologous PRL interacts transiently with, its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected. (C) 2000 Academic Press.