title: Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization.
abstract: The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-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 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% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine 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 nM. 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. Only 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 trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, 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.
authors.affiliation: Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel.
authors.forename: Y
authors.initials: Y
authors.lastname: Sandowski
authors.forename: Y
authors.initials: Y
authors.lastname: Cohen
authors.forename: P
authors.initials: P
authors.lastname: Le Rouzic
authors.forename: C
authors.initials: C
authors.lastname: Bignon
authors.forename: F
authors.initials: F
authors.lastname: Rentier-Delrue
authors.forename: J
authors.initials: J
authors.lastname: Djiane
authors.forename: P
authors.initials: P
authors.lastname: Prunet
authors.forename: A
authors.initials: A
authors.lastname: Gertler
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002645:Chickens; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019382:Human Growth Hormone / Q000378:metabolism; D007457:Iodine Radioisotopes; D017686:Oncorhynchus mykiss / Q000235:genetics*; D010928:Placental Lactogen / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011981:Receptors, Prolactin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins; D012756:Sheep; D014162:Transfection
chemical_list: D007457:Iodine Radioisotopes; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D011388:Prolactin; D010928:Placental Lactogen