Preparation of the extracellular domain of the rabbit prolactin receptor expressed in Escherichia coli and its interaction with lactogenic hormones
| Autor: | C, Bignon, E, Sakal, L, Belair, N, Chapnik-Cohen, J, Djiane, A, Gertler |
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| Rok vydání: | 1994 |
| Předmět: |
Protein Folding
Receptors Prolactin Swine Molecular Sequence Data Gene Expression Transfection Binding Competitive Polymerase Chain Reaction Mammary Glands Animal Organ Culture Techniques Escherichia coli Animals Humans Amino Acid Sequence Cloning Molecular Pseudopregnancy DNA Primers Binding Sites Base Sequence Caseins Peptide Fragments Recombinant Proteins Prolactin Molecular Weight Kinetics Culture Media Conditioned Growth Hormone Chromatography Gel Electrophoresis Polyacrylamide Gel Female Rabbits |
| Zdroj: | The Journal of biological chemistry. 269(5) |
| ISSN: | 0021-9258 |
| Popis: | The cDNA of the extracellular domain of the rabbit prolactin receptor (rbPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A to enable its expression in Escherichia coli after induction with isopropyl-1-thio-beta-D-galactopyranoside. 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 by stepwise elution with NaCl. The bioactive monomeric fraction was eluted in 0.05 M NaCl, yielding 15-20 mg/8 liters of induced culture. The purified protein was98% homogeneous, as shown by SDS-polyacrylamide gel electrophoresis in the presence or absence of reducing agent and by chromatography on a Superdex column. Its molecular mass was 25 kDa as determined by SDS-polyacrylamide gel electrophoresis in the absence of reducing agent and 22 kDa as determined by gel filtration. Binding experiments revealed remarkable differences between rabbit and porcine prolactins (PRLs) and the other tested lactogenic hormones. Gel filtration was used to determine the stoichiometry of the rbPRLR-ECD interaction with ovine, rabbit, and porcine PRLs, with human growth hormone and its truncated des-7 analogue, and with bovine placental lactogen (bPL) and des-13-bPL. The formation of only 1:1 complexes was indicated, except with bPL, for which a 2:1 complex was detected. Identical stoichiometry was also obtained using excess radiolabeled rbPRLR-ECD in gel filtration experiments. Interaction of 125I-labeled ovine PRL with rbPRLR-ECD secreted into conditioned medium by rbPRLR-ECD cDNA-transfected COS 7 cells also indicated formation of 1:1 molar complexes. Despite the differences in binding potency and stoichiometries of the interaction with rbPRLR-ECD, all seven tested hormones were biologically active in inducing PRL receptor-mediated casein synthesis in explants of rabbit mammary gland. We therefore propose that the formation of the 1:2 complexes with soluble rbPRLR-ECD is not predictive of biological activity of the different lactogenic hormones. |
| Databáze: | OpenAIRE |
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