Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity |
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| Authors: | Haim Leibovich Nina Raver Asael Herman Ewa L Gregoraszczuk Elisha Gootwine Arieh Gertler |
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| Institution: | a Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food, and Environmental Quality Sciences, Hebrew University, Rehovot, 76100, Israel;b Laboratory of Reproductive Physiology and Toxicology for Domestic Animals, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, Krakow, Poland;c Institute of Animal Science, ARO, Volcani Center, Bet Dagan, Israel |
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| Abstract: | Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb2 cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes. |
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| Keywords: | recombinant ovine prolactin bioactivity luteotropic effect galactopoiesis |
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