Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity.
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 approximately 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 Nb(2) 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.
Leibovich
H
H
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Raver
N
N
Herman
A
A
Gregoraszczuk
E L
EL
Gootwine
E
E
Gertler
A
A
D000818:Animals; D001681:Biological Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D013006:Growth Hormone / Q000235:genetics / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008892:Milk / Q000737:chemistry; D016296:Mutagenesis; D009268:Nalidixic Acid / Q000494:pharmacology; D010957:Plasmids; D011247:Pregnancy; D011374:Progesterone / Q000378:metabolism; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D009268:Nalidixic Acid; D011374:Progesterone; D011388:Prolactin; D013006:Growth Hormone; D008156:Luciferases