Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen.
Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta. The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector. E. coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa. Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL. Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected. The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.
Sakal
E
E
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Bignon
C
C
Chapnik-Cohen
N
N
Daniel
N
N
Paly
J
J
Belair
L
L
Djiane
J
J
Gertler
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001681:Biological Assay; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006041:Goats; D006801:Humans; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry*; D010928:Placental Lactogen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012756:Sheep
D011994:Recombinant Proteins; D010928:Placental Lactogen