TY - JOUR
T1 - Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen
AU - Sakal, E.
AU - Bignon, C.
AU - Chapnik-Cohen, N.
AU - Daniel, N.
AU - Paly, J.
AU - Belair, L.
AU - Djiane, J.
AU - Gertler, A.
PY - 1998
Y1 - 1998
N2 - 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-β-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 ~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 β-casein synthesis in rabbit and ovine mammary gland acini cultures.
AB - 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-β-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 ~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 β-casein synthesis in rabbit and ovine mammary gland acini cultures.
UR - http://www.scopus.com/inward/record.url?scp=0032441098&partnerID=8YFLogxK
U2 - 10.1677/joe.0.1590509
DO - 10.1677/joe.0.1590509
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C2 - 9834468
AN - SCOPUS:0032441098
SN - 0022-0795
VL - 159
SP - 509
EP - 518
JO - Journal of Endocrinology
JF - Journal of Endocrinology
IS - 3
ER -