Large-scale preparation and characterization of recombinant ovine placental lactogen.
To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/ PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate beta-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.
Sakal
E
E
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Bignon
C
C
Grosclaude
J
J
Kantor
A
A
Shapira
R
R
Leibovitch
H
H
Helman
D
D
Nespoulous
C
C
Shamay
A
A
Rowlinson
S W
SW
Djiane
J
J
Gertler
A
A
D000818:Animals; D001681:Biological Assay; D002850:Chromatography, Gel; D004926:Escherichia coli; D005260:Female; D010928:Placental Lactogen / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D010928:Placental Lactogen