LARGE-SCALE PREPARATION AND CHARACTERIZATION OF RECOMBINANT OVINE PLACENTAL-LACTOGEN

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep pla cental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, Nco I and PstI sites. The oPL cDNA fragment amplified between these two pr imers extended from A(-1) to the natural stop codon. The PCR product w as gel-purified and subcloned into a Puc vector and the insert was seq uenced 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 polym orphism, which has been described in detail for PLs of different speci es. The insert was subcloned into NcoI/PstI-digested pTrc99A procaryot ic 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 e xpression 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 homoge neity. Structural analysis using circular dichroism revealed a spectru m similar to that of human GH (hGH) thereby indicating proper refoldin g. 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 recept or. The purified oPL was biologically active in an Nb-2-11C cell proli feration bioassay, in Its ability to stimulate beta-casein synthesis i n explants of ovine and rabbit mammary gland and fat synthesis in expl ants oi bovine mammary gland, and in a proliferation assay using FDC-P 1 cells transfected with rabbit or hGH receptors.