Cloning, expression, purification, and immunocharacterization of placentalprotein-14

Human placental protein-14 (PP-14), a member of the lipocalin superfamily, shares homology at the level of the primary and secondary structures with b ovine beta-lactoglobulin. It is the most prominent endometrial protein synt hesized by the glandular cells of endometrium under estrogen priming and pr ogesterone stimulation. The temporal and spatial expression of PP-14 in the female reproductive tract combined with its biological activities ex vivo suggest that this glycoprotein probably plays an essential physiological ro le in the regulation of fertilization, implantation, and maintenance of pre gnancy. We proposed to elucidate the molecular mechanisms involved in the f unction of this protein. A prerequisite to such investigations on any prote in is the availability of sufficient amounts of the same in a homogenous fo rm. Therefore, recombinant DNA technology was employed. The PP-14 cDNA was obtained from the first-trimester endometrial tissue RNA by RT-PCR using un ique primers. After confirming the identity of the gene, the protein was ex pressed in Escherichia coli and purified to homogeneity. The gene was also cloned and expressed in Pichia pastoris to obtain the protein product in a glycosylated form. The recombinant proteins were immunocharacterized using a cross-reactive antibody raised to bovine beta-lactoglobulin. Polyclonal a ntiserum raised to the E coli expressed PP-14 also bound to the native PP-1 4 from amniotic fluid suggesting that recombinant PP-14 may be exploited to elucidate functional aspects of the protein. (C) 1998 Academic Press.