EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RAT PLACENTAL LACTOGEN-I - A COMPARISON WITH THE NATIVE HORMONE

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from t his source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (C HO) cells and in these studies have compared the recombinant protein w ith the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-tra nsfected CHO cells, 2) nonglycosylated recombinant PL-I produced by ad ding tunicamycin (10 mu M/ml medium) to rPL-I-transfected CHO cells, 3 ) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) se rum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensio nal polyacrylamide gel electrophoresis and Western blotting revealed n ine subforms with increasing mol wt [similar to 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (similar to 23 kDa) were evident in the nongl ycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (similar to 37 kDa) and more acidic pI. When analyzed by the Nb-2 lymphoma cell bioas say, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipoten t with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as act ive as ovine PRL in the Nb-2 assay. A RIA was established for rPL-I. T he variant rPL-I-v, displayed nonparallel displacement of [I-125]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum level s of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recom binant protein compared to 7.8 min for the choriocarcinoma-derived hor mone. In conclusion, we have shown that although CHO cells will glycos ylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to tho se of the native, placenta cell-derived hormone.