title: Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone.
abstract: 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 this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with 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-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels 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 recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
authors.forename: M C
authors.initials: MC
authors.lastname: Robertson
authors.forename: H
authors.initials: H
authors.lastname: Cosby
authors.forename: A
authors.initials: A
authors.lastname: Fresnoza
authors.forename: P A
authors.initials: PA
authors.lastname: Cattini
authors.forename: R P
authors.initials: RP
authors.lastname: Shiu
authors.forename: H G
authors.initials: HG
authors.lastname: Friesen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002822:Choriocarcinoma / Q000378:metabolism / Q000473:pathology; D002846:Chromatography, Affinity; D006224:Cricetinae; D003470:Culture Media; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D010928:Placental Lactogen / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D003470:Culture Media; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014415:Tunicamycin; D010928:Placental Lactogen