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Titolo:
EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RAT PLACENTAL LACTOGEN-I - A COMPARISON WITH THE NATIVE HORMONE
Autore:
ROBERTSON MC; COSBY H; FRESNOZA A; CATTINI PA; SHIU RPC; FRIESEN HG;
Indirizzi:
UNIV MANITOBA,FAC MED,DEPT PHYSIOL WINNIPEG R3E 0W3 MB CANADA
Titolo Testata:
Endocrinology
fascicolo: 1, volume: 134, anno: 1994,
pagine: 393 - 400
SICI:
0013-7227(1994)134:1<393:EPACOR>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
COMPLEMENTARY DEOXYRIBONUCLEIC-ACID; GROWTH-HORMONE; POLYACRYLAMIDE GELS; MOLECULAR-CLONING; PROLACTIN FAMILY; HUMAN-SERUM; PROTEINS; BIOASSAY; VARIANT; RADIOIMMUNOASSAY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
41
Recensione:
Indirizzi per estratti:
Citazione:
M.C. Robertson et al., "EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RAT PLACENTAL LACTOGEN-I - A COMPARISON WITH THE NATIVE HORMONE", Endocrinology, 134(1), 1994, pp. 393-400

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 amountof 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 monoclonalantibody 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 mu M/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 [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 nonglycosylated rPL-I, suggesting additional peptide cleavage sites. SerumrPL-I contained four additional forms of higher mol wt (similar to 37kDa) and more acidic pI. When analyzed by the Nb-2 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 Nb-2 assay. A RIA was established for rPL-I. The 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 levels of rPL-I by RIA after the injection of recombinant-rPL-I into adultfemale 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 11:13:17