UTILIZATION OF CHO CELLS CULTIVATED IN PRESENCE OF CYCLOHEXIMIDE FOR OBTAINMENT AND CHARACTARIZATION OF RECOMBINANT HUMAN GLYCOSYLATED PROLACTIN (G-hPRL) / Utilização de células CHO cultivadas na presença de cicloheximida para obtenção e caracterização de prolactina humana glicosilada (G-hPRL) recombinante

AUTOR(ES)

Susana da Rocha Heller

DATA DE PUBLICAÇÃO

2008

RESUMO

Human prolactin hPRL is a 199 aminoacid protein hormone (MM ~23.000 Da) with a wide spectrum of biological activities being, however, best known for its stimulation of lactation and development of mammary gland. Besides proteolytic cleavage, the majority of prolactin variants can be the result of other posttranslational processing of the mature molecule in the anterior pituitary gland or the plasma. These include polymerization, phosphorylation, deamidation, sulfation, and glycosylation. This protein contains only one potential asparagine-linked glycosylation site which is partially ( ~10%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found ~ 4-fold lower compared to that of hPRL, its physiological function is not well defined yet, and the carbohydrate moiety seems to play an important role in the biosynthesis, secretion, biological activity, and plasma clearance of the hormone. In order to better characterize and study this hormone variant, we carried out its laboratory scale synthesis and purification from genetically modified CHO cells medium that had been supplemented with cycloheximide, increasing thus ~4-fold its absolute concentration and ~10-fold the glycosylated versus non-glycosylated hPRL concentration ratio. G-hPRL purification was carried via a simple and effective two-step process based on a cationic exchanger and a preparative size-exclusion HPLC column (HPSEC). Characterization was carried out by reversed-phase and size-exclusion HPLC, SDS-PAGE, western blotting, MALDI-TOF MS and in vitro bioassay utilizing Nb2 and Ba/F3-LLP cells. Ours results show that cycloheximide can be an important tool to increase the production of glycosylated proteins hPRL facilitating the purification and characterization of these isoforms.

ASSUNTO(S)

cho ciclohexímida prolactina glicosilada prolactina glicosilada cicloheximida cho

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