Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

Autor(es)

Heller, S. R. ^[1] ; Goulart, H. Rodrigues ^[1] ; Arthuso, F. S. ^[1] ; Oliveira, T. L. ^[1] ; Bartolini, P. ^[1] ; Soares, C. R. J. ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN, Dept Biotechnol, BR-05508900 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Biotechnology; v. 145, n. 4, p. 334-340, FEB 15 2010.
• Citações Web of Science: 5

Resumo

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be similar to 4-fold lower than that of hPRL. its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL similar to 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by similar to 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms. (C) 2010 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 06/52973-9 - Sintese, purificacao e caracterizacao da prolactina humana glicosilada produzida em celulas de ovario de hamster chines.
• Beneficiário: Susana da Rocha Heller
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 05/60826-3 - Desenvolvimento de bioprocesso para produção de larga escala de hormônios recombinantes e seus antagonistas em cultura de células CHO em biorreatores
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 07/59709-8 - Síntese e caracterização de prolactina de camundongo (mPRL) em Escherichia coli
• Beneficiário: Fernanda dos Santos Arthuso
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 07/59540-3 - Síntese e caracterização de prolactina de camundongos (mPRL) e de seu análogo (S177D-mPRL) em Escherichia coli
• Beneficiário: Carlos Roberto Jorge Soares
• Modalidade de apoio: Auxílio à Pesquisa - Regular