Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.
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 approximately 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 approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 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.
Heller
S R
SR
Biotechnology Department, IPEN-CNEN, Av. Lineu Prestes, 2242, Cidade Universitária, 05508-900 São Paulo, Brazil.
Rodrigues Goulart
H
H
Arthuso
F S
FS
Oliveira
T L
TL
Bartolini
P
P
Soares
C R J
CR
D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D051379:Mice; D011388:Prolactin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; C043928:prolactin, glycosylated; D011388:Prolactin; D003513:Cycloheximide