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

Paper ID Volume ID Publish Year Pages File Format Full-Text
24374 43510 2010 7 PDF Available
Title
Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells
Abstract

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 ∼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 ∼4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by ∼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.

Keywords
CHO, Chinese hamster ovary; CRS, chemical reference standard; hPRL, human prolactin; HPSEC, size-exclusion HPLC; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Mr, relative molecular mass; PAGE, polyacrylamide-
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Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 145, Issue 4, 15 February 2010, Pages 334–340
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us