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Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

Paper ID Volume ID Publish Year Pages File Format Full-Text
23034 43413 2014 9 PDF Available
Title
Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations
Abstract

•All glycosylation genes insensitive to ammonium as analyzed by DNA microarray.•Five glycosylation genes insensitive to ammonium as analyzed by real-time qRT-PCR.•Translational or substrate-level control must be responsible for the decreased glycosylation.

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, these genes were analyzed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analyzed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium.

Keywords
Actb, Actin, beta; B4galt1, UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1; CHO, cells Chinese hamster ovary cells; Dpm1, dolichol-phosphate (beta-D) mannosyltransferase 1; Fut4, fucosyltransferase 4; Gapdh, glyceraldehyde-3-phosphate de
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Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 187, 10 October 2014, Pages 78–86
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