Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting

Biotechnology and Bioengineering
Tae Kwang HaGyun Min Lee

Abstract

To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs sign...Continue Reading

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Citations

Mar 27, 2016·Trends in Biotechnology·Sha ShaSeongkyu Yoon
Oct 23, 2016·Biotechnology Progress·Thomas K VilligerMiroslav Soos
Jan 14, 2019·Biotechnology and Bioengineering·Jong Hyun LeeGyun Min Lee
Feb 27, 2019·Bioprocess and Biosystems Engineering·Johannes MöllerRalf Pörtner

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