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Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells
Author(s) -
Zhang Min,
Koskie Kerry,
Ross James S.,
Kayser Kevin J.,
Caple Matthew V.
Publication year - 2010
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22633
Subject(s) - sialic acid , rna interference , sialidase , recombinant dna , glycoprotein , gene silencing , chinese hamster ovary cell , small interfering rna , biochemistry , biology , small hairpin rna , transfection , microbiology and biotechnology , gene , chemistry , rna , enzyme , neuraminidase , receptor
Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short‐hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.