Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells.
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 (hIFNgamma) 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 hIFNgamma 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.
Zhang
Min
M
Cell Sciences & Development, SAFC Biosciences (a member of Sigma-Aldrich Group), 2909 Laclede Avenue, Saint Louis, MO 63103, USA. zhang_min@lilly.com
Koskie
Kerry
K
Ross
James S
JS
Kayser
Kevin J
KJ
Caple
Matthew V
MV
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins; D016415:Sequence Alignment
D006023:Glycoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D007371:Interferon-gamma; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid