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Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases

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Abstract

Natural human interferon-γ (hIFN-γ) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN-γ in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: α1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN-γ having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN-γ having GlcNAcβ1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Galβ1-4GlcNAcβ1-3)n was observed. In the present study, we introduced mouse Galβ1-3/4GlcNAc-R α2,3-sialyltransferase (ST3Gal IV) and/or rat Galβ1-4GlcNAc-R α2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of α2,3-ST and/or α2,6-ST. The extent of sialylation of hIFN-γ from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of α2,3- and α2,6-sialic acid corresponded to the activity ratio of α2,3-ST to α2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.

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Authors and Affiliations

  1. Life Science Laboratory, Mitsui Chemicals, Inc., 1144 Togo, Mobara, Chiba, 297-0017, Japan

    Kazuhiro Fukuta, Tomoko Yokomatsu, Reiko Abe, Mineko Asanagi & Tadashi Makino

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  1. Kazuhiro Fukuta
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  2. Tomoko Yokomatsu
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  3. Reiko Abe
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  4. Mineko Asanagi
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  5. Tadashi Makino
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Fukuta, K., Yokomatsu, T., Abe, R. et al. Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases. Glycoconj J 17, 895–904 (2000). https://doi.org/10.1023/A:1010977431061

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