期刊
BIOTECHNOLOGY JOURNAL
卷 11, 期 3, 页码 399-414出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201500331
关键词
Chinese hamster ovary (CHO) cells; Fluorescence-activated cell sorting (FACS); Fucose-free antibodies; GDP-fucose transporter gene (Slc35c1); Genome editing technologies
资金
- Strategic Positioning Fund (GlycoSing) from Biomedical Research Council (BMRC) of Agency for Science, Technology and Research (A*STAR), Singapore [SPF2013/001)]
- A*STAR's Joint Council (JCO) Visiting Investigator Programme (HighGlycoART)
Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies.
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