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Murray, Thomas V.; Kozakowska-McDonnell, Kasia; Tibbles, Adam; Taylor, Annabel; Higazi, Daniel; Rossy, Emmanuel; Rossi, Alessandra; Genapathy, Sivaneswary; Tamburrino, Giulia; Rath, Nicola; Tigue, Natalie; Lindo, Vivian; Vaughan, Tristan; Papworth, Monika A.
mAbs, 11/2021, Letnik: 13, Številka: 1Journal Article
Bioconjugates are an important class of therapeutic molecules. To date, O- glycan-based metabolic glycoengineering has had limited use in this field, due to the complexities of the endogenous O- glycosylation pathway and the lack of an O- glycosylation consensus sequence. Here, we describe the development of a versatile on-demand O- glycosylation system that uses a novel, widely applicable 5 amino acid O- glycosylation tag, and a metabolically engineered UDP-galactose-4-eperimase (GALE) knock-out cell line. Optimization of the primary sequence of the tag enables the production of Fc-based proteins with either single or multiple O- glycans with complexity fully controlled by media supplementation. We demonstrate how the uniformly labeled proteins containing exclusively N-azido-acetylgalactosamine are used for CLICK chemistry-based bioconjugation to generate site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and bioactive Fc-peptides for applications in basic research and drug discovery. To our knowledge, this is the first description of generating a site-specific O- glycosylation system by combining an O- glycosylation tag and a metabolically engineered cell line.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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in: SICRIS
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