An efficient system for bioconjugation based on a widely applicable engineered O-glycosylation tag <subtitle>Short title: Controlled bioconjugation via O-glycan engineering</subtitle>

• Main Authors: Thomas V. Murray (Author), Kasia Kozakowska-McDonnell (Author), Adam Tibbles (Author), Annabel Taylor (Author), Daniel Higazi (Author), Emmanuel Rossy (Author), Alessandra Rossi (Author), Sivaneswary Genapathy (Author), Giulia Tamburrino (Author), Nicola Rath (Author), Natalie Tigue (Author), Vivian Lindo (Author), Tristan Vaughan (Author), Monika A. Papworth (Author)
• Format: Book
• Published: Taylor & Francis Group, 2021-01-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

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.

Item Description

10.1080/19420862.2021.1992068
1942-0870
1942-0862