Immobilized enzyme cascade for targeted glycosylation Makrydaki, Elli Donini, Roberto Krueger, Anja Royle, Kate Moya-Ramírez, Ignacio Kuntz, Douglas A. Rose, David R. Haslam, Stuart M. Polizzi, Karen M. Kontoravdi, Cleo Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation. 2024-05-24T08:44:08Z 2024-05-24T08:44:08Z 2024-02-06 info:eu-repo/semantics/article Makrydaki, E., Donini, R., Krueger, A. et al. Immobilized enzyme cascade for targeted glycosylation. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01539-4 https://hdl.handle.net/10481/92044 10.1038/s41589-023-01539-4 eng http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess Atribución 4.0 Internacional Springer Nature