biostudies-literatureMakrydaki ERCUK | Engineering and Physical Sciences Research Council (EPSRC)RCUK | Biotechnology and Biological Sciences Research CouncilRCUK | Engineering and Physical Sciences Research CouncilBiotechnology and Biological Sciences Research CouncilEngineering and Physical Sciences Research Council732-741https://www.ebi.ac.uk/biostudies/studies/S-EPMC11142912biostudies-literatureUnknown20(6)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 thrNature chemical biologyImmobilized enzyme cascade for targeted glycosylation.PMC11142912EP/N509486/1BB/P02789X/1EP/K038648/1, EP/H04986X/1 and EP/K038648/1EP/H04986X/1Donini RMakrydaki EKuntz DARoyle KRose DRMoya Ramirez IPolizzi KMKontoravdi CKrueger AHaslam SMfalseImmobilized enzyme cascade for targeted glycosylation.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 thr2024-01-01T00:00:00Z2024 Jun2026-04-29T14:12:14.267Z2026-04-14T03:06:54.834ZS-EPMC111429123832120910.1038/s41589-023-01539-4