Immobilized enzyme cascade for targeted glycosylation

  • Nat Chem Biol. 2024 Jun;20(6):732-741. doi: 10.1038/s41589-023-01539-4.
Elli Makrydaki  1 ,  Roberto Donini  2 ,  Anja Krueger  2 ,  Kate Royle  1 ,  Ignacio Moya Ramirez  1  3 ,  Douglas A Kuntz  4 ,  David R Rose  4  5 ,  Stuart M Haslam  2 ,  Karen M Polizzi  6 ,  Cleo Kontoravdi  7
Affiliations
  • 1. Department of Chemical Engineering, Imperial College London, London, UK.
  • 2. Department of Life Sciences, Imperial College London, London, UK.
  • 3. Departamento de Ingeniería Química, Universidad de Granada, Granada, Spain.
  • 4. Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
  • 5. Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
  • 6. Department of Chemical Engineering, Imperial College London, London, UK. [email protected].
  • 7. Department of Chemical Engineering, Imperial College London, London, UK. [email protected].
PMID: 38321209 DOI: 10.1038/s41589-023-01539-4
Abstract

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.