GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins

Ariel Helms Thames, Sam J. Moons, Derek A. Wong, Thomas J. Boltje, Bruce S. Bochner, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.

Original languageEnglish (US)
Pages (from-to)1264-1274
Number of pages11
JournalACS synthetic biology
Issue number4
StatePublished - Apr 21 2023


  • bacterial glycoengineering
  • cell-free protein synthesis
  • click chemistry
  • lectins
  • noncanonical glycans

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Biomedical Engineering


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