Glycosylation of Structured Protein Domains in Cell-Free Reaction Environments

The production of N -linked glycoproteins in genetically tractable bacterial hosts and their cell-free extracts holds great promise for low-cost, customizable, and distributed biomanufacturing of glycoconjugate vaccines and glycoprotein therapeutics. In nearly all bacterial N -linked protein glycosylation systems described so far, a single-subunit, transmembrane oligosaccharyltransferase (OST) is employed which favors acceptor sites in flexible, solvent-exposed motifs of the glycoprotein substrate. Yet despite this preference, acceptor sites in structured domains can also be glycosylated in living bacteria, presumably by a mechanism where the site is presented to the OST in a flexible form during or after the membrane translocation step but prior to