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 folding being completed. While N-glycoprotein biosynthesis can also be accomplished using cell-free extracts derived from glycosylation-competent bacteria, it remains to be determined whether the cell-free reaction environment involves a similar mechanism for glycosylation of structured domains. Using an Escherichia coli-based cell-free glycoprotein synthesis (CFGpS) system, we observed efficient glycosylation of two eukaryotic glycoproteins, namely ribonuclease A (RNase A) and the fragment crystallizable (Fc) region of human immunoglobulin G (IgG), whose acceptor sites occur in structurally constrained regions that were not glycosylated when the proteins were already folded. Because this cell-free glycosylation depended on ribosomal translation but not on signal peptide-mediated translocation, we propose the existence of a unique co-translational, but not co-translocational, glycosylation mechanism in CFGpS. Collectively, these findings reveal the potential for CFGpS to become a viable platform for producing complex eukaryotic glycoprotein targets.