(610e) Cell-Free Synthesis of Glycoproteins Bearing Complex-Type Human N-Glycans

Protein glycosylation is an important post-translational modification that occurs in all domains of life and is estimated to impact over half of all naturally occurring proteins in eukaryotes. Of the different types of protein glycosylation, asparagine-linked (N-linked) glycosylation is the most common and is known to significantly impact the structure and function of many proteins including valuable biopharmaceutical products like monoclonal antibodies. Given the importance of N-glycans for protein structure and function, there is an urgent need for technologies that enable reliable biosynthesis of proteins with controllable glycosylation. To this end, we previously developed a cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrated protein biosynthesis with N-linked protein glycosylation. However, a limitation of this earlier system was its inability to execute human-type glycosylation. Here, we sought to overcome this deficiency by engineering a modified CFGpS platform capable of site-specifically installing complex-type human N-glycans onto proteins of interest. This involved engineering a glyco-optimized Escherichia coli strain to source cell-free extracts that were selectively enriched with required glycosylation components, namely lipid-linked oligosaccharides (LLOs) bearing human N-glycan structures such as GlcNAc₂Man₃GlcNAc₂ (known as G0) and oligosaccharyltransferase (OST) enzymes for transferring these lipid-linked glycan structures onto target proteins. To our knowledge this is the first demonstration of an entirely cell-free synthesis of glycoprotein decorated with human N-glycan structures. By enabling eukaryotic glycosylation in CFGpS, we not only increase the range of therapeutic proteins that are capable of being produced in this format but also create a toolbox to explore how eukaryotic glycosylation affects the efficacy and biomanufacturability of N-glycoprotein therapeutics, generating insights that could be harnessed to intentionally remodel glycans in ways that improve product potency and stability.