Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N -GlcNAcylation.

N -linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N -linked glycans is the difficulty of installing a single N -acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step ( N -GlcNAcylation). Here, we develop an in vitro method for N -GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni . We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N -GlcNAc and successfully determine that PglB with an N311V mutation (PglB N311V ) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglB N311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.