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

• Published in: ACS chemical biology 2024-07, Vol.19 (7), p.1570-1582
• Main Authors: DeWinter, Madison A., Wong, Derek A., Fernandez, Regina, Kightlinger, Weston, Thames, Ariel Helms, DeLisa, Matthew P., Jewett, Michael C.
• Format: Article
• Language: English
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.4c00228

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 (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V 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.