Protein glycosylation is the covalent attachment of sugar moieties to proteins. Glycosylation is ubiquitous and is key for protein stability, activity, and immunogenicity. Glycosylated proteins are important molecules in biotechnology, comprising the majority of FDA-approved biologics, and can be used as vaccines against pathogens. However, controlling glycosylation for biomanufacturing defined glycoproteins, and for understanding the roles and regulations of glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of complex, pre-built glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (&gt;70 kDa) and contain many (&gt;10) transmembrane helices. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained soluble yields of up to 420 ug/ml for the single-subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional bacterial PglB homologs. Importantly, the cell-free derived enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed, and the ability to tightly control concentrations and ratios of glycosylation components in the in vitro system allowed us to quickly optimize the system for full glycosylation of target proteins. Since the publication of this work, we have extended the approach to various OST homologs. We anticipate that this broadly applicable production method will advance glycoengineering efforts and beyond, allowing for the facile synthesis and testing of complex membrane proteins.

Cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.