RT Journal Article SR Electronic T1 A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases JF bioRxiv FD Cold Spring Harbor Laboratory SP 145227 DO 10.1101/145227 A1 Jennifer A. Schoborg A1 Jasmine Hershewe A1 Jessica C. Stark A1 Weston Kightlinger A1 James E. Kath A1 Thapakorn Jaroentomeechai A1 Aravind Natarajan A1 Matthew P. DeLisa A1 Michael C. Jewett YR 2017 UL http://biorxiv.org/content/early/2017/06/05/145227.abstract AB Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific 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 glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. 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 yields of up to 440 µg/mL for the single-subunit OST enzyme, ‘Protein glycosylation B’ (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.