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A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

View ORCID ProfileJennifer A. Schoborg, Jasmine Hershewe, Jessica C. Stark, Weston Kightlinger, James E. Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P. DeLisa, Michael C. Jewett
doi: https://doi.org/10.1101/145227
Jennifer A. Schoborg
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
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  • ORCID record for Jennifer A. Schoborg
Jasmine Hershewe
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
3Master of Biotechnology Program, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
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Jessica C. Stark
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
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Weston Kightlinger
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
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James E. Kath
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
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Thapakorn Jaroentomeechai
4Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA
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Aravind Natarajan
5Department of Microbiology, Cornell University, Ithaca, New York 14853, USA
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Matthew P. DeLisa
4Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA
5Department of Microbiology, Cornell University, Ithaca, New York 14853, USA
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Michael C. Jewett
1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL 60208–3120, USA
3Master of Biotechnology Program, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
6Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 676 N. St Clair St, Suite 1200, Chicago, IL 60611–3068, USA
7Simpson Querrey Institute, Northwestern University, 303 E. Superior St, Suite 11–131, Chicago, IL 60611–2875, USA
8Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
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Abstract

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.

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A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases
Jennifer A. Schoborg, Jasmine Hershewe, Jessica C. Stark, Weston Kightlinger, James E. Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P. DeLisa, Michael C. Jewett
bioRxiv 145227; doi: https://doi.org/10.1101/145227
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A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases
Jennifer A. Schoborg, Jasmine Hershewe, Jessica C. Stark, Weston Kightlinger, James E. Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P. DeLisa, Michael C. Jewett
bioRxiv 145227; doi: https://doi.org/10.1101/145227

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