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Nat Commun. 2018 Jul 12;9(1):2686. doi: 10.1038/s41467-018-05110-x.

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery.

Author information

1
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA.
2
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA.
3
Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL, 60208-3120, USA.
4
Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208-3120, USA.
5
Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA.
6
Department of Mechanical Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute B224, Evanston, IL, 60208-3120, USA.
7
Department of Chemistry, Northwestern University, Evanston, IL, 60208, USA.
8
Department of Cell and Molecular Biology, Northwestern University, Chicago, IL, 60611, USA.
9
Department of Biomedical Engineering, Northwestern University, Evanston, IL, 60208, USA.
10
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA. m-jewett@northwestern.edu.
11
Chemistry of Life Processes Institute, 2170 Campus Drive, Evanston, IL, 60208-3120, USA. m-jewett@northwestern.edu.
12
Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208-3120, USA. m-jewett@northwestern.edu.
13
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.
14
Department of Microbiology, Cornell University, Ithaca, NY, 14853, USA. md255@cornell.edu.

Abstract

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

PMID:
30002445
PMCID:
PMC6043479
DOI:
10.1038/s41467-018-05110-x
[Indexed for MEDLINE]
Free PMC Article

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