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Nat Commun. 2019 Nov 27;10(1):5404. doi: 10.1038/s41467-019-12024-9.

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways.

Author information

1
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E136, Evanston, IL, 60208, USA.
2
Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Tech B486, Evanston, IL, 60208, USA.
3
Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL, 60208, USA.
4
Medical Scientist Training Program, Feinberg School of Medicine, Northwestern University, Morton Building 1-670, 303 E. Chicago Avenue, Chicago, IL, 60611, USA.
5
Department of Microbiology, Cornell University, 123 Wing Drive, Ithaca, NY, 14853, USA.
6
Department of Chemistry, Northwestern University, Evanston, IL, 60208, USA.
7
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA.
8
Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Weill Hall, Ithaca, NY, 14853, USA.
9
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E136, Evanston, IL, 60208, USA. m-jewett@northwestern.edu.
10
Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Tech B486, Evanston, IL, 60208, USA. m-jewett@northwestern.edu.

Abstract

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

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