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Biotechnol Bioeng. 2018 Mar;115(3):739-750. doi: 10.1002/bit.26502. Epub 2017 Dec 26.

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

Author information

1
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois.
2
Chemistry of Life Processes Institute, Evanston, Illinois.
3
Master of Biotechnology Program, Northwestern University, Evanston, Illinois.
4
Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York.
5
Department of Microbiology, Cornell University, Ithaca, New York.
6
Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois.
7
Simpson Querrey Institute, Northwestern University, Chicago, Illinois.
8
Center for Synthetic Biology, Northwestern University, Evanston, Illinois.

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 420 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.

KEYWORDS:

PglB; asparagine-linked protein glycosylation; cell-free protein synthesis; membrane protein; nanodisc; oligosaccharyltransferase; post-translational modification; synthetic biology

PMID:
29178580
DOI:
10.1002/bit.26502
[Indexed for MEDLINE]

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