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Nat Chem Biol. 2014 Oct;10(10):816-22. doi: 10.1038/nchembio.1609. Epub 2014 Aug 17.

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity.

Author information

1
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
2
Proteomics and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York, USA.
3
Glycobia Inc., Ithaca, New York, USA.

Abstract

The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-linked glycosylation. This machinery has been functionally transferred into Escherichia coli, enabling convenient mechanistic dissection of the N-linked glycosylation process in this genetically tractable host. Here we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the -2 position relative to asparagine. This involved creation of a genetic assay, glycosylation of secreted N-linked acceptor proteins (glycoSNAP), that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that could glycosylate an array of noncanonical acceptor sequences, including one in a eukaryotic N-glycoprotein. These results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-linked glycosylation and for engineering designer N-linked glycosylation biocatalysts.

PMID:
25129029
PMCID:
PMC4575499
DOI:
10.1038/nchembio.1609
[Indexed for MEDLINE]
Free PMC Article

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