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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.

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


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.

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