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Bioconjug Chem. 2014 Apr 16;25(4):788-95. doi: 10.1021/bc500061s. Epub 2014 Apr 7.

Chemoenzymatic Fc glycosylation via engineered aldehyde tags.

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1
Departments of Chemistry and ‡Molecular and Cell Biology and §Howard Hughes Medical Institute, ∥QB3/Chemistry Mass Spectrometry Facility, University of California , Berkeley, California 94720, United States.

Abstract

Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that was efficiently converted by recombinant formylglycine generating enzyme in vitro, thereby introducing aldehyde groups for subsequent chemical elaboration. Oxime-linked glycoconjugates were synthesized by conjugating aminooxy N-acetylglucosamine to the modified Fc followed by enzymatic transfer of complex N-glycans from corresponding glycan oxazolines by an EndoS-derived glycosynthase. In this manner we generated specific Fc glycoforms without relying on natural protein glycosylation machineries.

PMID:
24702330
PMCID:
PMC4004622
DOI:
10.1021/bc500061s
[Indexed for MEDLINE]
Free PMC Article
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