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Site-selective glycosylation of proteins: creating synthetic glycoproteins

Nature Protocols volume 2, pages 31853194 (2007) | Download Citation

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Abstract

In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs). Glycosylation is by far the most diverse of the PTM processes. Natural protein production methods typically produce PTM or glycoform mixtures within which function is difficult to dissect or control. Chemical tagging methods allow the precise attachment of multiple glycosylation modifications to bacterially expressed (bare) protein scaffolds, allowing reconstitution of functionally effective mimics of glycoproteins in higher organisms. In this way combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein–PTM interactions. This protocol describes the modification of Cys residues in proteins using glycomethanethiosulfonates and glycoselenenylsulfides and the modification of azidohomoalanine residues, introduced by Met replacement using auxotrophic Met(−) Escherichia coli strains, with glycoalkynes and the combination of these techniques for the creation of dual-tagged proteins. Each glycosylation procedure outlined in this protocol can be achieved in half a day.

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Acknowledgements

S.I.v.K. and H.B.K. contributed equally to this work.

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Author notes

    • Sander I van Kasteren
    •  & Holger B Kramer

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.

    • Sander I van Kasteren
    • , Holger B Kramer
    • , David P Gamblin
    •  & Benjamin G Davis

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Competing interests

The glycol-SeS, glycol-MTS and glycol-CCHC methods are registered in patents held by the University of Oxford. These may afford authors of this work royalties from subsequent licensing and/or other arrangements in line with standard university practice.

Corresponding author

Correspondence to Benjamin G Davis.

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DOI

https://doi.org/10.1038/nprot.2007.430

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