Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Site-selective glycosylation of proteins: creating synthetic glycoproteins

Nature Protocols volume 2, pages 3185–3194 (2007)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Glyco-CCHC reaction of SSβG-Aha43 with GlcNAc-O-CH2-alkyne.
Figure 3: Glyco-SeS reaction of SBL-Cys156 with GlcNAc-SH.
Figure 4: Double differential site-selective chemical glycosylation of SSβG-Aha43-Cys439 using glyco-CCHC with Gal-C-alkyne and glyco-MTS with Glc-O-CH2CH2-MTS.
Figure 5: The two parallel complementary modes of glyco-SeS.

Similar content being viewed by others

References

  1. Wold, F. In vivo chemical modification of proteins (post-translational modification). Ann. Rev. Biochem. 50, 783–814 (1981).

    Article  CAS  Google Scholar 

  2. Walsh, C.T., Garneau-Tsodikova, S. & Gatto, G.J. Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. 44, 7342–7372 (2005).

    Article  CAS  Google Scholar 

  3. Walsh, C.T. Posttranslational Modification of Proteins: Expanding Nature's Inventory (Roberts and Co. Press, Englewood, Colorado, 2005).

    Google Scholar 

  4. Rademacher, T.W., Parekh, R.B. & Dwek, R.A. Glycobiology. Annu. Rev. Biochem. 57, 785–838 (1988).

    Article  CAS  Google Scholar 

  5. Dwek, R.A. Glycobiology: toward understanding the function of sugars. Chem. Rev. 6, 683–720 (1996).

    Article  Google Scholar 

  6. Rudd, P.M. et al. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry 33, 17–22 (1994).

    Article  CAS  Google Scholar 

  7. Hamilton, S.R. et al. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443 (2006).

    Article  CAS  Google Scholar 

  8. Li, H. et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24, 210–215 (2006).

    Article  CAS  Google Scholar 

  9. Bobrowicz, P. et al. Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: Production of complex humanized glycoproteins with terminal galactose. Glycobiology 14, 757–766 (2004).

    Article  CAS  Google Scholar 

  10. Hamilton, S.R. et al. Production of complex human glycoproteins in yeast. Science 301, 1244–1246 (2003).

    Article  CAS  Google Scholar 

  11. Xie, J. & Schultz, P.G. A chemical toolkit for proteins—an expanded genetic code. Nat. Rev. Mol. Cell Biol. 7, 775–782 (2006).

    Article  CAS  Google Scholar 

  12. Zhang, Z. et al. A new strategy for the synthesis of glycoproteins. Science 303, 371–373 (2004).

    Article  CAS  Google Scholar 

  13. Xu, R. et al. Site-specific incorporation of the mucin-type N-acetylgalactosamine-β-O-threonine into protein in Escherichia coli. J. Am. Chem. Soc. 126, 15654–15655 (2004).

    Article  CAS  Google Scholar 

  14. van Kasteren, S.I., Garnier, P.G. & Davis, B.G. Chemical methods for mimicking post-translational modifications. in Protein Engineering (Springer Verlag, Berlin and Heidelberg, 2007).

    Google Scholar 

  15. Davis, B.G. Synthesis of glycoproteins. Chem. Rev. 102, 579–601 (2002).

    Article  CAS  Google Scholar 

  16. Liu, L., Bennett, C.S. & Wong, C.H. Advances in glycoprotein synthesis. Chem. Commun. 21–33 (2006).

  17. Muir, T.W. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 72, 249–289 (2003).

    Article  CAS  Google Scholar 

  18. Saxon, E., Armstrong, J.I. & Bertozzi, C.R. A 'traceless' Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2, 2141–2143 (2000).

    Article  CAS  Google Scholar 

  19. van Kasteren, S.I. et al. Expanding the diversity of chemical protein modification allows post-translational mimicry. Nature 446, 1105–1109 (2007).

    Article  CAS  Google Scholar 

  20. Gamblin, D.P. et al. Glycosyl phenylthiosulfonates (Glyco-PTS): novel reagents for glycoprotein synthesis. Org. Biomol. Chem. 1, 3642–3644 (2003).

    Article  CAS  Google Scholar 

  21. Gamblin, D.P. et al. Glyco-SeS: selenenylsulfide-mediated protein glycoconjugation—a new strategy in post-translational modification. Angew. Chem. Int. Ed. 43, 828–833 (2004).

    Article  CAS  Google Scholar 

  22. Davis, B.G., Lloyd, R.C. & Jones, J.B. Controlled site-selective glycosylation of proteins by a combined site- directed mutagenesis and chemical modification approach. J. Org. Chem. 63, 9614–9615 (1998).

    Article  CAS  Google Scholar 

  23. Hermanson, G.T. Bioconjugate Techniques 1st edn. (Academic Press, San Diego, California, 1996).

    Google Scholar 

  24. Davis, B.G., Maughan, M.A.T., Green, M.P., Ullman, A. & Jones, J.B. Glycomethanethiosulfonates: powerful reagents for protein glycosylation. Tetrahedron Asymmetry 11, 245–262 (2000).

    Article  CAS  Google Scholar 

  25. Swanwick, R.S., Daines, A.M., Flitsch, S.L. & Allemann, R.K. Synthesis of homogenous site-selectively glycosylated proteins. Org. Biomol. Chem. 3, 572–574 (2005).

    Article  CAS  Google Scholar 

  26. MacMillan, D., Bill, R.M., Sage, K.A., Fern, D. & Flitsch, S.L. Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin. Chem. Biol. 8, 133–145 (2001).

    Article  CAS  Google Scholar 

  27. Davis, B.G., Lloyd, R.C. & Jones, J.B. Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships. Bioorg. Med. Chem. 8, 1527–1535 (2000).

    Article  CAS  Google Scholar 

  28. Tornøe, C.W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    Article  Google Scholar 

  29. Demko, Z.P. & Sharpless, K.B. A click chemistry approach to tetrazoles by huisgen 1,3-dipolar cycloaddition: synthesis of 5-acyltetrazoles from azides and acyl cyanides. Angew. Chem. Int. Ed. 41, 2113–2116 (2002).

    Article  CAS  Google Scholar 

  30. Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2005–2021 (2001).

    Google Scholar 

  31. Deiters, A. & Schultz, P.G. In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorg. Med. Chem. Lett. 15, 1521–1524 (2005).

    Article  CAS  Google Scholar 

  32. Deiters, A. et al. Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J. Am. Chem. Soc. 125, 11782–11783 (2003).

    Article  CAS  Google Scholar 

  33. Lin, P.C. et al. Site-specific protein modification through Cu-I-catalyzed 1,2,3-triazole formation and its implementation in protein microarray fabrication. Angew. Chem. Int. Ed. 45, 4286–4290 (2006).

    Article  CAS  Google Scholar 

  34. Yang, W., Hendrickson, W.A., Crouch, R.J. & Satow, Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science 249, 1398–1405 (1990).

    Article  CAS  Google Scholar 

  35. Kiick, K.L., Weberskirch, R. & Tirrell, D.A. Identification of an expanded set of translationally active methionine analogues in Escherichia coli. FEBS Lett. 502, 25–30 (2001).

    Article  CAS  Google Scholar 

  36. Kiick, K.L. & Tirrell, D.A. Protein engineering by in vivo incorporation of non-natural amino acids: control of incorporation of methionine analogues by methionyl-tRNA synthetase. Tetrahedron 56, 9487–9493 (2000).

    Article  CAS  Google Scholar 

  37. Kiick, K.L., Saxon, E., Tirrell, D.A. & Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).

    Article  CAS  Google Scholar 

  38. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  CAS  Google Scholar 

  39. Bernardes, G.J., Gamblin, D.P. & Davis, B.G. The direct formation of glycosyl thiols from reducing sugars allows one-pot protein glycoconjugation. Angew. Chem. Int. Ed. 45, 4007–4011 (2006).

    Article  CAS  Google Scholar 

  40. Bernardes, G.J. & Davis, B.G. Direct thionation of reducing sugars. Nat. Protoc. (2007) doi: 10.1038/nprot.2007.439.

  41. Drummer, O.H., Routley, L. & Christophidis, N. Reversibility of disulfide formation. Biochem. Pharmacol. 36, 1197–1201 (1987).

    Article  CAS  Google Scholar 

  42. Lee, B. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–380 (1971).

    Article  CAS  Google Scholar 

  43. Glocker, M.O., Borchers, C., Fiedler, W., Suckau, D. & Przybylski, M. Molecular characterization of surface topology in protein tertiary structures by amino-acylation and mass spectrometric peptide mapping. Bioconjug. Chem. 5, 583–590 (1994).

    Article  CAS  Google Scholar 

  44. Stabile, M.R. et al. Probing the specificity of the S1 binding site of M222 mutants of subtilisin B. lentus with boronic acid inhibitors. Bioorg. Med. Chem. Lett. 6, 2501–2506 (1996).

    Article  CAS  Google Scholar 

  45. Van Hest, J.C., Kiick, K.L. & Tirrell, D.A. Efficient incorporation of unsaturated methionine analogues into proteins in vivo. J. Am. Chem. Soc. 122, 1282–1288 (2000).

    Article  CAS  Google Scholar 

  46. Perrin, D.D. & Armarego, W.L.F. Purification of Laboratory Chemicals (Pergamon Press, Oxford, 1988).

    Google Scholar 

  47. Chan, T.R., Hilgraf, R., Sharpless, K.B. & Fokin, V.V. Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org. Lett. 6, 2853–2855 (2004).

    Article  CAS  Google Scholar 

  48. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Author information

Author notes

  1. Sander I van Kasteren and Holger B Kramer: These authors contributed equally to this work.

Authors and 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

Authors
  1. Sander I van Kasteren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Holger B Kramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David P Gamblin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Benjamin G Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Benjamin G Davis.

Ethics declarations

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.

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Kasteren, S., Kramer, H., Gamblin, D. et al. Site-selective glycosylation of proteins: creating synthetic glycoproteins. Nat Protoc 2, 3185–3194 (2007). https://doi.org/10.1038/nprot.2007.430

Download citation

  • Published:

  • Issue Date:

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

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing