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.

  • Article
  • Published:

Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

Nature Chemistry volume 4, pages 298–304 (2012)Cite this article

Subjects

Abstract

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit ‘turn-on’ fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Scheme to label proteins via an inverse electron-demand Diels–Alder cycloaddition, and structural formulae of relevant compounds.
Figure 2: Efficient, genetically-encoded incorporation of 2 using the PylRS/tRNACUA pair in E. coli.
Figure 3: Characterization of tetrazine–norbornene reactions.
Figure 4: Site-specific incorporation of 2 into proteins in mammalian cells and the specific labelling of EGFR-GFP on the cell surface with 9.

Similar content being viewed by others

References

  1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    Article  CAS  Google Scholar 

  2. Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, 12501–12504 (1994).

    Article  CAS  Google Scholar 

  3. Giepmans, B. N., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  Google Scholar 

  4. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  5. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  Google Scholar 

  6. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnol. 21, 86–89 (2003).

    Article  CAS  Google Scholar 

  7. Kosaka, N. et al. In vivo stable tumor-specific painting in various colors using dehalogenase-based protein-tag fluorescent ligands. Bioconjug. Chem. 20, 1367–1374 (2009).

    Article  CAS  Google Scholar 

  8. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  Google Scholar 

  9. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  Google Scholar 

  10. Zhou, Z., Koglin, A., Wang, Y., McMahon, A. P. & Walsh, C. T. An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo. J. Am. Chem. Soc. 130, 9925–9930 (2008).

    Article  CAS  Google Scholar 

  11. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815–158120 (2005).

    Article  CAS  Google Scholar 

  12. Fernandez-Suarez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nature Biotechnol. 25, 1483–1487 (2007).

    Article  CAS  Google Scholar 

  13. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl Acad. Sci. USA 107, 10914–10919 (2010).

    Article  CAS  Google Scholar 

  14. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nature Chem. Biol. 3, 707–78 (2007).

    Article  CAS  Google Scholar 

  15. Antos, J. M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).

    Article  CAS  Google Scholar 

  16. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  17. Halo, T. L., Appelbaum, J., Hobert, E. M., Balkin, D. M. & Schepartz, A. Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J. Am. Chem. Soc. 131, 438–439 (2009).

    Article  CAS  Google Scholar 

  18. Hinner, M. J. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766–776 (2010).

    Article  CAS  Google Scholar 

  19. Chin, J. W. et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 9026–9027 (2002).

    Article  CAS  Google Scholar 

  20. Zhang, Z., Wang, L., Brock, A. & Schultz, P. G. The selective incorporation of alkenes into proteins in Escherichia coli. Angew. Chem. Int. Ed. 41, 2840–2842 (2002).

    Article  CAS  Google Scholar 

  21. Chin, J. W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003).

    Article  CAS  Google Scholar 

  22. 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 

  23. Deiters, A., Cropp, T. A., Summerer, D., Mukherji, M. & Schultz, P. G. Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg. Med. Chem. Lett. 14, 5743–5745 (2004).

    Article  CAS  Google Scholar 

  24. Mehl, R. A. et al. Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935–939 (2003).

    Article  CAS  Google Scholar 

  25. Wang, L., Zhang, Z., Brock, A. & Schultz, P. G. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 100, 56–61 (2003).

    Article  CAS  Google Scholar 

  26. Carrico, Z. M., Romanini, D. W., Mehl, R. A. & Francis, M. B. Oxidative coupling of peptides to a virus capsid containing unnatural amino acids. Chem. Commun. 1205–1207 (2008).

  27. Fekner, T., Li, X., Lee, M. M. & Chan, M. K. A pyrrolysine analogue for protein click chemistry. Angew. Chem. Int. Ed. 48, 1633–1635 (2009).

    Article  CAS  Google Scholar 

  28. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009).

    Article  CAS  Google Scholar 

  29. Wang, Y., Song, W., Hu, W. J. & Lin, Q. Fast alkene functionalization in vivo by photoclick chemistry: HOMO lifting of nitrile imine dipoles. Angew. Chem. Int. Ed. 48, 5330–5333 (2009).

    Article  CAS  Google Scholar 

  30. Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A. & Bertozzi, C. R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 1, 644–648 (2006).

    Article  CAS  Google Scholar 

  31. Wang, J. et al. A biosynthetic route to photoclick chemistry on proteins. J. Am. Chem. Soc. 132, 14812–14818 (2010).

    Article  CAS  Google Scholar 

  32. Nguyen, D. P., Elliott, T., Holt, M., Muir, T. W. & Chin, J. W. Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J. Am. Chem. Soc. 133, 11418–11421 (2011).

    Article  CAS  Google Scholar 

  33. Laughlin, S. T. & Bertozzi, C. R. Imaging the glycome. Proc. Natl Acad. Sci. USA 106, 12–17 (2009).

    Article  CAS  Google Scholar 

  34. Prescher, J. A. & Bertozzi, C. R. Chemical technologies for probing glycans. Cell 126, 851–854 (2006).

    Article  CAS  Google Scholar 

  35. Johnson, J. A., Lu, Y. Y., Van Deventer, J. A. & Tirrell, D. A. Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Biotechnol. 14, 774–780 (2010).

    Article  CAS  Google Scholar 

  36. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  Google Scholar 

  37. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 2297–2299 (2008).

    Article  CAS  Google Scholar 

  38. Devaraj, N. K. & Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816–827 (2011).

    Article  CAS  Google Scholar 

  39. Mukai, T. et al. Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

    Article  CAS  Google Scholar 

  40. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nature Chem. Biol. 4, 232–234 (2008).

    Article  CAS  Google Scholar 

  41. Hancock, S. M., Uprety, R., Deiters, A. & Chin, J. W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    Article  CAS  Google Scholar 

  42. Greiss, S. & Chin, J. W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 14196–14199 (2011).

    Article  CAS  Google Scholar 

  43. Polycarpo, C. R. et al. Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 580, 6695–6700 (2006).

    Article  CAS  Google Scholar 

  44. Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem. Int. Ed. 48, 9184–9187 (2009).

    Article  CAS  Google Scholar 

  45. Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically encoding N(epsilon)-methyl-L-lysine in recombinant histones. J. Am. Chem. Soc. 131, 14194–14195 (2009).

    Article  CAS  Google Scholar 

  46. Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. J. Am. Chem. Soc. 132, 4086–4088 (2010).

    Article  CAS  Google Scholar 

  47. Wijinen, J. W., Zavarise, S., Engberts, J. B. F. N, Cahrton, Ml. J. Substituent effects on an inverse electron demand hetero Diels–Alder reaction in aqueous solution and organic solvents: cycloaddition of substituted styrenes to di(2-pyridyl)-1,2,4,5-tetrazine. J. Org. Chem. 61, 2001–2005 (1996).

    Article  Google Scholar 

  48. Devaraj, N. K., Hilderbrand, S., Upadhyay, R., Mazitschek, R. & Weissleder, R. Bioorthogonal turn-on probes for imaging small molecules inside living cells. Angew. Chem. Int. Ed. 49, 2869–2872 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Medical Research Council (U105181009, UD99999908), the European Research Council and the National Institutes for Health (GM079114) for funding. J.T.K. was supported by the National Science Foundation Graduate Research Fellowship under Grant No. NSF 0750733. We thank R. Mehl, who pointed out developments in inverse electron-demand Diels–Alder reactions while on sabbatical in the Chin lab.

Author information

Author notes

  1. Kathrin Lang and Lloyd Davis: These authors contributed equally to this work

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

    Kathrin Lang, Lloyd Davis & Jason W. Chin

  2. Department of Chemistry, North Carolina State University, Raleigh, 27695-8204, North Carolina, USA

    Jessica Torres-Kolbus, Chungjung Chou & Alexander Deiters

Authors
  1. Kathrin Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lloyd Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jessica Torres-Kolbus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chungjung Chou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexander Deiters
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jason W. Chin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.L, L.D. J.T.K., C.C., A.D. & J.W.C. designed the research and analysed the data. K.L, L.D., J.T.K. and C.C. performed the experiments. K.L. and J.W.C co-wrote the paper with input from the co-workers.

Corresponding authors

Correspondence to Alexander Deiters or Jason W. Chin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 4112 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lang, K., Davis, L., Torres-Kolbus, J. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem 4, 298–304 (2012). https://doi.org/10.1038/nchem.1250

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1250

This article is cited by

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