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:

Protein engineering through tandem transamidation

Nature Chemistry volume 11, pages 737–743 (2019)Cite this article

Subjects

Abstract

Semisynthetic proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating biological processes. Here, we introduce a one-pot, chemoenzymatic method that allows broad access to chemically modified proteins. The approach involves a tandem transamidation reaction cascade that integrates intein-mediated protein splicing with enzyme-mediated peptide ligation. We show that this approach can be used to introduce PTMs and biochemical probes into a range of proteins including Cas9 nuclease and the transcriptional regulator MeCP2, which causes Rett syndrome when mutated. The versatility of the approach is further illustrated through the chemical tailoring of histone proteins within a native chromatin setting. We expect our approach will extend the scope of semisynthesis in protein engineering.

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

Fig. 1: Protein semisynthesis by TAIL.
Fig. 2: Identification of mutant inteins for use in TAIL.
Fig. 3: C-terminal protein engineering using TAIL (C-TAIL).
Fig. 4: N-terminal protein engineering using TAIL (N-TAIL).
Fig. 5: Application of TAIL to protein modification in increasingly complex environments.

Similar content being viewed by others

Data availability

All relevant data are included in the Supplementary Information and are available from the authors. Plasmids for the following vectors have been deposited, along with maps and sequences, in Addgene: pET30-His6-Sumo-IntNtr-eSrt2A, Addgene plasmid #126015; pET30-His6-Sumo-MBP-CfaN, Addgene plasmid #126016; pET30-MBP-CfaN-His6, Addgene plasmid #126017; pET30-His6-Sumo-CfaC-MBP, Addgene plasmid #126018; pTXB1-His6-IntCtr-GyrA-His6, Addgene plasmid #126019. All other plasmids reported in this manuscript will be available upon request.

References

  1. Boutureira, O. & Bernardes, G. J. L. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).

    Article  CAS  Google Scholar 

  2. Debelouchina, G. T. & Muir, T. W. A molecular engineering toolbox for the structural biologist. Q. Rev. Biophys. 50, 1–41 (2017).

    Article  Google Scholar 

  3. Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806 (2014).

    Article  CAS  Google Scholar 

  4. Wang, L. & Schultz, P. G. Expanding the genetic code. Angew. Chem. Int. Ed. 44, 34–66 (2005).

    Article  CAS  Google Scholar 

  5. Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nat. Commun. 5, 14 (2014).

    Article  Google Scholar 

  6. Tarrant, M. K. & Cole, P. A. The chemical biology of protein phosphorylation. Annu. Rev. Biochem. 78, 797–825 (2009).

    Article  CAS  Google Scholar 

  7. Davis, L. & Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nat. Rev. Mol. Cell Biol. 13, 168–182 (2012).

    Article  CAS  Google Scholar 

  8. Muller, M. M. & Muir, T. W. Histones: at the crossroads of peptide and protein chemistry. Chem. Rev. 115, 2296–2349 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Hackenberger, C. P. R. & Schwarzer, D. Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 47, 10030–10074 (2008).

    Article  CAS  Google Scholar 

  11. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  CAS  Google Scholar 

  12. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  Google Scholar 

  13. Henager, S. H. et al. Enzyme-catalyzed expressed protein ligation. Nat. Methods 13, 925–927 (2016).

    Article  CAS  Google Scholar 

  14. Jackson, D. Y. et al. A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science 266, 243–247 (1994).

    Article  CAS  Google Scholar 

  15. Nguyen, G. K. T. et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732–738 (2014).

    Article  CAS  Google Scholar 

  16. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707–708 (2007).

    Article  CAS  Google Scholar 

  17. Weeks, A. M. & Wells, J. A. Engineering peptide ligase specificity by proteomic identification of ligation sites. Nat. Chem. Biol. 14, 50–57 (2018).

    Article  CAS  Google Scholar 

  18. Yang, R. L. et al. Engineering a catalytically efficient recombinant protein ligase. J. Am. Chem. Soc. 139, 5351–5358 (2017).

    Article  CAS  Google Scholar 

  19. Schmidt, M., Toplak, A., Quaedflieg, P. & Nuijens, T. Enzyme-mediated ligation technologies for peptides and proteins. Curr. Opin. Chem. Biol. 38, 1–7 (2017).

    Article  CAS  Google Scholar 

  20. Kent, S. Chemical protein synthesis: inventing synthetic methods to decipher how proteins work. Bioorg. Med. Chem. 25, 4926–4937 (2017).

    Article  CAS  Google Scholar 

  21. Bondalapati, S., Jbara, M. & Brik, A. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nat. Chem. 8, 407–418 (2016).

    Article  CAS  Google Scholar 

  22. David, Y., Vila-Perello, M., Verma, S. & Muir, T. W. Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nat. Chem. 7, 394–402 (2015).

    Article  CAS  Google Scholar 

  23. Mootz, H. D. Split inteins as versatile tools for protein semisynthesis. ChemBioChem 10, 2579–2589 (2009).

    Article  CAS  Google Scholar 

  24. Shah, N. H. & Muir, T. W. Inteins: nature’s gift to protein chemists. Chem. Sci. 5, 446–461 (2014).

    Article  CAS  Google Scholar 

  25. Stevens, A. J. et al. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138, 2162–2165 (2016).

    Article  CAS  Google Scholar 

  26. Dorr, B. M., Ham, H. O., An, C. H., Chaikof, E. L. & Liu, D. R. Reprogramming the specificity of sortase enzymes. Proc. Natl Acad. Sci. USA 111, 13343–13348 (2014).

    Article  CAS  Google Scholar 

  27. Stevens, A. J. et al. A promiscuous split intein with expanded protein engineering applications. Proc. Natl Acad. Sci. USA 114, 8538–8543 (2017).

    Article  CAS  Google Scholar 

  28. Brocchieri, L. & Karlin, S. Protein length in eukaryotic and prokaryotic proteomes. Nucleic Acids Res. 33, 3390–3400 (2005).

    Article  CAS  Google Scholar 

  29. Harshman, S. W., Young, N. L., Parthun, M. R. & Freitas, M. A. H1 histones: current perspectives and challenges. Nucleic Acids Res. 41, 9593–9609 (2013).

    Article  CAS  Google Scholar 

  30. Wan, Q. & Danishefsky, S. J. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 46, 9248–9252 (2007).

    Article  CAS  Google Scholar 

  31. Guy, J., Cheval, H., Selfridge, J. & Bird, A. The role of MeCP2 in the brain. Annu. Rev. Cell Dev. Biol. 27, 631–652 (2011).

    Article  CAS  Google Scholar 

  32. Amir, R. E. et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188 (1999).

    Article  CAS  Google Scholar 

  33. Bellini, E. et al. MeCP2 post-translational modifications: a mechanism to control its involvement in synaptic plasticity and homeostasis?. Front. Cell. Neurosci. 8, 236 (2014).

    Article  Google Scholar 

  34. Chen, W. G. et al. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science 302, 885–889 (2003).

    Article  CAS  Google Scholar 

  35. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    Article  CAS  Google Scholar 

  36. Liszczak, G. P. et al. Genomic targeting of epigenetic probes using a chemically tailored Cas9 system. Proc. Natl Acad. Sci. USA 114, 681–686 (2017).

    Article  CAS  Google Scholar 

  37. Lockless, S. W. & Muir, T. W. Traceless protein splicing utilizing evolved split inteins. Proc. Natl Acad. Sci. USA 106, 10999–11004 (2009).

    Article  CAS  Google Scholar 

  38. Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D. & Schmidt, S. R. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J. Biol. Chem. 287, 28686–28696 (2012).

    Article  CAS  Google Scholar 

  39. Nguyen, G. K. et al. Butelase 1: a versatile ligase for peptide and protein macrocyclization. J. Am. Chem. Soc. 137, 15398–15401 (2015).

    Article  CAS  Google Scholar 

  40. Nikghalb, K. D. et al. Expanding the scope of sortase-mediated ligations by using sortase homologues. ChemBioChem 19, 185–195 (2018).

    Article  CAS  Google Scholar 

  41. Schmohl, L., Bierlmeier, J., Gerth, F., Freund, C. & Schwarzer, D. Engineering sortase A by screening a second-generation library using phage display. J. Pept. Sci. 23, 631–635 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank current members of the Muir laboratory (in particular G. Liszczak, M. Haugbro, A. Burton and J. Gramespacher) for discussions and comments. The authors also thank T. Srikumar of Princeton University Mass Spectrometry Facility. This work was supported by National Institutes of Health (NIH) grants R37 GM086868 and P01 CA196539. R.E.T. was supported by a Charles H. Revson Foundation postdoctoral fellowship and A.J.S. by a National Science Foundation graduate research fellowship (DGE-1148900). We dedicate this paper to R.T. Raines on the occasion of his 60th birthday.

Author information

Author notes

  1. Adam J. Stevens

    Present address: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

Authors and Affiliations

  1. Department of Chemistry, Princeton University, Frick Chemistry Laboratory, Princeton, NJ, USA

    Robert E. Thompson, Adam J. Stevens & Tom. W. Muir

Authors
  1. Robert E. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adam J. Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tom. W. Muir
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.E.T. and T.W.M. conceived the project. R.E.T., A.J.S. and T.W.M. designed all experiments and analysed all data. R.E.T. performed all experiments. R.E.T. and T.W.M. wrote the manuscript.

Corresponding author

Correspondence to Tom. W. Muir.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

The Supplementary Information file contains methods, protein sequences and characterization, as well as supplementary figures and data.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thompson, R.E., Stevens, A.J. & Muir, T.W. Protein engineering through tandem transamidation. Nat. Chem. 11, 737–743 (2019). https://doi.org/10.1038/s41557-019-0281-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-019-0281-2

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