Protein engineering through tandem transamidation


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.

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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.

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.


  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 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).

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 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).

  12. 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).

  13. 13.

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

  14. 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).

  15. 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).

  16. 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).

  17. 17.

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

  18. 18.

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

  19. 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).

  20. 20.

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

  21. 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).

  22. 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).

  23. 23.

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

  24. 24.

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

  25. 25.

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

  26. 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).

  27. 27.

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

  28. 28.

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

  29. 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).

  30. 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).

  31. 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).

  32. 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).

  33. 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).

  34. 34.

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

  35. 35.

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

  36. 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).

  37. 37.

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

  38. 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).

  39. 39.

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

  40. 40.

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

  41. 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).

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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

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.

Correspondence to Tom. W. Muir.

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Supplementary information

Supplementary Information

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

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