Ligation of Synthetic Peptides to Proteins Using Semisynthetic Protein trans-Splicing

  • Julian C. J. Matern
  • Anne-Lena Bachmann
  • Ilka V. Thiel
  • Gerrit Volkmann
  • Alexandra Wasmuth
  • Jens Binschik
  • Henning D. Mootz
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)


Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

Key words

Protein semisynthesis Protein labeling Intein Protein splicing Peptide ligation Synthetic label Peptide synthesis Protein expression Flurophore 



We thank Xiang-Qin Liu (Dalhousie University, Canada) for collaboration on initial work on the M86 mutant and Shmuel Pietrokovski (Weizmann Institute, Israel) for collaboration on the AceL-TerL intein. We acknowledge funding by DFG (grants MO1073/3-2; SPP1623, MO1073/5-1, and Cells-in-Motion excellence cluster, EXC1003) and Fonds der Chemischen Industrie.


  1. 1.
    Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779PubMedCrossRefGoogle Scholar
  2. 2.
    Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci U S A 95(12):6705–6710PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Nilsson BL, Kiessling LL, Raines RT (2000) Staudinger ligation: a peptide from a thioester and azide. Org Lett 2(13):1939–1941PubMedCrossRefGoogle Scholar
  4. 4.
    Saxon E, Armstrong JI, Bertozzi CR (2000) A “traceless” Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett 2(14):2141–2143PubMedCrossRefGoogle Scholar
  5. 5.
    Bode JW, Fox RM, Baucom KD (2006) Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and alpha-ketoacids. Angew Chem Int Ed 45(8):1248–1252. doi: 10.1002/anie.200503991 CrossRefGoogle Scholar
  6. 6.
    Mao HY, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126(9):2670–2671. doi: 10.1021/Ja039915e PubMedCrossRefGoogle Scholar
  7. 7.
    Noren CJ, Wang JM, Perler FB (2000) Dissecting the chemistry of protein splicing and its applications. Angew Chem Int Ed 39(3):450–466CrossRefGoogle Scholar
  8. 8.
    Volkmann G, Mootz HD (2013) Recent progress in intein research: from mechanism to directed evolution and applications. Cell Mol Life Sci 70(7):1185–1206. doi: 10.1007/s00018-012-1120-4 PubMedCrossRefGoogle Scholar
  9. 9.
    Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5:446–461. doi: 10.1039/C3SC52951G PubMedCrossRefGoogle Scholar
  10. 10.
    Shi J, Muir TW (2005) Development of a tandem protein trans-splicing system based on native and engineered split inteins. J Am Chem Soc 127(17):6198–6206PubMedCrossRefGoogle Scholar
  11. 11.
    Shah NH, Eryilmaz E, Cowburn D, Muir TW (2013) Extein residues play an intimate role in the rate-limiting step of protein trans-splicing. J Am Chem Soc 135(15):5839–5847PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Schwarzer D, Ludwig C, Thiel IV, Mootz HD (2012) Probing intein-catalyzed thioester formation by unnatural amino acid substitutions in the active site. Biochemistry 51(1):233–242PubMedCrossRefGoogle Scholar
  13. 13.
    Appleby-Tagoe JH, Thiel IV, Wang Y, Mootz HD, Liu XQ (2011) Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. J Biol Chem 286(39):34440–34447PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Binschik J, Zettler J, Mootz HD (2011) Photocontrol of protein activity mediated by the cleavage reaction of a split intein. Angew Chem Int Ed Engl 50(14):3249–3252PubMedCrossRefGoogle Scholar
  15. 15.
    Giriat I, Muir TW (2003) Protein semi-synthesis in living cells. J Am Chem Soc 125(24):7180–7181. doi: 10.1021/ja034736i PubMedCrossRefGoogle Scholar
  16. 16.
    Mootz HD (2009) Split inteins as versatile tools for protein semisynthesis. Chembiochem 10(16):2579–2589. doi: 10.1002/cbic.200900370 PubMedCrossRefGoogle Scholar
  17. 17.
    Volkmann G, Liu XQ (2009) Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein. PLoS One 4(12):e8381. doi: 10.1371/journal.pone.0008381 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Appleby JH, Zhou K, Volkmann G, Liu X-Q (2009) Novel split intein for trans-splicing synthetic peptide onto C terminus of protein. J Biol Chem 284(10):6194–6199PubMedCrossRefGoogle Scholar
  19. 19.
    Thiel IV, Volkmann G, Pietrokovski S, Mootz HD (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angew Chem Int Ed Engl. doi: 10.1002/anie.201307969 Google Scholar
  20. 20.
    Aranko AS, Zuger S, Buchinger E, Iwai H (2009) In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins. PLoS One 4(4):e5185. doi: 10.1371/journal.pone.0005185 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Oeemig JS, Aranko AS, Djupsjobacka J, Heinamaki K, Iwai H (2009) Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett 583(9):1451–1456. doi: 10.1016/j.febslet.2009.03.058 PubMedCrossRefGoogle Scholar
  22. 22.
    Borra R, Dong D, Elnagar AY, Woldemariam GA, Camarero JA (2012) In-cell fluorescence activation and labeling of proteins mediated by FRET-quenched split inteins. J Am Chem Soc 134(14):6344–6353. doi: 10.1021/ja300209u PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Ludwig C, Pfeiff M, Linne U, Mootz HD (2006) Ligation of a synthetic peptide to the N terminus of a recombinant protein using semisynthetic protein trans-splicing. Angew Chem Int Ed Engl 45(31):5218–5221PubMedCrossRefGoogle Scholar
  24. 24.
    Ludwig C, Schwarzer D, Mootz HD (2008) Interaction studies and alanine scanning analysis of a semi-synthetic split intein reveal thiazoline ring formation from an intermediate of the protein splicing reaction. J Biol Chem 283(37):25264–25272PubMedCrossRefGoogle Scholar
  25. 25.
    Wasmuth A, Ludwig C, Mootz HD (2013) Structure–activity studies on the upstream splice junction of a semisynthetic intein. Bioorg Med Chem 21(12):3495–3503PubMedCrossRefGoogle Scholar
  26. 26.
    Wu H, Xu M-Q, Liu X-Q (1998) Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein. Biochim et Biophys Acta 1387(1–2):422–432CrossRefGoogle Scholar
  27. 27.
    Sun W, Yang J, Liu X-Q (2004) Synthetic two-piece and three-piece split inteins for protein trans-splicing. J Biol Chem 279(34):35281–35286PubMedCrossRefGoogle Scholar
  28. 28.
    Brenzel S, Kurpiers T, Mootz HD (2006) Engineering artificially split inteins for applications in protein chemistry: biochemical characterization of the split Ssp DnaB intein and comparison to the split Sce VMA intein. Biochemistry 45(6):1571–1578PubMedCrossRefGoogle Scholar
  29. 29.
    Cheriyan M, Pedamallu CS, Tori K, Perler F (2013) Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. J Biol Chem 288(9):6202–6211PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Amitai G, Callahan BP, Stanger MJ, Belfort G, Belfort M (2009) Modulation of intein activity by its neighboring extein substrates. Proc Natl Acad Sci U S A 106(27):11005–11010. doi: 10.1073/pnas.0904366106 PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Kurpiers T, Mootz HD (2007) Regioselective cysteine bioconjugation by appending a labeled cystein tag to a protein by using protein splicing in trans. Angew Chem Int Ed Engl 46(27):5234–5237PubMedCrossRefGoogle Scholar
  32. 32.
    Zettler J, Schutz V, Mootz HD (2009) The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett 583(5):909–914. doi: 10.1016/j.febslet.2009.02.003 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Julian C. J. Matern
    • 1
  • Anne-Lena Bachmann
    • 1
  • Ilka V. Thiel
    • 1
  • Gerrit Volkmann
    • 1
  • Alexandra Wasmuth
    • 1
  • Jens Binschik
    • 1
  • Henning D. Mootz
    • 1
  1. 1.Department of Chemistry and Pharmacy, Institute of BiochemistryUniversity of MuensterMünsterGermany

Personalised recommendations