Chemical-Tag Labeling of Proteins Using Fully Recombinant Split Inteins

  • Anne-Lena Bachmann
  • Julian C. J. Matern
  • Vivien Schütz
  • Henning D. Mootz
Part of the Methods in Molecular Biology book series (MIMB, volume 1266)


Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.

Key words

Bioconjugation Protein labeling Intein Protein splicing Click chemistry Synthetic label Protein expression Fluorophore 



We thank Peter G. Schultz (The Scripps Research Institute) for providing plasmids for AzF incorporation. Financial support was kindly provided by the DFG (SPP1623).


  1. 1.
    Hermanson GT (2010) Bioconjugate techniques, 2nd edn. Elsevier Science, BurlingtonGoogle Scholar
  2. 2.
    Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7(12):1381–1386PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Stephanopoulos N, Francis MB (2011) Choosing an effective protein bioconjugation strategy. Nat Chem Biol 7(12):876–884PubMedCrossRefGoogle Scholar
  4. 4.
    Lin YA, Chalker JM, Davis BG (2009) Olefin metathesis for site-selective protein modification. Chembiochem 10(6):959–969PubMedCrossRefGoogle Scholar
  5. 5.
    Chalker JM, Bernardes GJ, Lin YA, Davis BG (2009) Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chem Asian J 4(5):630–640PubMedCrossRefGoogle Scholar
  6. 6.
    Griffin BA, Adams SR, Jones J, Tsien RY (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol 327:565–578PubMedCrossRefGoogle Scholar
  7. 7.
    Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15(2):128–136PubMedCrossRefGoogle Scholar
  8. 8.
    Los GV, Wood K (2007) The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol Biol 356:195–208PubMedGoogle Scholar
  9. 9.
    Chen Z, Jing C, Gallagher SS, Sheetz MP, Cornish VW (2012) Second-generation covalent TMP-tag for live cell imaging. J Am Chem Soc 134(33):13692–13699PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Mizukami S, Watanabe S, Hori Y, Kikuchi K (2009) Covalent protein labeling based on noncatalytic beta-lactamase and a designed FRET substrate. J Am Chem Soc 131(14):5016–5017PubMedCrossRefGoogle Scholar
  11. 11.
    Mao H, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc 126(9):2670–2671PubMedCrossRefGoogle Scholar
  12. 12.
    Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(11):707–708PubMedCrossRefGoogle Scholar
  13. 13.
    Chen I, Howarth M, Lin W, Ting AY (2005) Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods 2(2):99–104PubMedCrossRefGoogle Scholar
  14. 14.
    Uttamapinant C, Sanchez MI, Liu DS, Yao JZ, Ting AY (2013) Site-specific protein labeling using PRIME and chelation-assisted click chemistry. Nat Protoc 8(8):1620–1634PubMedCrossRefGoogle Scholar
  15. 15.
    Yin J, Straight PD, McLoughlin SM, Zhou Z, Lin AJ, Golan DE, Kelleher NL, Kolter R, Walsh CT (2005) Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc Natl Acad Sci U S A 102(44):15815–15820PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779PubMedCrossRefGoogle Scholar
  17. 17.
    Muir TW (2003) Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 72:249–289PubMedCrossRefGoogle Scholar
  18. 18.
    Hackenberger CP, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl 47(52):10030–10074PubMedCrossRefGoogle Scholar
  19. 19.
    Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444PubMedCrossRefGoogle Scholar
  20. 20.
    Davis L, Chin JW (2012) Designer proteins: applications of genetic code expansion in cell biology. Nat Rev Mol Cell Biol 13(3):168–182PubMedGoogle Scholar
  21. 21.
    Schütz V, Mootz HD (2014) Click-tag and amine-tag: new chemical tag approaches for efficient protein labeling in vitro and on live cells using the naturally split Npu DnaE intein. Angew Chem Int Ed Engl 53:4113–4117PubMedCrossRefGoogle Scholar
  22. 22.
    Volkmann G, Mootz HD (2013) Recent progress in intein research: from mechanism to directed evolution and applications. Cell Mol Life Sci 70(7):1185–1206PubMedCrossRefGoogle Scholar
  23. 23.
    Noren CJ, Wang J, Perler FB (2000) Dissecting the chemistry of protein splicing and its applications. Angew Chem Int Ed Engl 39(3):450–466PubMedCrossRefGoogle Scholar
  24. 24.
    Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5:446–461PubMedCrossRefGoogle Scholar
  25. 25.
    Dhar T, Kurpiers T, Mootz HD (2011) Extending the scope of site-specific cysteine bioconjugation by appending a prelabeled cysteine tag to proteins using protein trans-splicing. Methods Mol Biol 751:131–142PubMedCrossRefGoogle Scholar
  26. 26.
    Kurpiers T, Mootz HD (2008) Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein. Chembiochem 9(14):2317–2325PubMedCrossRefGoogle Scholar
  27. 27.
    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
  28. 28.
    Brenzel S, Cebi M, Reiss P, Koert U, Mootz HD (2009) Expanding the scope of protein trans-splicing to fragment ligation of an integral membrane protein: towards modulation of porin-based ion channels by chemical modification. Chembiochem 10(6):983–986PubMedCrossRefGoogle Scholar
  29. 29.
    Dassa B, London N, Stoddard BL, Schueler-Furman O, Pietrokovski S (2009) Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family. Nucleic Acids Res 37(8):2560–2573PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Carvajal-Vallejos P, Pallisse R, Mootz HD, Schmidt SR (2012) Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem 287(34):28686–28696PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    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 53(5):1306–1310PubMedCrossRefGoogle Scholar
  32. 32.
    Shah NH, Dann GP, Vila-Perello M, Liu Z, Muir TW (2012) Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering. J Am Chem Soc 134(28):11338–11341PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Iwai H, Zuger S, Jin J, Tam PH (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett 580(7):1853–1858PubMedCrossRefGoogle Scholar
  34. 34.
    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):909–914PubMedCrossRefGoogle Scholar
  35. 35.
    Shah NH, Vila-Perello M, Muir TW (2011) Kinetic control of one-pot trans-splicing reactions by using a wild-type and designed split intein. Angew Chem Int Ed Engl 50(29):6511–6515PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Mohlmann S, Bringmann P, Greven S, Harrenga A (2011) Site-specific modification of ED-B-targeting antibody using intein-fusion technology. BMC Biotechnol 11:76PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Dhar T, Mootz HD (2011) Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein. Chem Commun (Camb) 47(11):3063–3065CrossRefGoogle Scholar
  38. 38.
    Vila-Perello M, Liu Z, Shah NH, Willis JA, Idoyaga J, Muir TW (2013) Streamlined expressed protein ligation using split inteins. J Am Chem Soc 135(1):286–292PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    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):6344–6353PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Subramanyam P, Chang DD, Fang K, Xie W, Marks AR, Colecraft HM (2013) Manipulating l-type calcium channels in cardiomyocytes using split-intein protein transsplicing. Proc Natl Acad Sci U S A 110(38):15461–15466PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Ramirez M, Valdes N, Guan D, Chen Z (2013) Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification. Protein Eng Des Sel 26(3):215–223PubMedCrossRefGoogle Scholar
  42. 42.
    Matern CJ, Bachmann A-L, Thiel IV, Volkmann G, Wasmuth A, Binschik J, Mootz HD (2014) Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing. In: Gautier A, Hinner M (eds) Site-specific protein labeling. Methods Mol Biol 1266:129–143Google Scholar
  43. 43.
    Mootz HD (2009) Split inteins as versatile tools for protein semisynthesis. Chembiochem 10(16):2579–2589PubMedCrossRefGoogle Scholar
  44. 44.
    Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of p-azido-l-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124(31):9026–9027PubMedCrossRefGoogle Scholar
  45. 45.
    Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395(2):361–374PubMedCrossRefGoogle Scholar
  46. 46.
    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
  47. 47.
    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
  48. 48.
    Tornoe CW, Christensen C, Meldal M (2002) 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(9):3057–3064PubMedCrossRefGoogle Scholar
  49. 49.
    Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002) A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl 41(14):2596–2599PubMedCrossRefGoogle Scholar
  50. 50.
    de Almeida G, Sletten EM, Nakamura H, Palaniappan KK, Bertozzi CR (2012) Thiacycloalkynes for copper-free click chemistry. Angew Chem Int Ed Engl 51(10):2443–2447PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Agard NJ, Baskin JM, Prescher JA, Lo A, Bertozzi CR (2006) A comparative study of bioorthogonal reactions with azides. ACS Chem Biol 1(10):644–648PubMedCrossRefGoogle Scholar
  52. 52.
    Yang H, Srivastava P, Zhang C, Lewis JC (2014) A general method for artificial metalloenzyme formation through strain-promoted azide-alkyne cycloaddition. Chembiochem 15(2):223–227PubMedCrossRefGoogle Scholar
  53. 53.
    Shah NH, Eryilmaz E, Cowburn D, Muir TW (2013) Naturally split inteins assemble through a “capture and collapse” mechanism. J Am Chem Soc 135(49):18673–18681PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anne-Lena Bachmann
    • 1
  • Julian C. J. Matern
    • 1
  • Vivien Schütz
    • 1
  • Henning D. Mootz
    • 1
  1. 1.Department of Chemistry and Pharmacy, Institute of BiochemistryUniversity of MuensterMünsterGermany

Personalised recommendations