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Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids

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Noncanonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1728))

Abstract

Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.

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References

  1. Hackenberger CP, Schwarzer D (2008) Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed 47(52):10030–10074. https://doi.org/10.1002/anie.200801313

    Article  CAS  Google Scholar 

  2. Massa S, Xavier C, De Vos J, Caveliers V, Lahoutte T, Muyldermans S, Devoogdt N (2014) Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging. Bioconjug Chem 25(5):979–988. https://doi.org/10.1021/bc500111t

    Article  CAS  PubMed  Google Scholar 

  3. Schumacher D, Hackenberger CP (2014) More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins. Curr Opin Chem Biol 22:62–69. https://doi.org/10.1016/j.cbpa.2014.09.018

    Article  CAS  PubMed  Google Scholar 

  4. Pleiner T, Bates M, Trakhanov S, Lee C-T, Erik Schliep JE, Chug H, Böhning M, Stark H, Urlaub H, Görlich D (2015) Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. elife 4:e11349. https://doi.org/10.7554/eLife.11349

    Article  PubMed  PubMed Central  Google Scholar 

  5. Schumacher D, Hackenberger CP, Leonhardt H, Helma J (2016) Current status: site-specific antibody drug conjugates. J Clin Immunol 36:100. https://doi.org/10.1007/s10875-016-0265-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Massa S, Xavier C, Muyldermans S, Devoogdt N (2016) Emerging site-specific bioconjugation strategies for radioimmunotracer development. Expert Opin Drug Deliv 13(8):1149–1163. https://doi.org/10.1080/17425247.2016.1178235

    Article  CAS  PubMed  Google Scholar 

  7. Agrawal D, Hackenberger CPR (2013) Site-specific chemical modifications of proteins. Indian J Chem A 52(8–9):973–991

    Google Scholar 

  8. Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779

    Article  CAS  PubMed  Google Scholar 

  9. Muir TW (2003) Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 72:249–289. https://doi.org/10.1146/annurev.biochem.72.121801.161900

    Article  CAS  PubMed  Google Scholar 

  10. Budisa N (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew Chem Int Ed Engl 43(47):6426–6463. https://doi.org/10.1002/anie.200300646

    Article  CAS  PubMed  Google Scholar 

  11. Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444. https://doi.org/10.1146/annurev.biochem.052308.105824

    Article  CAS  PubMed  Google Scholar 

  12. Lotze J, Reinhardt U, Seitz O, Beck-Sickinger AG (2016) Peptide-tags for site-specific protein labelling in vitro and in vivo. Mol BioSyst 12:1731. https://doi.org/10.1039/c6mb00023a

    Article  CAS  PubMed  Google Scholar 

  13. Rashidian M, Dozier JK, Distefano MD (2013) Enzymatic labeling of proteins: techniques and approaches. Bioconjugate Chem 24(8):1277–1294. https://doi.org/10.1021/Bc400102w

    Article  CAS  Google Scholar 

  14. Schumacher D, Helma J, Mann FA, Pichler G, Natale F, Krause E, Cardoso MC, Hackenberger CP, Leonhardt H (2015) Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew Chem Int Ed Engl 54(46):13787–13791. https://doi.org/10.1002/anie.201505456

    Article  CAS  PubMed  Google Scholar 

  15. Patterson DM, Nazarova LA, Prescher JA (2014) Finding the right (bioorthogonal) chemistry. ACS Chem Biol 9(3):592–605. https://doi.org/10.1021/Cb400828a

    Article  CAS  PubMed  Google Scholar 

  16. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797. https://doi.org/10.1146/annurev-biochem-063011-092449

    Article  CAS  PubMed  Google Scholar 

  17. Baskin JM, Prescher JA, Laughlin ST, Agard NJ, Chang PV, Miller IA, Lo A, Codelli JA, Bertozzi CR (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A 104(43):16793–16797. https://doi.org/10.1073/pnas.0707090104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Serwa R, Wilkening I, Del Signore G, Muhlberg M, Claussnitzer I, Weise C, Gerrits M, Hackenberger CP (2009) Chemoselective staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem Int Ed Engl 48(44):8234–8239. https://doi.org/10.1002/anie.200902118

    Article  CAS  PubMed  Google Scholar 

  19. Bohrsch V, Serwa R, Majkut P, Krause E, Hackenberger CP (2010) Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites. Chem Commun (Camb) 46(18):3176–3178. https://doi.org/10.1039/b926818a

    Article  Google Scholar 

  20. Hoffmann E, Streichert K, Nischan N, Seitz C, Brunner T, Schwagerus S, Hackenberger CP, Rubini M (2016) Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites. Mol BioSyst 12(6):1750–1755. https://doi.org/10.1039/c5mb00857c

    Article  CAS  PubMed  Google Scholar 

  21. Nischan N, Chakrabarti A, Serwa RA, Bovee-Geurts PH, Brock R, Hackenberger CP (2013) Stabilization of peptides for intracellular applications by phosphoramidate-linked polyethylene glycol chains. Angew Chem Int Ed Engl 52(45):11920–11924. https://doi.org/10.1002/anie.201303467

    Article  CAS  PubMed  Google Scholar 

  22. Nischan N, Kasper MA, Mathew T, Hackenberger CP (2016) Bis(arylmethyl)-substituted unsymmetrical phosphites for the synthesis of lipidated peptides via Staudinger-phosphite reactions. Org Biomol Chem 14(31):7500–7508. https://doi.org/10.1039/c6ob00843g

    Article  CAS  PubMed  Google Scholar 

  23. Vallee MR, Artner LM, Dernedde J, Hackenberger CP (2013) Alkyne phosphonites for sequential azide-azide couplings. Angew Chem Int Ed Engl 52(36):9504–9508. https://doi.org/10.1002/anie.201302462

    Article  CAS  PubMed  Google Scholar 

  24. Vallee MR, Majkut P, Krause D, Gerrits M, Hackenberger CP (2015) Chemoselective bioconjugation of triazole phosphonites in aqueous media. Chemistry 21(3):970–974. https://doi.org/10.1002/chem.201404690

    Article  CAS  PubMed  Google Scholar 

  25. Vallee MR, Majkut P, Wilkening I, Weise C, Muller G, Hackenberger CP (2011) Staudinger-phosphonite reactions for the chemoselective transformation of azido-containing peptides and proteins. Org Lett 13(20):5440–5443. https://doi.org/10.1021/ol2020175

    Article  CAS  PubMed  Google Scholar 

  26. Dirksen A, Dawson PE (2008) Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjug Chem 19(12):2543–2548. https://doi.org/10.1021/bc800310p

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li N, Lim RK, Edwardraja S, Lin Q (2011) Copper-free sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells. J Am Chem Soc 133(39):15316–15319. https://doi.org/10.1021/ja2066913

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, Pellis M, Muyldermans S, Casas-Delucchi CS, Cardoso MC, Leonhardt H, Hopfner KP, Rothbauer U (2010) Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17(1):133–138. https://doi.org/10.1038/nsmb.1727

    Article  CAS  PubMed  Google Scholar 

  29. Jung ME, Lazarova TI (1997) Efficient synthesis of selectively protected L-Dopa derivatives from L-tyrosine via Reimer-Tiemann and Dakin reactions. J Org Chem 62(5):1553–1555. https://doi.org/10.1021/Jo962099r

    Article  CAS  Google Scholar 

  30. Banerjee A, Panosian TD, Mukherjee K, Ravindra R, Gal S, Sackett DL, Bane S (2010) Site-specific orthogonal labeling of the carboxy terminus of alpha-tubulin. ACS Chem Biol 5(8):777–785. https://doi.org/10.1021/cb100060v

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chalker JM, Wood CS, Davis BG (2009) A convenient catalyst for aqueous and protein Suzuki-Miyaura cross-coupling. J Am Chem Soc 131(45):16346–16347. https://doi.org/10.1021/ja907150m

    Article  CAS  PubMed  Google Scholar 

  32. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96(1):23–28

    Article  CAS  PubMed  Google Scholar 

  33. Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP1623) to C.P.R.H. (HA 4468/9-1), and H.L. (LE 721/13-1), the Nano- systems Initiative Munich (NIM) to H.L., the Einstein Foundation Berlin (Leibniz-Humboldt Professorship) and the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H. and the Fonds der Chemischen Industrie (FCI) to C.P.R.H. and to D.S. (Kekulé-scholarship).

Author information

Authors and Affiliations

  1. Department of Biology II, Center for Integrated Protein Science Munich, Ludwig Maximilians Universität München, Planegg-Martinsried, Germany

    Jonas Helma & Heinrich Leonhardt

  2. Department of Chemical-Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

    Christian P. R. Hackenberger & Dominik Schumacher

  3. Department of Chemistry, Humboldt Universität zu Berlin, Berlin, Germany

    Christian P. R. Hackenberger & Dominik Schumacher

Authors
  1. Jonas Helma
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  2. Heinrich Leonhardt
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  3. Christian P. R. Hackenberger
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  4. Dominik Schumacher
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Corresponding author

Correspondence to Dominik Schumacher .

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Editors and Affiliations

  1. Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany

    Edward A. Lemke

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Helma, J., Leonhardt, H., Hackenberger, C.P.R., Schumacher, D. (2018). Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7574-7_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7573-0

  • Online ISBN: 978-1-4939-7574-7

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