Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction

  • Mathis Baalmann
  • Marcel Best
  • Richard WombacherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1728)


Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplAW37V) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif—a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DAinv). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

Key words

Site-specific protein labeling Bioorthogonal reactions Fluorescent probes Inverse-electron-demand Diels–Alder cycloaddition Ligases Tetrazines 



This work was supported by funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623, WO 1888/1-2). Furthermore, Mathis Baalmann acknowledges support from the Landesgradiuertenförderung Baden-Württemberg (LGF BW). The authors thank Stefanie Kühn, Hagen Sparka, and Tobias T. Schmidt for technical and experimental support.


  1. 1.
    Plass T, Milles S, Koehler C, Szymański J, Mueller R, Wießler M, Schultz C, Lemke EA (2012) Amino acids for Diels–Alder reactions in living cells. Angew Chem Int Ed 51:4166–4170CrossRefGoogle Scholar
  2. 2.
    Kaya E, Vrabel M, Deiml C, Prill S, Fluxa VS, Carell T (2012) A genetically encoded norbornene amino acid for the mild and selective modification of proteins in a copper-free click reaction. Angew Chem Int Ed 51:4466–4469CrossRefGoogle Scholar
  3. 3.
    Lang K, Davis L, Torres-Kolbus J, Chou C, Deiters A, Chin JW (2012) Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat Chem 4:298–304CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nakamura Y, Ito K, Isaksson LA (1996) Emerging understanding of translation termination. Cell 87:147–150CrossRefPubMedGoogle Scholar
  5. 5.
    Cronan JE, Zhao X, Jiang Y (2005) Function, attachment and synthesis of lipoic acid in Escherichia coli. In: Poole RK (ed) Advances in microbial physiology, vol 50. Academic Press, Cambridge, pp 103–146CrossRefGoogle Scholar
  6. 6.
    Fujiwara K, Toma S, Okamura-Ikeda K, Motokawa Y, Nakagawa A, Taniguchi H (2005) Crystal structure of lipoate-protein ligase a from Escherichia coli determination of the lipoic acid-binding site. J Biol Chem 280:33645–33651CrossRefPubMedGoogle Scholar
  7. 7.
    Fernandez-Suarez M, Baruah H, Martinez-Hernandez L, Xie KT, Baskin JM, Bertozzi CR, Ting AY (2007) Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotech 25:1483–1487CrossRefGoogle Scholar
  8. 8.
    Green D, Morris T, Green J, Cronan J, Guest J (1995) Purification and properties of the lipoate protein ligase of Escherichia coli. Biochem J 309:853–862CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Uttamapinant C, Sanchez MI, Liu DS, Yao JZ, White KA, Grecian S, Clark S, Gee KR, Ting AY (2013) Site-specific protein labeling using PRIME and chelation-assisted click chemistry. Nat Protoc 8:1620–1634CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Uttamapinant C, Tangpeerachaikul A, Grecian S, Clarke S, Singh U, Slade P, Gee KR, Ting AY (2012) Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling. Angew Chem Int Ed 51:5852–5856CrossRefGoogle Scholar
  11. 11.
    Yao JZ, Uttamapinant C, Poloukhtine A, Baskin JM, Codelli JA, Sletten EM, Bertozzi CR, Popik VV, Ting AY (2012) Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition. J Am Chem Soc 134:3720–3728CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cohen JD, Zou P, Ting AY (2012) Site-specific protein modification using lipoic acid ligase and bis-aryl hydrazone formation. Chembiochem 13:888–894CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Liu DS, Tangpeerachaikul A, Selvaraj R, Taylor MT, Fox JM, Ting AY (2012) Diels–Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J Am Chem Soc 134:792–795CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Karver MR, Weissleder R, Hilderbrand SA (2011) Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation. Bioconjug Chem 22:2263–2270CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Yang J, Šečkutė J, Cole CM, Devaraj NK (2012) Live-cell imaging of cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew Chem Int Ed 51:7476–7479CrossRefGoogle Scholar
  16. 16.
    Uttamapinant C, White KA, Baruah H, Thompson S, Fernández-Suárez M, Puthenveetil S, Ting AY (2010) A fluorophore ligase for site-specific protein labeling inside living cells. Proc Natl Acad Sci 107:10914–10919CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Puthenveetil S, Liu DS, White KA, Thompson S, Ting AY (2009) Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. J Am Chem Soc 131:16430–16438CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hauke S, Best M, Schmidt TT, Baalmann M, Krause A, Wombacher R (2014) Two-step protein labeling utilizing Lipoic acid ligase and Sonogashira cross-coupling. Bioconjug Chem 25:1632–1637CrossRefPubMedGoogle Scholar
  19. 19.
    Best M, Degen A, Baalmann M, Schmidt TT, Wombacher R (2015) Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction. Chembiochem 16:1158–1162CrossRefPubMedGoogle Scholar
  20. 20.
    Hermes FAM, Cronan JE (2009) Scavenging of cytosolic octanoic acid by mutant LplA lipoate ligases allows growth of Escherichia coli strains lacking the LipB octanoyltransferase of lipoic acid synthesis. J Bacteriol 191:6796–6803CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wombacher R, Cornish VW (2011) Chemical tags: applications in live cell fluorescence imaging. J Biophotonics 4:391–402CrossRefPubMedGoogle Scholar
  22. 22.
    Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1:13–21CrossRefPubMedGoogle Scholar
  23. 23.
    Patterson DM, Nazarova LA, Prescher JA (2014) Finding the right (bioorthogonal) chemistry. ACS Chem Biol 9:592–605CrossRefPubMedGoogle Scholar
  24. 24.
    Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction. Science 287:2007–2010CrossRefPubMedGoogle Scholar
  25. 25.
    Agard NJ, Prescher JA, Bertozzi CR (2004) A strain-promoted [3 + 2] Azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126:15046–15047CrossRefPubMedGoogle Scholar
  26. 26.
    Tornøe 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 67:3057–3064CrossRefPubMedGoogle Scholar
  27. 27.
    Chalker JM, Wood CSC, Davis BG (2009) A convenient catalyst for aqueous and protein Suzuki−Miyaura cross-coupling. J Am Chem Soc 131:16346–16347CrossRefPubMedGoogle Scholar
  28. 28.
    Kodama K, Fukuzawa S, Nakayama H, Sakamoto K, Kigawa T, Yabuki T, Matsuda N, Shirouzu M, Takio K, Yokoyama S (2007) Site-specific functionalization of proteins by Organopalladium reactions. Chembiochem 8:232–238CrossRefPubMedGoogle Scholar
  29. 29.
    Ourailidou ME, van der Meer JY, Baas BJ, Jeronimus-Stratingh M, Gottumukkala AL, Poelarends GJ, Minnaard AJ, Dekker FJ (2014) Aqueous oxidative heck reaction as a protein-labeling strategy. Chembiochem 15:209–212CrossRefPubMedGoogle Scholar
  30. 30.
    Blackman ML, Royzen M, Fox JM (2008) Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels−Alder reactivity. J Am Chem Soc 130:13518–13519CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Devaraj NK, Weissleder R, Hilderbrand SA (2008) Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug Chem 19:2297–2299CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Thalhammer F, Wallfahrer U, Sauer J (1990) Reaktivität einfacher offenkettiger und cyclischer dienophile bei Diels-Alder-reaktionen mit inversem elektronenbedarf. Tetrahedron Lett 31:6851–6854CrossRefGoogle Scholar
  33. 33.
    Rossin R, van den Bosch SM, ten Hoeve W, Carvelli M, Versteegen RM, Lub J, Robillard MS (2013) Highly reactive trans-Cyclooctene tags with improved stability for Diels–Alder chemistry in living systems. Bioconjug Chem 24:1210–1217CrossRefPubMedGoogle Scholar
  34. 34.
    Zeglis BM, Sevak KK, Reiner T, Mohindra P, Carlin SD, Zanzonico P, Weissleder R, Lewis JS (2013) A pretargeted PET imaging strategy based on bioorthogonal Diels–Alder click chemistry. J Nucl Med 54:1389–1396CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Knight JC, Richter S, Wuest M, Way JD, Wuest F (2013) Synthesis and evaluation of an 18F-labelled norbornene derivative for copper-free click chemistry reactions. Org Biomol Chem 11:3817–3825CrossRefPubMedGoogle Scholar
  36. 36.
    Schoch J, Staudt M, Samanta A, Wiessler M, Jäschke A (2012) Site-specific one-pot dual labeling of DNA by orthogonal cycloaddition chemistry. Bioconjug Chem 23:1382–1386CrossRefPubMedGoogle Scholar
  37. 37.
    Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333CrossRefPubMedGoogle Scholar
  38. 38.
    Zeglis BM, Emmetiere F, Pillarsetty N, Weissleder R, Lewis JS, Reiner T (2014) Building blocks for the construction of bioorthogonally reactive peptides via solid-phase peptide synthesis. ChemistryOpen 3:48–53CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Mathis Baalmann
    • 1
  • Marcel Best
    • 1
  • Richard Wombacher
    • 1
    Email author
  1. 1.Institute of Pharmacy and Molecular BiotechnologyRuprecht-Karls-Universität HeidelbergHeidelbergGermany

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