Site-Specific Protein Labeling pp 81-92

Part of the Methods in Molecular Biology book series (MIMB, volume 1266) | Cite as

2-Cyanobenzothiazole (CBT) Condensation for Site-Specific Labeling of Proteins at the Terminal Cysteine Residues

Protocol

Abstract

Site specificity is pivotal in obtaining homogeneously labeled proteins without batch-to-batch variations. More importantly, precisely controlled modification at specific sites avoids potential pitfalls that could otherwise interfere with protein folding, structure, and function. Inspired by the chemical synthesis of d-luciferin, we have developed an efficient strategy (second-order rate constant k2 = 9.2 M−1 s−1) for labeling of proteins containing 1,2-aminothiol via reaction with 2-cyanobenzothiazole (CBT). In addition, the CBT condensation enjoys the convenience of protein engineering, as production of N-terminal cysteine-containing proteins has been well developed for native chemical ligation. This protocol describes the preparation of Renilla luciferase (rLuc) with 1,2-aminothiol at either its N- or C-terminus, and site-specific labeling of rLuc with fluorescein or 18F via CBT condensation.

Key words

Site-specific protein labeling Terminal cysteine 1,2-Aminothiol 2-Cyanobenzothiazole (CBT) Fluorescence labeling Radiolabeling (18F) 

References

  1. 1.
    Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9(12):929–943. doi:10.1038/nrm2531 PubMedCrossRefGoogle Scholar
  2. 2.
    Carter P, Merchant AM (1997) Engineering antibodies for imaging and therapy. Curr Opin Biotechnol 8(4):449–454. doi:10.1016/s0958-1669(97)80067-5 PubMedCrossRefGoogle Scholar
  3. 3.
    Jing CR, Cornish VW (2011) Chemical tags for labeling proteins inside living cells. Acc Chem Res 44(9):784–792. doi:10.1021/ar200099f PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    White EH, McCapra F, Field GF, McElroy WD (1961) The structure and synthesis of firefly luciferin. J Am Chem Soc 83(10):2402–2403. doi:10.1021/ja01471a051 CrossRefGoogle Scholar
  5. 5.
    Ren HJ, Xiao F, Zhan K, Kim YP, Xie HX, Xia ZY, Rao J (2009) A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins. Angew Chem Int Ed 48(51):9658–9662. doi:10.1002/anie.200903627 CrossRefGoogle Scholar
  6. 6.
    Soellner MB, Nilsson BL, Raines RT (2006) Reaction mechanism and kinetics of the traceless staudinger ligation. J Am Chem Soc 128(27):8820–8828. doi:10.1021/ja060484k PubMedCrossRefGoogle Scholar
  7. 7.
    Dirksen A, Hackeng TM, Dawson PE (2006) Nucleophilic catalysis of oxime ligation. Angew Chem Int Ed 45(45):7581–7584. doi:10.1002/anie.200602877 CrossRefGoogle Scholar
  8. 8.
    Dirksen A, Dirksen S, Hackeng TM, Dawson PE (2006) Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J Am Chem Soc 128(49):15602–15603. doi:10.1021/ja067189k PubMedCrossRefGoogle Scholar
  9. 9.
    Sletten EM, Bertozzi CR (2011) From mechanism to mouse: a tale of two bioorthogonal reactions. Acc Chem Res 44(9):666–676. doi:10.1021/ar200148z PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Dawson PE, Muir TW, Clarklewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779PubMedCrossRefGoogle Scholar
  11. 11.
    Erlanson DA, Chytil M, Verdine GL (1996) The leucine zipper domain controls the orientation of AP-1 in the NFAT center dot AP-1 center dot DNA complex. Chem Biol 3(12):981–991. doi:10.1016/s1074-5521(96)90165-9 PubMedCrossRefGoogle Scholar
  12. 12.
    Tolbert TJ, Wong C-H (2002) New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation. Angew Chem Int Ed 41(12):2171–2174. doi:10.1002/1521-3773(20020617)41:12<2171::aid-anie2171>3.0.co;2-qCrossRefGoogle Scholar
  13. 13.
    Gentle IE, De Souza DP, Baca M (2004) Direct production of proteins with N-terminal cysteine for site-specific conjugation. Bioconjug Chem 15(3):658–663. doi:10.1021/bc049965o PubMedCrossRefGoogle Scholar
  14. 14.
    Nguyen DP, Elliott T, Holt M, Muir TW, Chin JW (2011) Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J Am Chem Soc 133(30):11418–11421. doi:10.1021/ja203111c PubMedCrossRefGoogle Scholar
  15. 15.
    Li X, Fekner T, Ottesen JJ, Chan MK (2009) A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem 121(48):9348–9351. doi:10.1002/ange.200904472 CrossRefGoogle Scholar
  16. 16.
    Muralidharan V, Muir TW (2006) Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Methods 3(6):429–438. doi:10.1038/nmeth886 PubMedCrossRefGoogle Scholar
  17. 17.
    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):6705–6710. doi:10.1073/pnas.95.12.6705 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Jeon J, Shen B, Xiong L, Miao Z, Lee KH, Rao J, Chin FT (2012) Efficient method for site-specific 18F-labeling of biomolecules using the rapid condensation reaction between 2-cyanobenzothiazole and cysteine. Bioconjug Chem 23(9):1902–1908. doi:10.1021/bc300273m PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Cheng Y, Peng H, Chen W, Ni N, Ke B, Dai C, Wang B (2013) Rapid and specific post-synthesis modification of DNA through a biocompatible condensation of 1,2-aminothiols with 2-cyanobenzothiazole. Chemistry 19(12):4036–4042. doi:10.1002/chem.201201677 PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Liang GL, Ren HJ, Rao JH (2010) A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat Chem 2(1):54–60. doi:10.1038/nchem.480 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Liang GL, Ronald J, Chen YX, Ye DJ, Pandit P, Ma ML, Rutt B, Rao JH (2011) Controlled self-assembling of gadolinium nanoparticles as smart molecular magnetic resonance imaging contrast agents. Angew Chem Int Ed 50(28):6283–6286. doi:10.1002/anie.201007018 CrossRefGoogle Scholar
  22. 22.
    Ye DJ, Liang GL, Ma ML, Rao JH (2011) Controlling intracellular macrocyclization for the imaging of protease activity. Angew Chem Int Ed 50(10):2275–2279. doi:10.1002/anie.201006140 CrossRefGoogle Scholar
  23. 23.
    Shen B, Jeon J, Palner M, Ye D, Shuhendler A, Chin FT, Rao J (2013) Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-triggered nanoaggregation probe. Angew Chem Int Ed 52(40):10511–10514. doi:10.1002/anie.201303422CrossRefGoogle Scholar
  24. 24.
    Ye D, Shuhendler AJ, Cui L, Tong L, Tee SS, Tikhomirov G, Felsher DW, Rao J (2014) Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in living mice. Nat Chem 6 (6):519–526. doi:10.1038/nchem.1920 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Ye D, Shuhendler AJ, Pandit P, Brewer KD, Tee SS, Cui L, Tikhomirov G, Rutt B, Rao J (2014) Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis. Chem Sci 5(10):3845–3852. doi:10.1039/c4sc01392a CrossRefGoogle Scholar
  26. 26.
    Van de Bittner GC, Bertozzi CR, Chang CJ (2013) Strategy for dual-analyte luciferin imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation. J Am Chem Soc 135(5):1783–1795. doi:10.1021/ja309078t PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Godinat A, Park HM, Miller SC, Cheng K, Hanahan D, Sanman LE, Bogyo M, Yu A, Nikitin GF, Stahl A, Dubikovskaya EA (2013) A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice. ACS Chem Biol. doi:10.1021/cb3007314 PubMedCentralPubMedGoogle Scholar
  28. 28.
    Loening AM, Fenn TD, Wu AM, Gambhir SS (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel 19(9):391–400. doi:10.1093/protein/gzl023 PubMedCrossRefGoogle Scholar
  29. 29.
    Schmidt MJ, Summerer D (2012) A need for speed: genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells. Chembiochem 13(11):1553–1557. doi:10.1002/cbic.201200321 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, School of MedicineStanford UniversityStanfordUSA

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