Advertisement

In Vivo Histone Labeling Using Ultrafast trans-Splicing Inteins

  • Nicholas A. Prescott
  • Yael DavidEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2133)

Abstract

The development of expressed protein ligation (EPL) widened the scope of questions that could be addressed by mechanistic biochemistry. Protein trans-splicing (PTS) relies on the same basic chemical principles, but utilizes split inteins to tracelessly ligate distinct peptide or polypeptide fragments together with native peptide bonds. Here we present a method to adapt PTS methodologies for their use in live cells, in order to deliver synthetic or native histone modifications. As an example, we provide a protocol to incorporate a small molecule fluorophore into chromatinized histones. The protocol should be easily adaptable to incorporate other modifications to chromatin in vivo.

Key words

Split inteins In cellulo labeling Synthetic biology Chromatin 

Notes

Acknowledgments

Work in the David lab is supported by the Josie Robertson Foundation, the Pershing Square Sohn Cancer Alliance, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Center for Experimental Therapeutics at Memorial Sloan Kettering Cancer Center, the CCSG core grant P30 CA008748, and the NIH CEBRA award # DA044767. N.A.P. is supported by the NIH T32 GM115327-Tan chemistry-biology interface training grant and the National Science Foundation Graduate Research Fellowship Grant Number 2017239554.

References

  1. 1.
    Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci U S A 95:6705–6710CrossRefGoogle Scholar
  2. 2.
    Vila-Perelló M, Muir TW (2010) Biological applications of protein splicing. Cell 143:191–200CrossRefGoogle Scholar
  3. 3.
    Perler FB (2002) InBase: the intein database. Nucleic Acids Res 30:383–384CrossRefGoogle Scholar
  4. 4.
    Shah NH, Dann GP, Vila-Perelló M et al (2012) Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering. J Am Chem Soc 134:11338–11341CrossRefGoogle Scholar
  5. 5.
    Shah NH, Muir TW (2011) Split inteins: nature’s protein ligases. Isr J Chem 51:854–861CrossRefGoogle Scholar
  6. 6.
    Mootz HD (2009) Split inteins as versatile tools for protein semisynthesis. Chembiochem 10:2579–2589CrossRefGoogle Scholar
  7. 7.
    Vila-Perelló M, Liu Z, Shah NH et al (2013) Streamlined expressed protein ligation using split inteins. J Am Chem Soc 135:286–292CrossRefGoogle Scholar
  8. 8.
    Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5:446–461CrossRefGoogle Scholar
  9. 9.
    Juillerat A, Gronemeyer T, Keppler A et al (2003) Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10:313–317CrossRefGoogle Scholar
  10. 10.
    Los GV, Encell LP, McDougall MG et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382CrossRefGoogle Scholar
  11. 11.
    Giriat I, Muir TW (2003) Protein semi-synthesis in living cells. J Am Chem Soc 125:7180–7181CrossRefGoogle Scholar
  12. 12.
    Mootz HD, Blum ES, Tyszkiewicz AB et al (2003) Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 125:10561–10569CrossRefGoogle Scholar
  13. 13.
    David Y, Vila-Perelló M, Verma S et al (2015) Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nat Chem 7:394–402CrossRefGoogle Scholar
  14. 14.
    Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10:310–315CrossRefGoogle Scholar
  15. 15.
    Hoye AT, Davoren JE, Wipf P et al (2008) Targeting mitochondria. Acc Chem Res 41:87–97CrossRefGoogle Scholar
  16. 16.
    Yu H-C, Lu M-C, Li C et al (2013) Targeted delivery of an antigenic peptide to the endoplasmic reticulum: application for development of a peptide therapy for ankylosing spondylitis. PLoS One 8:e77451CrossRefGoogle Scholar
  17. 17.
    Stevens AJ, Sekar G, Shah NH et al (2017) A promiscuous split intein with expanded protein engineering applications. Proc Natl Acad Sci 114:8538–8543CrossRefGoogle Scholar
  18. 18.
    Suhorutsenko J, Oskolkov N, Arukuusk P et al (2011) Cell-penetrating peptides, PepFects, show no evidence of toxicity and immunogenicity in vitro and in vivo. Bioconjug Chem 22:2255–2262CrossRefGoogle Scholar
  19. 19.
    Guidotti G, Brambilla L, Rossi D (2017) Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol Sci 38:406–424CrossRefGoogle Scholar
  20. 20.
    García-Martín F, Quintanar-Audelo M, García-Ramos Y et al (2006) ChemMatrix, a Poly(ethylene glycol)-based support for the solid-phase synthesis of complex peptides. J Comb Chem 8:213–220CrossRefGoogle Scholar
  21. 21.
    Stevens AJ, Brown ZZ, Shah NH et al (2016) Design of a split intein with exceptional protein splicing activity. J Am Chem Soc 138:2162–2165CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Tri-Institutional PhD Program in Chemical BiologyNew YorkUSA
  2. 2.Chemical Biology Program, Memorial Sloan Kettering Cancer CenterNew YorkUSA
  3. 3.Department of PharmacologyWeill Cornell MedicineNew YorkUSA
  4. 4.Department of Physiology, Biophysics and Systems BiologyWeill Cornell MedicineNew YorkUSA

Personalised recommendations