Skip to main content
SpringerLink
Log in

Conditional Toxin Splicing Using a Split Intein System

  • Protocol
  • First Online:
Split Inteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1495))

Abstract

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin–intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin–intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Araki K, Araki M, Yamamura K (2006) Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells. J Biochem 140(6):793–798

    Article  CAS  PubMed  Google Scholar 

  2. Fraser B, DuVal MG, Wang H, Allison WT (2013) Regeneration of cone photoreceptors when cell ablation is primarily restricted to a particular cone subtype. PLoS One 8(1):e55410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Schuldiner M, Itskovitz-Eldor J, Benvenisty N (2003) Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells 21(3):257–265

    Article  CAS  PubMed  Google Scholar 

  4. Tanoue S, Krishnan P, Krishnan B, Dryer SE, Hardin PE (2004) Circadian clocks in antennal neurons are necessary and sufficient for olfaction rhythms in Drosophila. Curr Biol 14(8):638–649

    Article  CAS  PubMed  Google Scholar 

  5. Cui W, Gusterson B, Clark AJ (1999) Nitroreductase-mediated cell ablation is very rapid and mediated by a p53-independent apoptotic pathway. Gene Ther 6(5):764–770

    Article  CAS  PubMed  Google Scholar 

  6. Denny WA (2003) Prodrugs for gene-directed enzyme-prodrug therapy (suicide gene therapy). J Biomed Biotechnol 2003(1):48–70

    Article  PubMed  PubMed Central  Google Scholar 

  7. Denny WA (2004) Tumor-activated prodrugs--a new approach to cancer therapy. Cancer Invest 22(4):604–619

    Article  CAS  PubMed  Google Scholar 

  8. Kirn D, Niculescu-Duvaz I, Hallden G, Springer CJ (2002) The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 8(4 Suppl):S68–S73

    Article  CAS  PubMed  Google Scholar 

  9. Malecki M (2012) Frontiers in suicide gene therapy of cancer. J Genet Syndr Gene Ther 2012(3):e114

    Google Scholar 

  10. Ciceri F, Bonini C, Gallo-Stampino C, Bordignon C (2005) Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation. Cytotherapy 7(2):144–149

    Article  CAS  PubMed  Google Scholar 

  11. Qasim W, Gaspar HB, Thrasher AJ (2005) T cell suicide gene therapy to aid haematopoietic stem cell transplantation. Curr Gene Ther 5(1):121–132

    Article  CAS  PubMed  Google Scholar 

  12. Alford SC, O’Sullivan C, Obst J, Christie J, Howard PL (2014) Conditional protein splicing of alpha-sarcin in live cells. Mol Biosyst 10(4):831–837

    Article  CAS  PubMed  Google Scholar 

  13. Eiklid K, Olsnes S, Pihl A (1980) Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells. Exp Cell Res 126(2):321–326

    Article  CAS  PubMed  Google Scholar 

  14. Jennings JC, Olson BH, Roga V, Junek AJ, Schuurmans DM (1965) Alpha sarcin, a new antitumor agent. II. Fermentation and antitumor spectrum. Appl Microbiol 13:322–326

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Endo Y, Huber PW, Wool IG (1983) The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates. J Biol Chem 258(4):2662–2667

    CAS  PubMed  Google Scholar 

  16. Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5(1):446–461

    Article  CAS  PubMed  Google Scholar 

  17. Elleuche S, Poggeler S (2010) Inteins, valuable genetic elements in molecular biology and biotechnology. Appl Microbiol Biotechnol 87(2):479–489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Eryilmaz E, Shah NH, Muir TW, Cowburn D (2014) Structural and dynamical features of inteins and implications on protein splicing. J Biol Chem 289(21):14506–14511

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Amitai G, Callahan BP, Stanger MJ, Belfort G, Belfort M (2009) Modulation of intein activity by its neighboring extein substrates. Proc Natl Acad Sci U S A 106(27):11005–11010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Xu MQ, Perler FB (1996) The mechanism of protein splicing and its modulation by mutation. EMBO J 15(19):5146–5153

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Appleby-Tagoe JH, Thiel IV, Wang Y, Wang Y, Mootz HD, Liu XQ (2011) Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. J Biol Chem 286(39):34440–34447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chong S, Williams KS, Wotkowicz C, Xu MQ (1998) Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J Biol Chem 273(17):10567–10577

    Article  CAS  PubMed  Google Scholar 

  23. Binschik J, Zettler J, Mootz HD (2011) Photocontrol of protein activity mediated by the cleavage reaction of a split intein. Angew Chem Int Ed Engl 50(14):3249–3252

    Article  CAS  PubMed  Google Scholar 

  24. Tyszkiewicz AB, Muir TW (2008) Activation of protein splicing with light in yeast. Nat Methods 5(4):303–305

    CAS  PubMed  Google Scholar 

  25. Buskirk AR, Ong YC, Gartner ZJ, Liu DR (2004) Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc Natl Acad Sci U S A 101(29):10505–10510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Peck SH, Chen I, Liu DR (2011) Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells. Chem Biol 18(5):619–630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yuen CM, Rodda SJ, Vokes SA, McMahon AP, Liu DR (2006) Control of transcription factor activity and osteoblast differentiation in mammalian cells using an evolved small-molecule-dependent intein. J Am Chem Soc 128(27):8939–8946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Berrade L, Kwon Y, Camarero JA (2010) Photomodulation of protein trans-splicing through backbone photocaging of the DnaE split intein. Chembiochem 11(10):1368–1372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wong CC, Traynor D, Basse N, Kay RR, Warren AJ (2011) Defective ribosome assembly in Shwachman-Diamond syndrome. Blood 118(16):4305–4312

    Article  CAS  PubMed  Google Scholar 

  30. Wood DW, Wu W, Belfort G, Derbyshire V, Belfort M (1999) A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol 17(9):889–892

    Article  CAS  PubMed  Google Scholar 

  31. Zeidler MP, Tan C, Bellaiche Y, Cherry S, Hader S, Gayko U, Perrimon N (2004) Temperature-sensitive control of protein activity by conditionally splicing inteins. Nat Biotechnol 22(7):871–876

    Article  CAS  PubMed  Google Scholar 

  32. Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A 95(16):9226–9231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mootz HD, Blum ES, Tyszkiewicz AB, Muir TW (2003) Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 125(35):10561–10569

    Article  CAS  PubMed  Google Scholar 

  34. Mootz HD, Muir TW (2002) Protein splicing triggered by a small molecule. J Am Chem Soc 124(31):9044–9045

    Article  CAS  PubMed  Google Scholar 

  35. Shi J, Muir TW (2005) Development of a tandem protein trans-splicing system based on native and engineered split inteins. J Am Chem Soc 127(17):6198–6206

    Article  CAS  PubMed  Google Scholar 

  36. Zettler J, Schütz 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):909–914

    Article  CAS  PubMed  Google Scholar 

  37. Kanno A, Ozawa T, Umezawa Y (2006) Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells. Anal Chem 78(2):556–560

    Article  CAS  PubMed  Google Scholar 

  38. Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y (2000) A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal Chem 72(21):5151–5157

    Article  CAS  PubMed  Google Scholar 

  39. Ozawa T, Takeuchi TM, Kaihara A, Sato M, Umezawa Y (2001) Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem 73(24): 5866–5874

    Article  CAS  PubMed  Google Scholar 

  40. Ozawa T, Kaihara A, Sato M, Tachihara K, Umezawa Y (2001) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem 73(11):2516–2521

    Article  CAS  PubMed  Google Scholar 

  41. Sonntag T, Mootz HD (2011) An intein-cassette integration approach used for the generation of a split TEV protease activated by conditional protein splicing. Mol Biosyst 7(6):2031–2039

    Article  CAS  PubMed  Google Scholar 

  42. Narayanan S, Surendranath K, Bora N, Surolia A, Karande AA (2005) Ribosome inactivating proteins and apoptosis. FEBS Lett 579(6):1324–1331

    Article  CAS  PubMed  Google Scholar 

  43. Alford SC, Pearson JD, Carette A, Ingham RJ, Howard PL (2009) Alpha-sarcin catalytic activity is not required for cytotoxicity. BMC Biochem 10:9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by an NSERC Discovery Grant to PLH. The NMBP and CHis constructs described were the kind gift of Dr. Tom Muir.

Author information

Author notes

    Authors and Affiliations

    1. Department of Bioengineering, Stanford University, Stanford, CA, USA

      Spencer C. Alford

    2. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada

      Spencer C. Alford, Connor O’Sullivan & Perry L. Howard

    Authors
    1. Spencer C. Alford
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Connor O’Sullivan
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Perry L. Howard
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Perry L. Howard .

    Editor information

    Editors and Affiliations

    1. Institute of Biochemistry Department of Chemistry and Pharmacy, University of Muenster, Münster, Germany

      Henning D. Mootz

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2017 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    Alford, S.C., O’Sullivan, C., Howard, P.L. (2017). Conditional Toxin Splicing Using a Split Intein System. In: Mootz, H. (eds) Split Inteins. Methods in Molecular Biology, vol 1495. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6451-2_13

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-6451-2_13

    • Published:

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6449-9

    • Online ISBN: 978-1-4939-6451-2

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation