Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed

Nature Protocols volume 1, pages 947–953 (2006)Cite this article

Abstract

The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectral characteristics of KillerRed.
Figure 2: Potential applications of KillerRed.
Figure 3: Light-induced killing of HeLa cell expressing mitochondrially targeted KillerRed.
Figure 4: Light-induced killing of HeLa cell expressing membrane-targeted KillerRed.
Figure 5: KillerRed-mediated CALI of PLC δ1 PH domain.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Lippincott-Schwartz, J. & Patterson, G.H. Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).

    Article  CAS  Google Scholar 

  2. Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 23, 605–613 (2005).

    Article  CAS  Google Scholar 

  3. Surrey, T. et al. Chromophore-assisted light inactivation and self-organization of microtubules and motors. Proc. Natl. Acad. Sci. USA 95, 4293–4298 (1998).

    Article  CAS  Google Scholar 

  4. Bulina, M.E. et al. A genetically encoded photosensitizer. Nat. Biotechnol. 24, 95–99 (2006).

    Article  CAS  Google Scholar 

  5. Eustace, B.K., Buchstaller, A. & Jay, D.G. Adapting chromophore-assisted laser inactivation for high throughput functional proteomics. Brief Funct. Genomic Proteomic 1, 257–265 (2002).

    Article  CAS  Google Scholar 

  6. Huang, Z. A review of progress in clinical photodynamic therapy. Technol. Cancer Res. Treat. 4, 283–293 (2005).

    Article  CAS  Google Scholar 

  7. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  8. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  9. Grate, D. & Wilson, C. Laser-mediated, site-specific inactivation of RNA transcripts. Proc. Natl. Acad. Sci. USA 96, 6131–6136 (1999).

    Article  CAS  Google Scholar 

  10. Tsien, R.Y. Imagining imaging's future. Nat. Rev. Mol. Cell Biol. Suppl: SS16–21 (2003).

  11. Grabenbauer, M. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods 2, 857–862 (2005).

    Article  CAS  Google Scholar 

  12. Filippin, L. et al. Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix. Cell Calcium 37, 129–136 (2005).

    Article  CAS  Google Scholar 

  13. Iinuma, S. et al. In vivo fluence rate and fractionation effects on tumor response and photobleaching: photodynamic therapy with two photosensitizers in an orthotopic rat tumor model. Cancer Res. 59, 6164–6170 (1999).

    CAS  PubMed  Google Scholar 

  14. Skene, J.H. & Virag, I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J. Cell Biol. 108, 613–624 (1989).

    Article  CAS  Google Scholar 

  15. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  16. Fradkov, A.F. et al. Far-red fluorescent tag for protein labelling. Biochem. J. 368, 17–21 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Howard Hughes Medical Institute (HHMI 55005618), molecular and cell biology program RAS and EC FP-6 integrated project LSHG-CT-2003-503259. D.M.C. is supported by grants of the president of Russian Federation MK-8236.2006.4 and Russian Science Support Foundation.

Author information

Authors and Affiliations

  1. Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow, 117997, Russia

    Maria E Bulina, Konstantin A Lukyanov, Olga V Britanova, Daria Onichtchouk, Sergey Lukyanov & Dmitriy M Chudakov

Authors
  1. Maria E Bulina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Konstantin A Lukyanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olga V Britanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daria Onichtchouk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sergey Lukyanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dmitriy M Chudakov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Konstantin A Lukyanov.

Ethics declarations

Competing interests

K.A.L. and D.M.C. have a pending patent application on KillerRed uses.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bulina, M., Lukyanov, K., Britanova, O. et al. Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed. Nat Protoc 1, 947–953 (2006). https://doi.org/10.1038/nprot.2006.89

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.89

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing