Skip to main content

Thank you for visiting 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.

A genetically encoded photosensitizer


Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation1. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to β-galactosidase and phospholipase Cδ1 pleckstrin homology domain.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Identification and characterization of the phototoxic properties of KillerRed.
Figure 2: Light-induced killing of eukaryotic cells expressing KillerRed.
Figure 3: KillerRed-mediated light-induced inactivation of the PLC δ1 PH domain.

Accession codes




  1. 1

    Liao, J.C., Roider, J. & Jay, D.G. Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals. Proc. Natl. Acad. Sci. USA 91, 2659–2663 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Beermann, A.E. & Jay, D.G. Chromophore-assisted laser inactivation of cellular proteins. Methods. Cell. Biol. 44, 715–732 (1994).

    CAS  Article  Google Scholar 

  3. 3

    Jay, D.G. Selective destruction of protein function by chromophore-assisted laser inactivation. Proc. Natl. Acad. Sci. USA 85, 5454–5458 (1988).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Wong, E.V., David, S., Jacob, M.H. & Jay, D.G. Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration. J. Neurosci. 23, 3112–3117 (2003).

    CAS  Article  Google Scholar 

  6. 6

    Rubenwolf, S. et al. Functional proteomics using chromophore-assisted laser inactivation. Proteomics 2, 241–246 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Rajfur, Z., Roy, P., Otey, C., Romer, L. & Jacobson, K. Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat. Cell Biol. 4, 286–293 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Hauptschein, R.S. et al. Functional proteomic screen identifies a modulating role for CD44 in death receptor-mediated apoptosis. Cancer Res. 65, 1887–1896 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Moor, A.C. Signaling pathways in cell death and survival after photodynamic therapy. J. Photochem. Photobiol. B 57, 1–13 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Plaetzer, K., Kiesslich, T., Krammer, B. & Hammerl, P. Characterization of the cell death modes and the associated changes in cellular energy supply in response to AlPcS4-PDT. Photochem. Photobiol. Sci. 1, 172–177 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Cho, K.S., Lee, E.H., Choi, J.S. & Joo, C.K. Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 40, 911–919 (1999).

    CAS  Google Scholar 

  12. 12

    Calin, M.A., Gruia, M., Herascu, N. & Coman, T. The monitoring of the accumulation of protoporphyrin IX in Walker tumours by subcutaneous administration of delta-aminolevulinic acid. J. Exp. Ther. Oncol. 4, 247–251 (2004).

    CAS  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    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).

    CAS  Article  Google Scholar 

  15. 15

    Marek, K.W. & Davis, G.W. Transgenically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron 36, 805–813 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Poskanzer, K.E., Marek, K.W., Sweeney, S.T. & Davis, G.W. Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo. Nature 426, 559–563 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. & Tsien, R.Y. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Greenbaum, L., Rothmann, C., Lavie, R. & Malik, Z. Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen. Biol. Chem. 381, 1251–1258 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Dixit, R. & Cyr, R. Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J. 36, 280–290 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Shagin, D.A. et al. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol. Biol. Evol. 21, 841–850 (2004).

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Loschenov, V.B., Konov, V.I. & Prokhorov, A.M. Photodynamic therapy and fluorescence diagnostics. Laser Physics 10, 1188–1207 (2000).

    Google Scholar 

  23. 23

    Zorov, D.B., Filburn, C.R., Klotz, L.O., Zweier, J.L. & Sollott, S.J. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J. Exp. Med. 192, 1001–1014 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Ferguson, K.M., Lemmon, M.A., Schlessinger, J. & Sigler, P.B. Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83, 1037–1046 (1995).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Fujii, M. et al. Real-time visualization of PH domain-dependent translocation of phospholipase C-delta1 in renal epithelial cells (MDCK): response to hypo-osmotic stress. Biochem. Biophys. Res. Commun. 254, 284–291 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Shimohama, S. et al. Mutation in the pleckstrin homology domain of the human phospholipase C-delta 1 gene is associated with loss of function. Biochem. Biophys. Res. Commun. 245, 722–728 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Foote, C.S. Definition of type I and type II photosensitized oxidation. Photochem. Photobiol. 54, 659 (1991).

    CAS  Article  Google Scholar 

  28. 28

    Milano, J. & Day, B.J. A catalytic antioxidant metalloporphyrin blocks hydrogen peroxide-induced mitochondrial DNA damage. Nucleic Acids Res. 28, 968–973 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Yu, Y.A., Timiryasova, T., Zhang, Q., Beltz, R. & Szalay, A.A. Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals. Anal. Bioanal. Chem. 377, 964–972 (2003).

    CAS  Article  Google Scholar 

  30. 30

    Sloan, K.E. et al. CD155/PVR plays a key role in cell motility during tumor cell invasion and migration. BMC Cancer 4, 73 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Kremser, L., Petsch, M., Blaas, D. & Kenndler, E. Labeling of capsid proteins and genomic RNA of human rhinovirus with two different fluorescent dyes for selective detection by capillary electrophoresis. Anal. Chem. 76, 7360–7365 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Thompson, A., Nigro, J. & Seliger, H.H. Efficient singlet oxygen inactivation of firefly luciferase. Biochem. Biophys. Res. Commun. 140, 888–894 (1986).

    CAS  Article  Google Scholar 

  33. 33

    Posner, G.H. et al. A chemiluminescent probe specific for singlet oxygen. Biochem. Biophys. Res. Commun. 123, 869–873 (1984).

    CAS  Article  Google Scholar 

  34. 34

    Horstkotte, E. et al. Toward understanding the mechanism of chromophore-assisted laser inactivation—evidence for the primary photochemical steps. Photochem. Photobiol. 81, 358–366 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Lukyanov, K.A. et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 25879–25882 (2000).

    CAS  Article  Google Scholar 

  36. 36

    Labas, Y.A. et al. Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. USA 99, 4256–4261 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Gurskaya, N.G., Savitsky, A.P., Yanushevich, Y.G., Lukyanov, S.A. & Lukyanov, K.A. Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2, 6 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references


We are grateful to Alexei V. Feofanov for valuable advice and help in light intensity measurements. This work was supported by Howard Hughes Medical Institute grant HHMI 55005618, the Russian Academy of Sciences for the program “Molecular and Cell Biology” and the EC FP-6 Integrated Project LSHG-CT-2003-503259.

Author information



Corresponding author

Correspondence to Konstantin A Lukyanov.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Absorption spectra of and superoxide generation by intact and prebleached KillerRed protein. (PDF 92 kb)

Supplementary Data (PDF 62 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bulina, M., Chudakov, D., Britanova, O. et al. A genetically encoded photosensitizer. Nat Biotechnol 24, 95–99 (2006).

Download citation


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