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Light Induced Cell Killing

KillerRed and KillerOrange Internal Photosensitizers for selective light-induced Cell Killing
• First fully genetically-encoded photosensitizer
• No exogenous chemical compounds or cofactors except oxygen required
• Direct expression in cells
• Induction by green light irradiation
• Allows selective light-induced cell killing (mammalian cells or bacteria)
• Unique tool for precise protein photoinactivation in vivo (in mammalian cells or in bacteria)

Evrogen´s KillerRed is the first complete genetically encoded photosensitizer developed from the Hydrozoa jellyfish chromoprotein anm2CP.
Evrogen´s KillerOrange represents a mutant of KillerRed with a bright orange fluorescence. In contrast to KillerRed, which becomes phototoxic after illumination with green or orange (540-580 nm) light, KillerOrange was shown to be phototoxic after illumination with blue (450-490 nm) or green (500-540 nm) light.

Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They can be used for precise inactivation of selected proteins in chromophore-assisted light inactivation (CALI) technique and for the light-induced cell killing, for example in photodynamic therapy. Besides KillerRed protein, all known to date photosensitizers are chemical compounds that must be introduced into living systems exogenously. Red fluorescent protein KillerRed is the first genetically-encoded photosensitizer [Bulina et al., 2006a]. Unlike chemical analogs, KillerRed can be directly expressed by target cells, both individually and in fusion with a target protein. It shows no cell toxic effects before light activation. Upon green-light irradiation, KillerRed generates ROS that damage the neighboring molecules.

Confocal image of HeLa cells expressing KillerRed in mitochondria (red) and TurboGFP in cytosol (green).
Lower left cells were pre-irradiated with green light (515-560 nm, 7W/cm2) light for ten minutes. This led to profound KillerRed photobleaching. Cells are shown in 60 minutes after irradiation. It is clearly visible that irradiated cells have abnormal shape and "bubbles", characteristic for apoptotic cell-death pathway.
Note: KillerRed-mediated ROS production is accompanied by profound KillerRed photobleaching. The resulting cell events (cell fate after irradiation, effect on protein localization) can be monitored using another fluorescent reporter, e.g. TurboGFP (see link below).

Available KillerRed and KillerOrange Vectors
KillerRed and KillerOrange expression vectors are developed to express KillerRed or KillerOrange in bacterial or eukaryotic cells and to generate KillerRed fusions with proteins of interest.

pKillerRed-mem and pKillerOrange-mem vectors provide expression of membrane-targeted KillerRed or KillerOrange. Irradiation of membrane-localized KillerRed/KillerOrange leads to high effective and fast cell death, presumably due to lipid oxidation.

pKillerRed-dMito and pKillerRed-dMito vectors provide expression of mitochondria-targeted KillerRed or KillerOrange. Mitochondrial localization of this photosensitizer increases its light-induced cell toxity and makes it highly effective for cell killing.

Recommended Filter Sets and Activating Parameters
Before light activation, KillerRed can be detected using TRITC filter set or similar. Recommended Omega Optical filter sets are QMAX-Red and XF174.
KillerRed phototoxicity is induced by green-light irradiation at 540-580 nm and depends on light intensity irradiation time and KillerRed concentration. Arc-lamp irradiation is strongly recommended; laser-light irradiation in confocal mode is much less efficient.
In CALI, mild illumination of KillerRed-tagged protein for a limited time results in precise inactivation of this protein only. Upon more prolonged and intensive irradiation, KillerRed can be effectively used for damaging the organelles and killing the target cells. Intensity of green light and irradiation time must be individually determined for particular biological system and instrumentation.
KillerOrange phototoxicity can be induced by blue (450-490 nm) or green (500-540 nm) light irradiation and depends on light intensity, irradiation time and KillerOrange concentration and localization. LEDs light is strongly recommended; laser-light irradiation in confocal mode is much less efficient.

Recommended Antibody
KillerRed and KillerOrange can be recognized using Anti-KillerRed antibody (Cat# AB961-EV), see link below.

For the use of KillerRed for Chromophore Assisted Light Inactivation (CALI) of proteins, please see link below.

Bulina, M.E. et al. (2006) A genetically encoded photosensitizer. Nature Biotechnology 24:95-99.
Bulina M.E. et al. (2006) Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed. Nature Protocols 1: 947-953.

Related Links

Detailed KillerRed Features and Applications
Detailed KillerOrange Features and Applications
Anti-KillerRed Antibody
Precise Protein Photoinactivation by KillerRed

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