Chromophore Assisted Light Inactivation (CALI) of Proteins

• KillerRed Photosensitizer Protein Expression Vector
• KillerRed Photosensitizer Protein Vectors for Protein Fusions

• First fully genetically-encoded photosensitizer

• No exogenous chemical compounds or cofactors except oxygen required

• Direct expression in cells

• Induction by green light irradiation

• Unique tool for precise protein photoinactivation in vivo (in mammalian cells or in bacteria)

• Allows selective light-induced cell killing (mammalian cells or bacteria)


Evrogen´s KillerRed is the first complete genetically encoded photosensitizer developed from the Hydrozoa jellyfish chromoprotein anm2CP.

KillerRed is a red fluorescent protein capable of reactive oxygen species (ROS) generation upon green light irradiation. While known photosensitizers are chemical compounds that must be added into living systems exogenously, KillerRed offers an upgrade, for the first time providing internal photosensitizer generation.

Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They find use 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.
All known to date photosensitizers are chemical compounds that must be introduced into living systems exogenously. This limitation severely constricts the area of their applications.
KillerRed is a first entirely genetically encoded photosensitizer. Unlike chemical photosensitizers, 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 reactive oxygen species (ROS) that damage the neighboring molecules.

KillerRed expression and fusion vectors are developed to express KillerRed in eukaryotic cells and to generate KillerRed fusions with proteins of interest.



Chromophore-assisted light inactivation (CALI) in mammalian cells

Pleckstrin homology (PH) domain of phospholipase C delta-1 (PLC delta-1) was used as a model protein for testing KillerRed-mediated CALI in mammalian cells. Due to its high affinity for membrane phospholipids the PH domain locates to the inner leaf of the plasma membrane. It was anticipated that in the event of direct protein inactivation by dye-generated ROS, the PH domain will lose its membrane affinity and become evenly distributed throughout the cell.
A triple EGFP-PH-KillerRed fusion protein that allows both protein visualization and CALI was constructed and transiently expressed in a mammalian cell line. Intracellular localization of EGFP signal was evaluated before and after CALI of the PH domain using confocal and fluorescence microscopy. As expected, light-induced damaging of the PH domain affected its membrane affinity dramatically.




KillerRed-mediated light-induced inactivation of PLC delta-1 PH domain.
(a) Schematic outline of the experimental system. (b,c) A confocal image of a cell expressing EGFP-PH-KillerRed triple fusion (EGFP green fluorescent signal) before (b) and after (c) 10-s irradiation with green light. Note the considerable increase in cytoplasmic signal.



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, for example a green fluorescent protein like TurboGFP (see link below).


For the use of KillerRed for selective light-induced cell killing, please see link below.


References
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.

Selective Light-Induced Cell Killing by KillerRed
Detailed KillerRed Features and Applications
TurboGFP