Genetically-encoded photosensitizer KillerOrange

- Blue and green light-induced production of reactive oxygen species
- Direct expression in cells, easy targeting to various subcellular compartments
- No exogenous chemical compounds required for chromophore maturation
- Not toxic before activation by blue or green light irradiation
- Recommended for selective light-induced protein inactivation and cell killing

KillerOrange represents a mutant of KillerRed with a bright orange fluorescence (excitation maximum at 512 nm and emission maximum at 555 nm) [Sarkisyan et al., 2015]. In contrast to KillerRed, which becomes phototoxic after illumination with green or orange (540-580 nm) light, KillerOrange was shown to be phototoxic to E.coli after illumination with blue or green (450-540 nm) light. The blue-shifted spectrum of KillerOrange makes it potentially more suitable for two-photon microscopy than the parental KillerRed. Also, the large Stokes shift of over 40 nm should make it possible to spectrally separate signals of KillerOrange from cyan and green fluorescent proteins when the proteins are excited simultaneously by blue light. One can thus use green and cyan indicators to observe the effects of phototoxicity in real time without the need to change the excitation light. KillerOrange-KillerRed pair can potentially be used to independently ablate two cell populations. This pair also promises the orthogonal optical control of the propagation of signaling cascades either by chromophore-assisted light inactivation of the participating proteins or by triggering cascades with hydrogen peroxide produced by KillerRed and likely by KillerOrange. KillerOrange-KillerRed tandem fusions or combination of various photosensitizers in one cassette may enhance phototoxicity under white light irradiation and may be useful as a research tool in biology.

Main properties

Blue line – excitation (em@600 nm), Orange line – emission (ex@510 nm) Download KillerOrange spectra (xls)

CHARACTERISTIC * Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 27 Polypeptide length, aa 239 Structure dimer Aggregation no Maturation rate at 37°C fast Activating light blue or green (e.g. 450-540 nm) Fluorescence color orange Excitation maximum, nm 512 Emission maximum, nm 555 Quantum yield 0.42 Extinction coefficient, M-1cm-1 41200 Brightness* 17.3

Recommended antibodies, filter sets, and activating parameters

KillerOrange can be recognized using Anti-KillerRed antibody (Cat.# AB961) available from Evrogen.

KillerOrange phototoxicity can be induced by blue or green light irradiation (450-540 nm) 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.

Performance and use

KillerOrange can be used for in vivo killing of selected cells. It can be expressed and induced in various experimental systems, including bacteria and mammalian cells.

KillerOrange's suitability for light-induced killing of prokaryotic cells has been demonstrated using E. coli XL1-Blue strain:

Aliquots of E. coli XL1 Blue cells transformed with vectors expressing EGFP (non-phototoxic control), KillerOrange (KO) and KillerRed (KR) were mixed together. The E. coli cells used for the experiment were incubated overnight at 4°C to increase the fraction of the mature protein. The suspension was than diluted to the optical density of 0.05 and aliquoted into transparent PCR tubes (40 μl of the suspension per tube). Samples of EGFP-KO-KR mixture were illuminated with one of LEDs of X-Cite XLED1 illuminator (Lumen Dynamics Group Inc.) The LEDs named "BDX" (450–495 nm), "BGX" (505-545 nm) and "GYX" (540-600 nm) were used. The power of all LEDs was set to be about 80 mW/cm². All aliquots were then spread over the Petri dishes, cultivated for 12-16 hours at 37°C, washed out and analyzed with fluorescence-activated cell sorter (FACS Aria III, BD Biosciences).

KillerOrange (KO) and KillerRed (KR) toxicity under light illumination at different wavelengths. Illumination with 540-600 nm light resulted in the selective removal of KillerRed-expressing cells, while the illumination with 450-495 nm light killed the majority of KillerOrange-expressing cells. Interestingly, 505–545 nm light illumination was almost equally efficient in killing both KillerOrange and KillerRed cells. Thus, by combining different light sources one can achieve precise control over cell populations expressing KillerOrange and KillerRed. Grey column – EGFP, orange column – KillerOrange, red column – KillerRed. Error bars represent SD, N=4.

KillerOrange-mediated killing of eukaryotic cells:

HEK 293 cells were grown in DMEM medium on 6-cm Petri dishes until about 70% confluency. They were then transiently transfected using FuGene HD (Roche) with one of the following plasmids: pKillerRed-dMito, pKillerOrange-dMito (Evrogen, Cat.# FP224) or pEGFP-N1 (Clontech). 24 hours after transfection cells were washed off from the dish, centrifuged, resuspended in PBS pH7.4 and mixed in pairs (KillerOrange with EGFP, KillerRed with EGFP) in polystyrene optical cuvettes (Sarstedt). The suspensions were then split in two halves, first one was illuminated with custom LEDs while keeping the second in the darkness for the same period of time. For blue light illumination, assembly of 7 Luxeon Star LXML-PR₀1-0500 Rebel LEDs, Lumileds (dominant wavelength is 447 nm, spectral half-width is 20 nm), for orange illumination we used 7 Luxeon Star 7 LXML-PL01-0040 Rebel LEDs, Lumileds (dominant wavelength is 590 nm, spectral half-width is 20 nm). The light power at the sample was set to be about 60 mW for 447 nm light and about 20 mW for 590 nm light. After illumination the cells were seeded back to Petri dishes, incubated for 24 hours, washed off again, and analyzed cell fluorescence with flow cytometer (Beckman Coulter, USA).

Phototoxicity of mitochondria-targeted versions of KillerOrange and KillerRed in mammalian cells. KillerOrange, KillerRed and EGFP-expressing cells were mixed and illuminated with 477 nm or 590 nm light. Y-axis depicts changes in fractions of KillerOrange (yellow lines) or KillerRed-expressing (red lines) cells in the population. Cell fractions were normalized to the fractions in non-illuminated sample. Red dashed line – KillerRed, 447 nm; orange dashed line – KillerOrange, 447 nm; red line – KillerRed, 590 nm; orange line – KillerOrange, 590 nm. Error bars represent SD, N=3.

A strong decrease in the fraction of KillerRed-expressing cells was seen in the population that passed through the orange light irradiation. In contrast, KillerOrange-expressing cells were substantially removed from the population of cells that was illuminated with the blue light. Taken together, these results show that KillerOrange is phototoxic to mammalian cells and that KillerRed and KillerOrange can be used simultaneously to independently control fates of two cell populations.

KillerOrange expression in mammalian cells. (A) Transiently transfected HeLa cells expressing mitochondria-targeted KillerOrange. (B) Transiently transfected HeLa cells expressing membrane-targeted KillerOrange.

Available variants and fusions

Variant	Description	Related vector	Cat.#
Humanized KillerOrange	KillerOrange is developed on the basis of the KillerRed [Bulina et al., 2006]. Its codon usage is optimized for high expression in mammalian cells [Haas et al., 1996], but it can be successfully expressed in many other heterological systems. Mammalian expression vectors allowing fusions to the KillerRed C-terminus / N-terminus respectively	pKillerOrange-C	FP221
		pKillerOrange-N	FP222
KillerOrange-dMito fusion	Mitochondrial targeting sequence (MTS) was derived from subunit VIII precursor of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. Two MTS were fused to KillerOrange N-terminus. When expressed in mammalian cells, this variant is localized in mitochondria.	pKillerOrange-dMito	FP224