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Photina® is the new and improved Ca2+activated
photoprotein for the development of precise Ca2+ mobilisation assays for High-Throughput Screening (HTS). Developed by Axxam researchers from their extensive experience in molecular biology and drug discovery, Photina® is validated for use with a wide range of plate readers and is commercially available from PerkinElmer through an exclusive license granted by Axxam for worldwide commercialization.
While the mechanism of action of Photina® is similar to other photoproteins, the total light release is higher and the reaction kinetic is slower - opening new opportunities for the application of flash
luminescence assays in HTS. The inherent flash luminescence of Photina® offers several advantages when compared to classical Ca2+ fluorescent
dyes:
- low background levels result in a large signal to noise ratio
- Ca2+ concentrations can be measured at specific cellular sites
- tested compounds only require short incubation periods
- reaction kinetics can be followed.
Axxam scientists tested the expression and functionality of Photina® in various cell lines most frequently used in HTS, such as CHO and HEK293. Results demonstrate that the expression of the protein is stable over time, does not affect cell growth and viability and no toxicity is detected - even after long periods of cell culture. Cells expressing Photina® and the target gene of interest are suited both for use in adhesion and in suspension.
Photina® has been targeted to different cell compartments such as plasma membranes and mitochondria. The choice between cytoplasmic or mitochondrial localization of Photina® greatly expands the range of targets addressable by the use of flash luminescence. Localization of a Ca2+ activated photoprotein within the mitochondria has the advantage of amplifying the luminescent response triggered by the activation of GPCRs coupled to the Gαq/PLC pathway.
While Photina® is well-suited to measure GPCRs activation, it also is ideal for any other cellular targets capable of increasing the intracellular Ca2+ concentration. Photina® has been validated for addressing other important drug discovery target classes, like Ca2+ permeable ion
channels or Na+/Ca2+ exchangers. Members of these target classes have been transfected in CHO cells that express either the mitochondrial or the cytoplasmic version of Photina® and results clearly demonstrate that the functional activation of these targets can be detected easily and reliably by Photina® using a range of flash luminescence plate readers - FLIPR3, FLIPRTETRA, CyBi® Lumax, Lumilux, CCD camera - and with different MTP formats (96, 384, 1536).
In all tested conditions, the very high light intensity generated by Photina® allows the use of very few cells/well versus the larger requirements of most other photoproteins and HTS protocols. The signal intensity obtained with 250 cells/well is strong enough to guarantee reliable signal detection. Photina® is therefore particularly attractive for HTS campaigns involving large compound collections since the required production quantity of the cellular assay material can be reduced significantly. An average Z’ factor value of 0.7 for most of the tested assays also provides the required robustness for application to HTS.
Photina® is covered under the European granted patent EP1413584 entitled "Photoprotein with Improved Bioluminescence" and corresponding patent applications and issued patents.
In May 2007, Axxam granted an exclusive global
license to PerkinElmer whereby PerkinElmer became
the exclusive provider of the Axxam Photina® photoprotein
technology to the drug discovery market.
Axxam retains the rights to use the technology for its discovery services to
third parties, as assay development, high-throughput
screening (HTS) and compound profiling, as well
as for any further application of development of
this technology.
The terms of the agreement also provide for a formal research and development
program between Axxam and PerkinElmer to develop
additional Photina® GPCR and ion channel cell lines
for use in high throughput screening and compound
profiling. |
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