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Variants of Cyan Fluorescent Protein with Improved Fluorescent Properties
Dr. Piston's group has generated a variant of the enhanced cyan fluorescent protein (ECFP) that has improved brightness, more resistance to photo-bleaching and a fluorescence lifetime that is best fit by a single exponential.
The green fluorescent protein GFP from jellyfish is extensively used in modernmolecular biology to tag and follow proteins of interest. In the last several years, many mutations in GFP have generated GFP variants with new colors, improved folding properties and increased brightness. The cyan fluorescence protein (CFP) is a commonly used GFP variant. For example, the enhanced CYP (ECFP) is commonly used as the donor fluorophore in Forster resonace energy transfer (FRET) experiments, where energy transfer between two fluorescent proteins is used to measure biochemical phenomena, such as conformational changes in proteins in living cells.
Although ECFP is commonly usedin FRET experiments, it is less than ideal due to the major disadvantage of its dim fluorescence, which results in low signal to noise ration. Further, ECFP is difficult to use in lifetime FRET measurements because the fluorescence lifetime of ECFP is not best fit by a single exponential. Finally, the fluorescence lifetime is reduced when two ECFP molecules are fused together. To overcome these limita- tions Dr. Piston's group has mutated ECFP to generate Cerulean.
Cerulean offers several advantagesover ECFP including:
1) It is 2.5 fold brighter the ECFP.
2) Its fluorescence lifetime is best fit by a single exponential.
3) It is more resistant to photo-bleaching.
Cerulean and ECFP have an equivalent pH stability and have similar absorption and fluorescence spectrum. These mutations make Cerulean a more suitable FRET donor than ECFP and simplify the interpretation of fluorescence lifetime measurementsto detect FRET. Cerulean should not only improve the usefulness of FRET based biosensors, but it would also improve CFP fluorescence in more traditional multi-colored imaging experiments.
Potential Market Size
Any researcher performing FRET experiments or multi-colored imaging experiments would benefit from thistechnology.Current Competitive Product(s)
Currently an ECFP is used. However, it is relatively dim and not ideal for FRET experiments.
Stage of Development
Cerulean is ready to be licensed for the life science research reagent market.
Cerulean is ideal for FRET experiments in that it is 2.5 fold brighter than the current dye ECFP, more resistant to photo-bleaching and its fluorescence lifetime is best fit by a single exponential.
United States patent pending.
Inventors:David PistonMark Rizzo