CONTEXT

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell bioassays and imaging. They have been originally obtained by the replacement of tyrosine 66 by a tryptophan in the chromophore of the green fluorescent protein AvGFP. Combined with a yellow acceptor from the same AvGFP family, it forms by far the most reliable and popular FRET pair currently in use in biosensors. CFPs have already a myriad of applications in the study of cell physiopathology, and might be ideal building blocks in the development of highly specific, non-toxic and biodegradable nanobiosensors for in vitro and in vivo molecular bioassays. 

TECHNICAL DESCRIPTION

This invention provides a unique method for the optimization of existing cyan fluorescent proteins. Strongly improved CFPs have been developed recently but these new variants carry many mutations away from their ancestors, due to the use of large scale random mutagenesis. Thus, a complete replacement of the fluorescent protein sequence is required in the biosensor gene, which involves sophisticated molecular biology techniques and raises hazards in the functionality of the final construct. Our invention provides a simpler method to reach at least equivalent performances. The method is applicable to the genes of ECFP, Cerulean and other widely distributed cyan fluorescent proteins plasmids from Addgene Bank or Tsien’s lab, and is based on a maximum of two rounds of single site-directed mutagenesis using standard commercial kits. The obtained construct is spectrally and structurally identical to the original version, and can directly replace it in standard protocols. 

BENEFITS

The new CFP variants obtained with our method have important advantages :

• Near ultimate brigthness, fluorescence quantum yield of 90%.
• Single & long emission lifetime (4.12 ns)
• Outstanding pH stability (pK=3.3)
• Reduced general sensitivity to environment
• Nearly undetectable photoswitching reactions

INDUSTRIAL APPLICATIONS

FRET-based biosensors are used in biological, pharmacological, and clinical research, as well as environmental sciences and biotechnology, to specifically assay chemical compounds and ions, enzymatic activities and protein-protein interactions. 

DEVELOPMENT STAGE

Accomplished tests:

• efficient expression demonstrated in various cell lines and as fusion to different proteins.
• can efficiently locate at the membrane and at gap junctions.
• bright intracellular fluorescence (improved by a factor 2 compared to ECFP)
• fluorescence insensitive to fusion and/or sub-cellular location and changes in intracellular pH
• performs efficiently as a FRET donor with usual yellow and orange acceptors
• major improvement of a biosensor of kinase activity (~3-times increase in DR/R)

Current developments
Evaluation of benefits in cell and tissue imaging:

• planned building of expression vectors for tissue imaging (brain slices, tumor models)
• study of protein-protein interactions in a multi-subunit enzyme complex involved in oxidative stress.

For further information, please contact us (Ref 03624-01)