Shibata, Akihiro C E Maebashi, Hiroshi K Nakahata, Yoshihisa Nabekura, Junichi Murakoshi, Hideji
Supportive Centre for Brain Research, National Institute for Physiological Science, Okazaki, Aichi, Japan, Division of Homeostatic Development, National Institute for Physiological Science, Okazaki, Aichi, Japan, Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan, Okazaki Institute for Integrative Bioscience, Okazaki, Aichi, Japan, Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan, Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.
Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.
… “475 nm LED; CoolLED) through an excitation filter (FF01-510/84;”…
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