mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.
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