2-oxogluatarate (2-OG), a metabolite from the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also acts as a signaling molecule in a variety of organisms. Tracing 2-OG would show great significance in related studies of cell metabolism and signal transduction while sensitive and specific detection in living cells has still been difficult. Here we develop a novel genetically encoded 2-oxoglutarate sensor based on fluorescent resonance energy transfer (FRET) using a functional 2-OG-binding domain GAF of NifA protein from Azotobacter vinelandii, and demonstrated its feasibility in the application of visualization of metabolite both in vitro , in vivo and imaging 2-OG in intact, individual cells with confocal microscope. We also attained a mono-fluorescent sensor with GAF domain inserted into YFP, which was found response to not only 2-OG but also glutamine by serendipity, promising a ratiometrical sensor for dual detection for these two significant metabolites.

Fluorescent protein based nanosensors for 2-oxoglutarate detection in E. <i>coli</i> cells

245th National Meeting (2013)

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Fluorescent protein based nanosensors for 2-oxoglutarate detection in E. coli cells