A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH

作者：Rupprecht Christian; Wingen Marcus; Potzkei Janko; Gensch Thomas; Jaeger Karl Erich; Drepper Thomas^*

来源：Journal of Biotechnology, 2017, 258: 25-32.

DOI：10.1016/j.jbiotec.2017.05.006

• FRET biosensor
• Flavin-binding fluorescent proteins (FbFP)
• Yellow fluorescent protein (YFP)
• Intracellular pH
• Bacterial acid stress response

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution.
We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pK(a) similar to 3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pK(a) values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coil cells during acid stress.

• 出版日期2017-9-20