期刊
JOURNAL OF BIOTECHNOLOGY
卷 258, 期 -, 页码 25-32出版社
ELSEVIER
DOI: 10.1016/j.jbiotec.2017.05.006
关键词
FRET biosensor; Flavin-binding fluorescent proteins (FbFP); Yellow fluorescent protein (YFP); Intracellular pH; Bacterial acid stress response
资金
- Federal Ministry of Education and Research [FKZ 031A167]
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.
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