Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany; GO-Bio Projekt SenseUP, Forschungszentrum Jülich, D-52425 Jülich GmbH, Germany.
J Biotechnol. 2017 Sep 20;258:25-32. doi: 10.1016/j.jbiotec.2017.05.006. Epub 2017 May 10.
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∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pK 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. coli cells during acid stress.
细胞内 pH 值是许多生物（技术）过程的重要调节剂，如代谢物的酶促转化或跨细胞膜的运输。由于质子分布的改变而导致的细胞内 pH 值的变化，可能会导致细胞过程的功能障碍。因此，准确监测细胞内 pH 值可以阐明（病理）生理和生物技术过程对 pH 值的依赖性。在这方面，基因编码的生物传感器代表了一种强大的工具，可以非侵入性地、以高时空分辨率来确定细胞内 pH 值。我们构建了一套新的基因编码基于 FRET 的 pH 生物传感器（称为荧光生物传感器用于 pH 值或 FluBpH）的工具包，该工具包利用 FMN 结合荧光蛋白 EcFbFP 作为供体结构域。与 GFP 家族的许多荧光蛋白不同，EcFbFP 对酸性 pH 值（pK∼3.2）具有显著的耐受性。为了覆盖广泛的生理相关 pH 值范围，使用了三个 pK 值为 5.7、6.1 和 7.5 的 EYFP 变体作为 pH 感应 FRET 受体结构域。所得的生物传感器 FluBpH 5.7、FluBpH 6.1 和 FluBpH 7.5 在体外和体内进行了校准，以准确评估其 pH 指示剂特性。为了证明 FluBpH 的体内适用性，在大肠杆菌细胞中测量了酸应激期间细胞内 pH 值的比率变化。
