Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025, India.
Department of Chemistry, Jamia Millia Islamia, New Delhi, 110025, India.
J Ind Microbiol Biotechnol. 2019 Dec;46(12):1669-1683. doi: 10.1007/s10295-019-02235-w. Epub 2019 Sep 17.
Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (K) of 5.09 × 10 M, thus providing a detection range for Hg quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
由于汞的潜在毒性，我们急需了解其在活细胞内的摄取、转运和流动情况。传统的汞分析技术具有侵入性、成本高且灵敏度较低。在本研究中，开发了一种高效的遗传编码汞荧光共振能量转移（FRET）传感器（MerFS），可实时痕量水平测量汞的细胞动力学。为构建 MerFS，将周质结合汞蛋白 MerP 夹在增强型青色荧光蛋白（ECFP）和 Venus 之间。MerFS 具有 pH 稳定性，提供可测量的荧光信号，对汞具有高灵敏度和选择性。突变体 MerFS-51 的表观亲和力（K）为 5.09×10 M，因此提供了 0.210 μM 至 1.196 μM 之间用于汞定量检测的检测范围。此外，MerFS-51 被靶向到大肠杆菌（E. coli）、酵母和人胚肾（HEK）-293T 细胞，允许用高响应性的饱和曲线动态测量细胞内汞浓度，证明了其在细胞系统中的潜在应用。
