Branchini Bruce R, Southworth Tara L
Connecticut College, New London, CT, United States.
Connecticut College, New London, CT, United States.
Methods Enzymol. 2017;589:351-364. doi: 10.1016/bs.mie.2017.01.004. Epub 2017 Feb 20.
Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications based on the detection of the enzymes or using the proteins to measure ATP levels. Based on studies of chimeric luciferases, we engineered a novel luciferase called PLG2 that has enhanced specific activity, and thermal and pH stability compared to the commonly used Photinus pyralis luciferase. We present here protocols for preparing a single assay mixture containing PLG2 that can be used to readily detect femtomole levels of ATP. Our methodology can be used with a variety of samples, including human and bacterial cells, where measurements of ATP can be used as a biosensor for the detection of viable cells.
萤火虫荧光素酶可在高度特异性的ATP依赖过程中发出可见光，基于对该酶的检测或利用这些蛋白质来测量ATP水平，它已被应用于多种用途。基于对嵌合荧光素酶的研究，我们设计了一种新型荧光素酶PLG2，与常用的萤火虫荧光素酶相比，它具有更高的比活性、热稳定性和pH稳定性。我们在此展示了制备含有PLG2的单一检测混合物的方案，该混合物可用于轻松检测飞摩尔水平的ATP。我们的方法可用于多种样品，包括人类和细菌细胞，其中ATP的测量可用作检测活细胞的生物传感器。
