Fujioka Mari, Asano Yumi, Nakada Shigeyuki, Ohba Yusuke
Department of Cell Physiology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo, 060-8638, Japan.
Tamano Technology Center, Research and Development Headquarters, Mitsui Engineering & Shipbuilding Co., Ltd., 3-16-1, Tamahara, Tamano, 706-0014, Japan.
Methods Mol Biol. 2017;1555:513-534. doi: 10.1007/978-1-4939-6762-9_30.
Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.
具有不同光谱的荧光蛋白（FPs）为广泛的生物学功能提供了线索。此外，经过精心设计含有单个或多个荧光蛋白的生物传感器，包括基于荧光共振能量转移（FRET）的生物传感器，能够时空揭示各种病理生理过程背后的分子机制。然而，它们在应用生命科学中的效用尚未得到充分探索。最近，我们的研究小组已开始将荧光蛋白的潜力从基础生物学研究扩展到临床。在此，我们描述一种方法，该方法使用基于荧光蛋白技术和荧光共振能量转移原理的生物传感器来评估患者白血病细胞对酪氨酸激酶抑制剂的反应性。当生物传感器的酪氨酸残基磷酸化时，SH2结构域与磷酸酪氨酸的结合会诱导生物传感器的构象变化，并使供体和受体荧光蛋白紧密靠近。因此，激酶活性和对激酶抑制剂的反应可以分别通过荧光共振能量转移效率的增加和降低来监测。与基础研究一样，这种生物传感器解决了迄今为止艰巨的任务，并可能在临床实验室检查中提供创新的技术进展。还介绍了实现这种创新的最先进检测设备。
