Romero Francisco, Santana-Calvo Carmen, Sánchez-Guevara Yoloxochitl, Nishigaki Takuya
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
FEBS Lett. 2017 Sep;591(18):2869-2878. doi: 10.1002/1873-3468.12760. Epub 2017 Aug 20.
The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs.
环核苷酸结合结构域（CNBD）作为许多参与环核苷酸信号传导的蛋白质的调节结构域发挥作用。我们基于荧光素标记的腺苷 - 3'，5'-环磷酸类似物与用青色荧光蛋白（CFP）标记的人EPAC1重组CNBD之间的分子间荧光共振能量转移（FRET），开发了一种直接且可靠的结合测定法。该方法的高FRET效率（约80%）使我们能够使用常规设备对纳摩尔范围的样品进行几种类型的结合实验。此外，CNBD上的CFP标签使我们能够使用未纯化的蛋白质进行特异性结合实验。考虑到这些优点，该技术对于研究特征不明确的CNBD很有用。
