Fernández Matilde, Ortega Álvaro, Rico-Jiménez Miriam, Martín-Mora David, Daddaoua Abdelali, Matilla Miguel A, Krell Tino
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Methods Mol Biol. 2018;1729:291-301. doi: 10.1007/978-1-4939-7577-8_23.
The majority of bacterial chemoreceptors remain functionally un-annotated. The knowledge of chemoreceptor function, however, is indispensable to understanding the evolution of the chemotaxis system in bacteria with different lifestyles. Significant progress in the annotation of chemoreceptor function has been made using experimental strategies that are based on the individual, genetically engineered ligand binding domain (LBD) of chemoreceptors. There is now evidence that all major classes of LBDs can be produced as individual domains that retain their ligand binding activity. Here, we provide a protocol for the combined use of high-throughput ligand screening using Differential Scanning Fluorimetry followed by Isothermal Titration Calorimetry to identify and characterize ligands that bind to recombinant chemoreceptor LBDs. This approach has been shown to be very efficient for determining the function of novel chemoreceptors.
大多数细菌化学感受器的功能仍未得到注释。然而，化学感受器功能的知识对于理解不同生活方式细菌趋化系统的进化是不可或缺的。利用基于化学感受器单个基因工程配体结合结构域（LBD）的实验策略，在化学感受器功能注释方面取得了重大进展。现在有证据表明，所有主要类别的LBD都可以作为保留其配体结合活性的单个结构域产生。在这里，我们提供了一个方案，用于结合使用差示扫描荧光法进行高通量配体筛选，然后进行等温滴定量热法，以鉴定和表征与重组化学感受器LBD结合的配体。这种方法已被证明对于确定新型化学感受器的功能非常有效。
