Unitat de Fisiologia, Departament de Ciències Fisiològiques, Genes Disease and Therapy Program, IDIBELL-Institute of Neurosciences, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain.
Centro de Investigación en red de enfermedades raras (CIBERER), ISCIII, Instituto de Salud Carlos III, Madrid, Spain.
Methods Mol Biol. 2021;2268:223-232. doi: 10.1007/978-1-0716-1221-7_15.
Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.
Split-TEV 测定法可用于鉴定哺乳动物细胞中的蛋白质-蛋白质相互作用。该方法基于烟草蚀纹病毒 (TEV) 蛋白酶的两个片段的拆分，其中每个片段都融合到预测相互作用的候选蛋白上。如果两个蛋白之间确实存在相互作用，TEV 蛋白酶会重新形成其蛋白水解活性，该活性用于诱导一些报告基因的表达。然而，由于膜蛋白更容易聚集，一些研究已经检测到其之间存在非特异性相互作用。在这里，我们描述了一种改良的 Split-TEV 方法，旨在提高 G 蛋白偶联受体 (GPCR) 相互作用蛋白研究的特异性。这种监测 GPCR 之间相互作用的方法是一种简单而稳健的测定方法，在药物发现方面具有良好的前景。
