Remy Ingrid, Ghaddar Galia, Michnick Stephen W
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada.
Nat Protoc. 2007;2(9):2302-6. doi: 10.1038/nprot.2007.356.
We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.
我们基于蛋白质片段互补分析（PCA）开发了一种通用实验策略，能够对完整活细胞中的动态蛋白质-蛋白质相互作用进行定量检测。在该方法中，蛋白质相互作用与同源片段中酶的重新折叠相偶联，其中酶活性的重建作为蛋白质相互作用的检测器。我们已经描述了许多具有不同报告读出方式的分析方法，但对于蛋白质相互作用动力学研究特别有价值的是基于催化产物生成的酶报告基因的分析方法，从而利用信号放大的优势。在此，我们描述了一种基于TEMβ-内酰胺酶作为报告基因在哺乳动物细胞中的PCA方法。β-内酰胺酶PCA包括将β-内酰胺酶的互补片段与两个感兴趣的蛋白质融合。如果蛋白质相互作用，片段就会聚集在一起并折叠成有活性的β-内酰胺酶。在此，我们描述了一种该PCA的方法，该方法可以在几个小时内完成，使用两种不同的底物，它们会被β-内酰胺酶转化为荧光或有色产物。
