European Molecular Biology Laboratory Australia (EMBL Australia) Node in Single Molecule Science, Sydney NSW 2031, Australia.
School of Medical Sciences, The University of New South Wales, Sydney NSW 2031, Australia.
Int J Mol Sci. 2017 Dec 11;18(12):2681. doi: 10.3390/ijms18122681.
The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar.
荧光标记蛋白在显微镜中的应用已成为常规手段，抗 GFP（绿色荧光蛋白）亲和基质在蛋白质组学方案中也越来越多地被使用。然而，一些蛋白质-蛋白质相互作用检测，如蛋白互补检测（PCA），需要将每个蛋白重新克隆为与互补系统不同部分的融合蛋白。在这里，我们描述了一个通用系统，其中互补作用与蛋白分离，可以直接与荧光标记蛋白一起使用。通过使用纳米抗体并在无细胞表达系统中进行测试，我们加速了多个报告基因的开发，可检测 GFP 或 mCherry 标记的异二聚体、同二聚体或寡聚体。我们证明该系统可以在从纳摩尔到微摩尔的广泛浓度范围内检测相互作用。
