Department of Chemistry, BMC, Uppsala, Sweden.
Methods Mol Biol. 2020;2141:529-552. doi: 10.1007/978-1-0716-0524-0_27.
The intrinsically disordered regions of the proteome are enriched in short linear motifs (SLiMs) that serve as binding sites for peptide binding proteins. These interactions are often of low-to-mid micromolar affinities and are challenging to screen for experimentally. However, a range of dedicated methods have been developed recently, which open for screening of SLiM-based interactions on large scale. A variant of phage display, termed proteomic peptide phage display (ProP-PD), has proven particularly useful for the purpose. Here, we describe a complete high-throughput ProP-PD protocol for screening intrinsically disordered regions for SLiMs. The protocol requires some basic bioinformatics skills for the design of the library and for data analysis but can be performed in a standard biochemistry lab. The protocol starts from the construction of a library, followed by the high-throughput expression and purification of bait proteins, the phage selection, and the analysis of the binding-enriched phage pools using next-generation sequencing. As the protocol generates rather large data sets, we also emphasize the importance of data management and storage.
蛋白质组中的无规则区域富含短线性基序 (SLiMs)，这些基序充当肽结合蛋白的结合位点。这些相互作用通常具有低至中微摩尔亲和力，并且难以通过实验筛选。然而，最近已经开发出一系列专门的方法，这些方法为大规模筛选基于 SLiM 的相互作用开辟了道路。噬菌体展示的一种变体，称为蛋白质组肽噬菌体展示 (ProP-PD)，已被证明对该目的特别有用。在这里，我们描述了一种完整的高通量 ProP-PD 筛选方案，用于筛选无规则区域中的 SLiMs。该方案需要一些基本的生物信息学技能来设计文库和进行数据分析，但可以在标准的生物化学实验室中进行。该方案从文库的构建开始，接着是诱饵蛋白的高通量表达和纯化、噬菌体选择以及使用下一代测序对结合富集噬菌体池进行分析。由于该方案生成了相当大的数据集，我们还强调了数据管理和存储的重要性。
