Abstract

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.

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