Scaling‐up a fragment‐based protein–protein interaction method using a human reference interaction set

Abstract

Protein-protein interactions (PPIs) are essential in understanding numerous aspects of protein function. Here, we significantly scaled and modified analyses of the recently developed all-vs-all sequencing (AVA-Seq) approach using a gold-standard human protein interaction set (hsPRS-v2) containing 98 proteins. Binary interaction analyses recovered 20 of 47 (43%) binary PPIs from this positive reference set (PRS), comparing favorably with other methods. However, the increase of 20Â in the interaction search space for AVA-Seq analysis in this manuscript resulted in numerous changes to the method required for future use in genome-wide interaction studies. We show that standard sequencing analysis methods must be modified to consider the possible recovery of thousands of positives among millions of tested interactions in a single sequencing run. The PRS data were used to optimize data scaling, autoactivator removal, rank interaction features (such as orientation and unique fragment pairs), and statistical cutoffs. Using these modifications to the method, AVA-Seq recovered >500 known and novel PPIs, including interactions between wild-type fragments of tumor protein p53 and minichromosome maintenance complex proteins 2 and 5 (MCM2 and MCM5) that could be of interest in human disease.

Identifying novel interactions of the colon-cancer related APC protein with Wnt-pathway nuclear transcription factors

“…Additionally, proteins containing an internal BstXI site are more likely to have poor or limited coverage. This restriction enzyme is required to ligate the fragment pairs into the pAVA plasmid [24]. The ORF filtering process (see methods) may also introduce a bias toward longer proteins and exacerbate poor coverage at the N- and C-termini [24].…”

“…This restriction enzyme is required to ligate the fragment pairs into the pAVA plasmid [24]. The ORF filtering process (see methods) may also introduce a bias toward longer proteins and exacerbate poor coverage at the N- and C-termini [24]. Nevertheless, coverage of the search space yielded significant and interesting novel interactions.…”

“…Data analysis was performed as previously [24]. Briefly, raw sequencing data of the plasmids containing the paired fragments grown in selective media were translated and aligned to the Wnt ORF clone database using DIAMOND Blastp [25].…”

“…The final volume of 120 µ L is split into two reactions, each with 50 µ L, and sheared using a Covaris focused-ultrasonicator. Sheared DNA was end-repaired (NEB E6050S) followed by Ampure cleaning and ligation (NEB M0202S) into pBORF filtering vectors, which were described previously [22, 24].…”

Identifying novel interactions of the colon-cancer related APC protein with Wnt-pathway nuclear transcription factors

“…Additionally, proteins containing an internal BstXI site are more likely to have poor or limited coverage. This restriction enzyme is required to ligate the fragment pairs into the pAVA plasmid [24]. The ORF ltering process (see methods) may also introduce a bias toward longer proteins and exacerbate poor coverage at the N-and C-termini [24].…”