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A growth-based platform for detecting domain-peptide interactions in the cytoplasm of mammalian cells
- Source :
- Scientific reports. 12(1)
- Publication Year :
- 2022
-
Abstract
- Development of a method for detecting protein–protein interactions (PPIs) in living cells is important for therapeutic drug screening against various diseases including infectious diseases. We have recently developed a method named SOS localization-based interaction screening (SOLIS), in which we designed membrane-anchored and SOS-fused chimeric proteins, whose PPI-dependent association triggers membrane localization of the SOS-fused chimeric protein, activates the Ras/MAPK pathway, and induces cell growth. While SOLIS was able to detect relatively strong PPIs, further sensitivity was required for detecting intracellular endogenous PPIs typically having a micromolar order of dissociation constant (Kd). Here we develop high-sensitive SOLIS (H-SOLIS) that could universally detect PPIs with lower affinities. In order to improve the sensitivity, H-SOLIS introduces a heterodimeric helper interaction, in which addition of a small-molecule helper ligand could accommodate association of the two chimeric proteins and regulate the sensitivity. Four types of domain–peptide interactions having known Kd values are employed to examine the versatility and detection limit of H-SOLIS. Consequently, the heterodimer-inducible helper ligand dramatically enhances detection sensitivity, lowering the detection limit to a ten-micromolar order of Kd. Thus, H-SOLIS could be a platform to detect disease-related domain–peptide interactions for drug discovery screening.
Details
- ISSN :
- 20452322
- Volume :
- 12
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Scientific reports
- Accession number :
- edsair.doi.dedup.....85911a0ea1e7046b6232d8adc1ef8a1b