Intervention
in protein–protein interactions (PPIs) has
tremendous effects in the molecular therapy of many diseases. To fulfill
the requirements for targeting intracellular proteins, here we develop
SOS-localization-based interaction screening (SOLIS), which elaborately
mimics signaling via the Ras-mitogen-activated protein
kinase pathway. SOLIS employs two chimeric proteins in which a membrane
localization motif (CaaX) is fused at the C-terminus of a protein
of interest and the catalytic domain of SOS is fused at the C-terminus
of another protein of interest. Interaction between the two proteins
of interest induces membrane localization of the SOS chimera and cell
proliferation. Thus, the SOLIS system enables enrichment of superior
binders based on cell proliferation in an intracellular PPI-dependent
manner. This was verified by three major modalities against intracellular
PPIs (small molecules, peptide aptamers, and intrabodies). The system
worked over a broad range of affinities (KD = 0.32–140 nM). In a screening of a site-directed randomized
library, novel intrabody clones were selected on the basis of the
potency of cell proliferation. Three other PPI detection methods (NanoBiT,
SPR, and pull-down assays) were employed to characterize the SOLIS
system, and several intrabody clones were judged as false negatives
in these assays. SOLIS signals would be less sensitive to the orientation/conformation
of the chimeric proteins, and this feature emerges as the advantage
of SOLIS as a mammalian cytosolic PPI detection system with few false
negatives.