Abstract
Chimeric proteins have been widely used to evaluate intracellular protein–protein interactions (PPIs) in living cells with various readouts. By combining an interleukin-3-dependent murine cells and chimeric proteins containing a receptor tyrosine kinase c-kit, we previously established a c-kit-based PPI screening (KIPPIS) system to evaluate and select protein binders. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. To prove this superiority, we apply KIPPIS to 6 peptide aptamer–polypeptide pairs, which are derived from endogenous, synthetic, and viral proteins. Consequently, all of the 6 peptide aptamer–polypeptide interactions are successfully detected by cell proliferation. The detection sensitivity can be modulated in a helper ligand-dependent manner. The assay results of KIPPIS correlate with the activation levels of Src, which is located downstream of c-kit-mediated signal transduction. Control experiments reveal that KIPPIS clearly discriminates interacting aptamers from non-interacting ones. Thus, KIPPIS proves to be a versatile platform for evaluating the binding properties of peptide aptamers.
Highlights
Chimeric proteins have been widely used to evaluate intracellular protein–protein interactions (PPIs) in living cells with various readouts
The intracellular domain (ICD) of a receptor tyrosine kinase c-kit is fused to the C-terminus of proteins of interest, resulting in cell proliferation induced by the c-kit ICD homodimerization in a PPI-dependent manner
The 6 peptide aptamer– polypeptide pairs tested in this study are grouped into endogenous proteins (p53–MDM218 and EZH2–EED19,20), artificial peptide aptamers (PA7–Id121,22 and DIEDML–KIX23), and viral peptides ( PB1N–PA24,25 and PB2–PB1C26,27)
Summary
Chimeric proteins have been widely used to evaluate intracellular protein–protein interactions (PPIs) in living cells with various readouts. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. There is a need for developing new methods that can spontaneously concentrate superior binders and efficiently eliminate inferior and non-specific ones To this end, we previously developed a cell proliferation-based PPI-screening system, represented as c-kitbased PPI screening (KIPPIS)[15]. By transducing the chimeric protein genes and hacking signaling pathways in interleukin-3 (IL-3)-dependent Ba/F3 cells, the transductants can proliferate in a PPI-dependent
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