Evolution of KIPPIS as a versatile platform for evaluating intracellularly functional peptide aptamers

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- ki t-bas...

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Bibliographic Details
Published in:Scientific reports 2021-06, Vol.11 (1), p.11758-11758, Article 11758
Main Authors: Kashima, Daiki, Kawahara, Masahiro
Format: Article
Language:English
Subjects:
631/61
631/61/17
631/61/32
631/61/338
Aptamers
Binders
c-Kit protein
Cell growth
Cell proliferation
Humanities and Social Sciences
Interleukin 3
Intracellular
Intracellular signalling
Kinases
multidisciplinary
Peptides
Polypeptides
Protein interaction
Protein-tyrosine kinase receptors
Proteins
Science
Science (multidisciplinary)
Signal transduction
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Summary: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- ki t-based PPI s creening (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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-021-91287-z