In vitro selection of Jun-associated proteins using mRNA display.

Kenichi Horisawa, Seiji Tateyama, Masamichi Ishizaka, Nobutaka Matsumura, Hideaki Takashima, Etsuko Miyamoto-Sato, Nobuhide Doi, Hiroshi Yanagawa

研究成果: Article

46 引用 (Scopus)

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Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.

元の言語English
ページ(範囲)e169
ジャーナルNucleic acids research
32
発行部数21
DOI
出版物ステータスPublished - 2004

ASJC Scopus subject areas

  • Genetics

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    Horisawa, K., Tateyama, S., Ishizaka, M., Matsumura, N., Takashima, H., Miyamoto-Sato, E., Doi, N., & Yanagawa, H. (2004). In vitro selection of Jun-associated proteins using mRNA display. Nucleic acids research, 32(21), e169. https://doi.org/10.1093/nar/gnh167