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

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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.

Original languageEnglish
JournalNucleic acids research
Volume32
Issue number21
DOIs
Publication statusPublished - Jan 1 2004
Externally publishedYes

Fingerprint

Messenger RNA
Proteins
Technology
In Vitro Techniques
Leucine Zippers
Two-Hybrid System Techniques
Poisons
Gene Library
Ribosomes
Immunoprecipitation
Proteomics
Sequence Analysis
Real-Time Polymerase Chain Reaction
Cultured Cells
Mass Spectrometry
Observation
Brain

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

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

In vitro selection of Jun-associated proteins using mRNA display. / Horisawa, Kenichi; Tateyama, Seiji; Ishizaka, Masamichi; Matsumura, Nobutaka; Takashima, Hideaki; Miyamoto-Sato, Etsuko; Doi, Nobuhide; Yanagawa, Hiroshi.

In: Nucleic acids research, Vol. 32, No. 21, 01.01.2004.

Research output: Contribution to journalArticle

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, vol. 32, no. 21. https://doi.org/10.1093/nar/gnh167
Horisawa K, Tateyama S, Ishizaka M, Matsumura N, Takashima H, Miyamoto-Sato E et al. In vitro selection of Jun-associated proteins using mRNA display. Nucleic acids research. 2004 Jan 1;32(21). https://doi.org/10.1093/nar/gnh167
Horisawa, Kenichi ; Tateyama, Seiji ; Ishizaka, Masamichi ; Matsumura, Nobutaka ; Takashima, Hideaki ; Miyamoto-Sato, Etsuko ; Doi, Nobuhide ; Yanagawa, Hiroshi. / In vitro selection of Jun-associated proteins using mRNA display. In: Nucleic acids research. 2004 ; Vol. 32, No. 21.
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