In vitro selection of Jun-associated proteins using mRNA display.
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.
Horisawa
Kenichi
K
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
Tateyama
Seiji
S
Ishizaka
Masamichi
M
Matsumura
Nobutaka
N
Takashima
Hideaki
H
Miyamoto-Sato
Etsuko
E
Doi
Nobuhide
N
Yanagawa
Hiroshi
H
D000818:Animals; D002460:Cell Line; D015723:Gene Library; D047468:Immunoprecipitation; D016350:Leucine Zippers; D051379:Mice; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000379:methods*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger