Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein-protein interactions and complexes

Etsuko Miyamoto-Sato, Masamichi Ishizaka, Kenichi Horisawa, Seiji Tateyama, Hideaki Takashima, Shinichiro Fuse, Kaori Sue, Naoya Hirai, Kazuyo Masuoka, Hiroshi Yanagawa

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37 Citations (Scopus)

Abstract

We have developed a simple and totally in vitro selection procedure based on cell-free cotranslation using a highly stable and efficient in vitro virus (IVV). Cell-free cotranslation of tagged bait and prey proteins is advantageous for the formation of protein complexes and allows high-throughput analysis of protein-protein interactions (PPI) as a result of providing in vitro instead of in vivo preparation of bait proteins. The use of plural selection rounds and a two-step purification of the IVV selection, followed by in vitro post-selection, is advantageous for decreasing false positives. In a single experiment using bait Fos, more than 10 interactors, including not only direct, but also indirect interactions, were enriched. Further, previously unidentified proteins containing novel leucine zipper (L-ZIP) motifs with minimal binding sites identified by sequence alignment as functional elements were detected as a result of using a randomly primed cDNA library. Thus, we consider that this simple IVV selection system based on cell-free cotranslation could be applicable to high-throughput and comprehensive analysis of PPI and complexes in large-scale settings involving parallel bait proteins.

Original languageEnglish
Pages (from-to)710-717
Number of pages8
JournalGenome Research
Volume15
Issue number5
DOIs
Publication statusPublished - May 2005

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)

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    Miyamoto-Sato, E., Ishizaka, M., Horisawa, K., Tateyama, S., Takashima, H., Fuse, S., Sue, K., Hirai, N., Masuoka, K., & Yanagawa, H. (2005). Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein-protein interactions and complexes. Genome Research, 15(5), 710-717. https://doi.org/10.1101/gr.3510505