Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein-protein interactions and complexes.
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.
Miyamoto-Sato
Etsuko
E
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
Ishizaka
Masamichi
M
Horisawa
Kenichi
K
Tateyama
Seiji
S
Takashima
Hideaki
H
Fuse
Shinichiro
S
Sue
Kaori
K
Hirai
Naoya
N
Masuoka
Kazuyo
K
Yanagawa
Hiroshi
H
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D017931:DNA Primers; D015723:Gene Library; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D016350:Leucine Zippers / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos