Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein-protein interactions and complexes.
Genome Res
; 15(5): 710-7, 2005 May.
Article
en En
| MEDLINE
| ID: mdl-15867431
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Virus del Mosaico del Tabaco
/
Proteínas
/
Leucina Zippers
/
Proteínas Proto-Oncogénicas c-fos
/
Mapeo de Interacción de Proteínas
Límite:
Animals
Idioma:
En
Revista:
Genome Res
Asunto de la revista:
BIOLOGIA MOLECULAR
/
GENETICA
Año:
2005
Tipo del documento:
Article
País de afiliación:
Japón