RESUMO
The yeast two-hybrid system (Y2H) is a powerful method to identify binary protein-protein interactions in vivo. Here we describe Y2H screening strategies that use defined libraries of open reading frames (ORFs) and cDNA libraries. The array-based Y2H system is well suited for interactome studies of small genomes with an existing ORFeome clones preferentially in a recombination based cloning system. For large genomes, pooled library screening followed by Y2H pairwise retests may be more efficient in terms of time and resources, but multiple sampling is necessary to ensure comprehensive screening. While the Y2H false positives can be efficiently reduced by using built-in controls, retesting, and evaluation of background activation; implementing the multiple variants of the Y2H vector systems is essential to reduce the false negatives and ensure comprehensive coverage of an interactome.
Assuntos
Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta , ProteomaAssuntos
Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas/normas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Técnicas do Sistema de Duplo-Híbrido/normasRESUMO
Yeast two-hybrid screens often produce vastly non-overlapping interaction data when the screens are conducted in different laboratories, or use different vectors, strains, or reporter genes. Here we investigate the underlying reasons for such inconsistencies and compare the effect of seven different vectors and their yeast two-hybrid interactions. Genome-wide array screens with 49 motility-related baits from Treponema pallidum yielded 77 and 165 interactions with bait vectors pLP-GBKT7 and pAS1-LP, respectively, including 21 overlapping interactions. In addition, 90 motility-related proteins from Escherichia coli were tested in all pairwise combinations and yielded 140 interactions when tested with pGBKT7g/pGADT7g vectors but only 47 when tested with pDEST32/pDEST22. We discuss the factors that determine these effects, including copy number, the nature of the fusion protein, and species-specific differences that explain non-conserved interactions among species. The pDEST22/pDEST32 vectors produce a higher fraction of interactions that are conserved and that are biologically relevant when compared with the pGBKT7/pGADT7-related vectors, but the latter appear to be more sensitive and thus detect more interactions overall.
