RESUMEN
Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein interactions at genome scales (that is, constructing an interactome), the yeast two-hybrid system has been extensively tested and is preferred over bacterial two-hybrid systems, given that users have created more resources such as a variety of vectors and other modifications. Each system has its own advantages and limitations and thus needs to be compared directly. For instance, the bacterial two-hybrid method seems a better fit than the yeast two-hybrid system to screen membrane-associated proteins. In this chapter, we provide detailed protocols for yeast and bacterial two-hybrid systems as well as a comparison of outcomes for each approach using our own and published data.
Asunto(s)
Técnicas del Sistema de Dos Híbridos , Escherichia coli , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Transformación BacterianaRESUMEN
Two-hybrid (Y2H) assays are available in a variety of different versions, including bacterial, yeast, and mammalian systems. However, even when done exclusively in yeast, multiple different host strains, vectors, reporter genes, or protocols can be used. Here we systematically compare protein-protein interactions (PPIs) from several previously published Y2H datasets. PPIs of a human gold-standard dataset were generated by Y2H assays as well as other methods such as LUMIER or protein fragment complementation assays (PCAs). Different Y2H methods detect substantially different subsets of these PPIs, even when protocols are standardized. In order to maximize the number of interactions found and to minimize the number of false positive interactions we recommend to combine multiple vectors and protocols. While the combined results of all 18 methods detected about 92% of a gold-standard interaction set, a combination of just three Y2H assays detected up to 78% of these protein pairs, or up to 83% when a fourth assay was included. These findings indicate that three or four separate assays may be sufficient to detect the majority of protein-protein interactions in many systems.