Coupled Yeast 2-Hybrid–Mammalian 2-Hybrid Reading-Frame-Independent and Site-Specific Recombinational Cloning Vector System

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The yeast 2-hybrid (Y2H) system is a powerful method for identifying protein-protein interactions (PPIs), requiring minimal prior information of the putative interactors. Currently available automated versions of the Y2H system are sufficiently developed to allow facile genome-wide PPI screening to compile extensive inter-actome data. A limitation of the Y2H approach, however, is that all primary hits have to be technically verified and biologically evaluated by complementary methods, which is time-consuming, costly, and laborious. Furthermore, the yeast intracellular environment can lead to spurious results for proteins of other organisms, for example because of differences in post-translational modifications or the presence/absence of bridging proteins. Many researchers now confirm PPIs found in the Y2H system by retesting the candidates in the mammalian 2-hybrid system (M2H). However, although such combined Y2H-M2H testing is desirable and perhaps necessary, recloning of Y2H candidates into M2H vectors, especially on a large scale, is time-consuming and costly. To address this shortcoming, we introduce here a new site-specific recombination-capable M2H vector system that is fully compatible with the site-specific Y2H system that we recently described in Biotechniques [2008;45(3):235-244]. The results show that the new vectors are: (a) Gateway®, compatible and suitable for fast cloning; (b) fully functional in the M2H system without influencing the capacity of the selection system or creating autoactivators; and (c) directly compatible with the existing site-specific Y2H vector system, as demonstrated by confirmation of Y2H PPI candidates in the M2H system.