DUAL membrane technology utilizes the separated ubiquitin system (split ubiquitin) to screen protein interactions based on the traditional yeast two hybrid system. Firstly, the 3-isoleucine of ubiquitin Nub was artificially mutated to glycine (NubI was mutated to NubG), which greatly reduces the affinity with Cub, avoiding the possibility of Cub and Nub self-binding or approaching each other. Secondly, the Cub was fused with the artificially synthesized LexA-VP16 transcription activator to form a fusion protein Cub-LexiA-VP16. Therefore, under normal conditions, NubG does not bind to Cub, UBPs cannot recognize isolated ubiquitins, and transcription activators are not cleaved. Finally, the proteins to be detected were fused separately with NubG and Cub to form Bait fusion protein (Bait-cube-LexA-VP16) and Prey fusion protein (Prey NubG). If Bait and Prey interact with each other, it will cause NubG and Cub to approach each other, resulting in the reconstruction of ubiquitin, which is recognized by UBPs and leads to the dissociation of LexA-VP16 into the nucleus, thereby activating the transcription of the reporter gene.
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