Abstract A novel yeast three-hybrid Y3H vector pBT was developed, which contains a tetracycline Tet -sensitive transactivator tTA expression unit and a Tet-responsive element TRE -driven 3rd protein expression unit within a single plasmid. To circumvent this problem, a tetracycline Tet -regulated expression system was chosen. From a practical point of view, the use of an inducible promoter for driving the 3rd protein makes it easy to isolate triplex interactions from duplexes formed only by bait and prey. It has been widely used to identify novel protein interactions and to analyze cell signaling networks 1. All other reagents were from Nacalai Tesque Kyoto, Japan. Western blotting Protein extraction from yeast cells was performed as described previously If the bait and prey—which do not form a complex by themselves—can interact with the addition of a 3rd protein, the reporter genes will be activated. Image-capture conditions were kept constant throughout the experiments.
Even in the case of two-protein interactions, a 3rd protein is often required to stabilize or facilitate the binding between the two partners. The isolated plasmids were used to retransform AH with the bait or control plasmids to verify the three-hybrid interaction. In conclusion, the pBT system is a useful tool for studying the structures of higher-order protein complexes. The 3rd protein is incorporated into the protein interaction between bait and prey through direct binding or protein modifications such as phosphorylation. For example, methionine depletion for the Met25 promoter hampers basal growth of the AH strain unpublished data. From a practical point of view, the use of an inducible promoter for driving the 3rd protein makes it easy to isolate triplex interactions from duplexes formed only by bait and prey. Plasmids pBridge was used as a backbone for all vectors. A low-copy-number version of the pBT vector, pBT L , completely eliminated the leakage activity of pBT found under low-stringency condition. In this Y3H system, a 3rd protein is expressed together with a DNA-binding domain BD -bait fusion protein and a transcription activating domain AD -prey fusion protein. The Met25 promoter has been used for this purpose 3 , 5 , and several other inducible promoters can be used as well 6—9. Extracted proteins were separated by SDS—polyacrylamide gel electrophoresis, blotted onto nitrocelulose membranes, and probed with HRP-conjugated anti-Flag M2 antibody 1: Originally reported in mammalian cells 10 , Tet-regulated gene expression systems can also be used in yeast 11— Western blotting Protein extraction from yeast cells was performed as described previously Reporter activation was assessed by colony formation and the X-gal overlay assay. Abstract A novel yeast three-hybrid Y3H vector pBT was developed, which contains a tetracycline Tet -sensitive transactivator tTA expression unit and a Tet-responsive element TRE -driven 3rd protein expression unit within a single plasmid. A total of 1. This simple-to-use pBT Y3H system will facilitate the high-throughput analysis of higher-order protein complexes. All other reagents were from Nacalai Tesque Kyoto, Japan. Image-capture conditions were kept constant throughout the experiments. To simultaneously achieve sufficient inducibility and decreased leakage, it is critical to maintain an adequate but not an excessive tTA expression level. It has been widely used to identify novel protein interactions and to analyze cell signaling networks 1. In this article, the construction of a novel Y3H vector pBT is reported, which has all the Tet-OFF components within a single plasmid with optimizations to minimize background leakage activity. Media preparation, growth, transformation and selection of yeast were carried out as described previously 18 , This article has been cited by other articles in PMC. To study this kind of higher-order protein complex, a yeast three-hybrid Y3H system was developed.
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