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Yeast 2 Hybrid Assay

The yeast 2 hybrid experiments study protein-protein interactions in a semi in vivo system. It involves the subcloning of the genes of the proteins in question into vectors with a portion of a transcriptional activator of a reporter gene (usually fluorescent gene like beta-Gal or Lex A). The DNA binding domain is on one vector while the activation domain is on the other vector. Each protein is subcloned into both vectors, and then these vectors are transformed in to yeast with the reporter gene already present. Important controls for this assay are autoactivation testing where gene+binding domain are transformed with empty activation domain to confirm the protein does not activate transcription itself. Autoactivation can be worked around by only having that gene in the activation domain vector. Autoactivation with the gene binding to the DNA sequence the binding domain interacts with is not tested due the sequence specificity of the DNA binding domain. Each vector will have a different growth advantage such that initial transforms will for example grow on -Leucine and -Tryptophan plates. Colonies are picked from these plates and transferred to a second reporter plate which is usually -Histidine. Only cells with the transformed proteins interacting will acquire the histidine growth advantage. These colonies are then picked and their fluorescence confirmed to insure no false positive growth.

This system works great for proteins you speculate will interact and you know their sequences. Large scale screen have been produced to test entire yeast genomes by creating a gene library fused to the separate domains, however comparison of lab's screen of the same yeast strain usually only overlap ~10%. It is important to remember protein localization to organelles, cytoplasm or membrane when evaluating data from these screens to remove all impossible in vivo interactions. This system also works great for testing how mutations in known protein interactions disrupt their binding. For mutation testing, the reporter gene is usually replaced by a death gene in which only non interacting protein systems leave the cell alive. Yeast 3 hybrids are also performed where a chaperone is necessary for the major protein-protein interaction, but it is far less popular a test. As expected it is very difficult to use yeast 2 hybrid to look at transcription factor interactions, and therefore is almost never done. This assay should be performed in conjunction will other interaction assays like GST pulldown or immunofluorescence before the interaction will be accepted as real.

Editor(s): John Bracht

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