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Details of Two hybrid screening

A method of screening cDNA libraries on the basis of protein-protein interaction (for a diagram look here). Briefly, this approach requires introducing three vectors into a cell:

• First, the cell (frequently yeast) is transfected with an expression vector designed so that the expression of a reporter gene is under the control of a well characterized promoter element.
• The cell is then transfected with another expression vector with a constitutive promoter. This second vector expresses a fusion protein containing two domains. One domain recognizes the promoter region of the reporter construct, and the second domain has a portion of the protein of interest. Generally this combination fails to activate transcription.
• These cells are then transfected with a library of expression vectors. These expression vectors express a collection of fusion proteins. One part of the fusion protein is a transcription activating domain, while the other domain includes different proteins in each member of the library.
• In those cases where there is an interaction between the fusion protein in the expression library (the 'fish') and the fusion protein containing a DNA binding region (the 'bait'), there is an activation of transcription. Activation of transcription of the reporter gene (frequently beta galactosidase) is preliminary evidence for the isolation of a cDNA for an interacting protein.
• Of course, this hypothesis must be further tested to rule out potential artifacts and to determine if the cloned protein really interacts with the target protein (the bait).