Y2H Transactivating Starter Kits

Description
Y2H Transactivating Starter kits enable you to screen proteins which are unsuitable for use in classical two-hybrid systems, such as transcription factors, nuclear proteins or acidic proteins. The bait protein is attached to the membrane via a small transmembrane domain and is screened outside of the nucleus for interaction with other proteins.
A bait is constructed by fusing a protein of interest at its N-terminus to a small membrane anchor (Ost4p) and its C-terminus to the C-terminal half of ubiquitin (Cub) and a transcription factor. The bait is inserted into the membranes of yeast. Since the transcription factor is fused to the membrane protein, it is immobilized at the membrane and cannot reach the nucleus. Consequently, the reporter genes integrated into the yeast genome remain silent. (Fig. 1 A)
A prey is constructed by fusing a second protein of interest to the N-terminal half of ubiquitin carrying a I3G point mutation (NubG). (Fig. 1 A)
An interaction between bait and prey results in assembly of split-ubiquitin from Cub and NubG. The split-ubiquitin is recognized by ubiquitin-specific proteases present in yeast, resulting in proteolytic cleavage of the transcription factor from the bait. The liberated transcription factor moves to the nucleus and activates the reporter genes in the yeast genome. The protein interaction is thus converted into reporter gene activity (Fig. 1 B). Commonly used reporter genes are either auxotrophic markers which allow selection on a minimal medium or color reporters like lacZ.

The defined prey can be replaced by a collection of different preys (i.e. different proteins fused to the activation domain) expressed from a cDNA library. Each yeast cell expresses the same bait and a particular prey for the cDNA library. If bait and prey interact, the yeast cell multiplies and gives rise to a visible colony on minimal medium. All yeast cells which do not express an interacting bait/prey pair fail to grow and are discarded. Finally, yeast colonies are picked and the prey plasmid is isolated and sequenced, yielding the information on the protein which interacts with the bait. The final result of an Y2H Transactivating screen is a list of proteins that interact with your protein of interest.