Screening transactivating proteins: problems and pitfalls

Due to their ease of use, genetic screening systems such as the yeast two-hybrid (y2h) system  have been especially successful in identifying novel protein interactions. However, the y2h system is biased against certain classes of proteins, such as transcription factors . When fused to a DNA-binding domain, these proteins autonomously activate transcription because they contain sequence stretches that act as transcriptional activators for the RNA polymerase II complex. Since the output of the y2h system is based on transcriptional activation of a reporter gene, these proteins activate the reporter genes on their own, in the absence of a protein interaction. Such proteins are termed "self-activating" and they cannot be  used in a classical yeast two-hybrid system.

Unfortunately, many important proteins belong to this class, including transcription factors, regulatory proteins associated with DNA, checkpoint proteins like p53 or p65, and many other proteins.

Screening transactivating proteins: the DUALhunter system

The DUALhunter system is a protein interaction assay which has been especially designed for transcriptionally active proteins. It detects the protein interaction outside of the nucleus and therefore works with all transcriptionally active proteins. In addition, it can also be used to screen any soluble protein, domain or protein fragment to uncover novel protein interactions.

The Figure below shows the mechanism of the DUALhunter system. A protein of interest (the bait) is inserted between the membrane protein Ost4p and the C-terminal half of ubiquitin (Cub), followed by the artificial transcription factor LexA-VP16.

A second protein (the prey) is fused to the mutated N-terminal half of ubiquitin (NubG).

The detection of a protein interaction is based on the split-ubiquitin system. If bait and prey interact, Cub and NubG complement to form split-ubiquitin, followed by cleavage and translocation of LexA-VP16 to the nucleus and transcriptional activation of endogenous reporter genes. The protein interaction between bait and prey is detected using the output of the reporter genes, either via a growth selection on minimal medium, or via the color marker lacZ.

Replacing the plasmid encoding the prey by a cDNA library expressing millions of different proteins or protein fragments allows the uncovery of novel protein interactions.