System to detect protein-protein interactions

#1. Field of the Invention

#The present invention relates to a method for detecting the interaction of proteins in an in vivo system through the use of fused genes encoding hybrid proteins.

#2. Background of the Related Art

#1A-C schematically represents transcriptional activation by reconstitution of GAL4 activity.

#2A-E schematically represents plasmid constructions used in Example 1.

#A method for detecting the interaction between a first test protein and a second test protein is provided in accordance to the present invention. The method includes providing a host cell, preferably a yeast cell, most preferably Saccharomyces cerevisiae or Schizosaccharomyces pombe. The host cell contains a detectable gene having a binding site for the DNA-binding domain of the transcriptional activator, such that the detectable gene expresses a detectable protein when the detectable gene is transcriptionally activated. Such activation occurs when the transcriptional activation domain of a transcriptional activator is brought into sufficient proximity to the DNA-binding domain of the transcriptional activator.

#A first chimeric gene is provided which is capable of being expressed in the host cell. The first chimeric gene may be present in the chromosome of the host cell. The first chimeric gene comprises a DNA sequence that encodes a first hybrid protein. The first hybrid protein contains a DNA-binding domain that recognizes the binding site on the detectable gene in the host cell. The first hybrid protein also contains a first test protein or protein fragment which is to be tested for interaction with a second test protein or protein fragment.

#A second chimeric gene is provided which is capable of being expressed in the host cell. In one embodiment, both the first and the second chimeric genes are introduced into the host cell in the form of plasmids. Preferably, however, the first chimeric gene is present in a chromosome of the host cell and the second chimeric gene is introduced into the host cell as part of a plasmid. The second chimeric gene contains a DNA sequence that encodes a second hybrid protein. The second hybrid protein contains a transcriptional activation domain. The second hybrid protein also contains a second test protein or a protein fragment which is to be tested for interaction with the first test protein or protein fragment. Preferably, the DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein are derived from transcriptional activators having separate DNA-binding and transcriptional activation domains. These separate DNA-binding and transcriptional activation domains are also known to be found in the yeast GAL4 protein, and are also known to be found in the yeast GCN4 and ADR1 proteins. Many other proteins involved in transcription also have separable binding and transcriptional activation domains which make them useful for the present invention. In another embodiment, the DNA-binding domain and the transcriptional activation domain may be from different transcriptional activators. The second hybrid protein may be encoded on a library of plasmids that contain genomic, cDNA or synthetically generated DNA sequences fused to the DNA sequence encoding the transcriptional activation domain.