Detection of protein to protein interactions
AssigneeThe Governing Council of the University of Toronto
InventorsIgor Stagljar, Julia Petschnigg, Bella Groisman
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Abstract
The present invention relates to methods and kits for detecting the interaction between a first membrane protein and a second protein in mammalian cells as well as to identify molecules that can disrupt protein to protein interactions. The invention relies on the functional reconstitution of an active human ubiquitin by two inactive fragments upon the interaction of two proteins attached through a linker to the inactive fragments. The reconstituted ubiquitin is then cleaved by human ubiquitin proteases resulting in the release of an artificial transcription factor, which in turn activates a reporter gene transcription. Activation of the reporter gene is indicative of the interaction between the two proteins.
Claims23
A method for detecting the interaction between a first membrane protein or part thereof and a second membrane or soluble protein or part thereof comprising:
- (a) providing a host mammalian cell including at least one detectable gene (reporter gene) having a binding site for a transcription factor, such that the detectable gene expresses a detectable product;
- (b) expressing in the host mammalian cell the first protein or part thereof, the first protein or part thereof being attached through a suitable first linker to a C-terminal sub-domain of a human ubiquitin (Cub) and a transcription factor;
- (c) expressing in the host mammalian cell the second protein or part thereof, the second protein or part thereof being attached to a wild-type N-terminal sub-domain of the human ubiquitin protein (Nub) through a second suitable linker;
- and (d) determining whether the detectable product is detected, detection of the detectable product being indicative that the first protein and the second protein interact, wherein the first linker and the second linker is a peptide comprising the amino acid sequence (GGGGS)n, wherein “n” is an integer equal to or larger than 1, and wherein the detectable gene includes a fluorescent reporter gene and the detectable product is luciferase.
A method for quantitatively measuring strength and affinity of an interaction between a first membrane protein or part thereof and a second membrane or soluble protein or part thereof comprising:
- (a) providing a host mammalian cell including a detectable gene (reporter gene) having a binding site for a transcription factor, such that the detectable gene expresses a measurable detectable product when the detectable gene is transcriptionally activated;
- (b) expressing in the host mammalian cell the first protein or part thereof, the first protein or part thereof being attached through a suitable first linker to a C-terminal sub-domain of a human ubiquitin (Cub) and the transcription factor;
- (c) expressing in the host mammalian cell the second protein or part thereof, the second protein or part thereof being attached to a wild-type N-terminal sub-domain of the human ubiquitin protein (Nub) through a second suitable linker;
- and (d) measuring an expression output of the detectable product as a measure of the amount of interaction between the first and the second proteins, wherein the first linker and the second linker is a peptide comprising the amino acid sequence (GGGGS)n, wherein “n” is an integer equal to or larger than 1, and wherein the detectable gene includes a fluorescent reporter gene and the detectable product is luciferase.
A kit of reagents for quantitatively measuring strength and affinity binding between a first membrane protein or part thereof and a second membrane or soluble protein or part thereof comprising:
- (a) a host cell including at least one detectable gene (reporter gene) having a binding site for a transcription factor, such that the detectable gene expresses a detectable product when the detectable gene is transcriptionally activated, the at least one detectable gene including a fluorescent reporter gene and the detectable product is luciferase;
- (b) a first vector (bait) comprising a first site that can receive a first nucleic acid coding for the first protein or part thereof such that when the first nucleic acid is inserted it becomes attached to the DNA sequence of a first module encoding inter alia a C-terminal sub-domain of a human ubiquitin protein (Cub), a first suitable linker between the first protein and the Cub, the first module further comprising a nucleic acid for the transcription factor, and a promoter, the first linker being a peptide comprising the amino acid sequence (GGGGS)n, wherein “n” is an integer equal to or larger than 1;
- (c) a second vector (prey) comprising a second site that can receive a second nucleic acid coding for the second protein or part thereof such that when the second nucleic acid is inserted it becomes attached to the DNA sequence of a second module encoding inter alia a wild-type N-terminal sub-domain of the human ubiquitin protein (Nub) and a second suitable linker between the second protein and the Nub, wherein the second module further comprises a promoter, the second linker being a peptide comprising the amino acid sequence (GGGGS)n, wherein “n” is an integer equal to or larger than 1.
Dependencies are inferred from the claim text.