Compositions, methods and kits for identying protein-protein interaction disrupting agents
First Claim
1. A mammalian reverse two-hybrid system for identifying a change in protein-protein interaction comprising:
- a suppressor construct comprising a constitutive promoter operably linked to a repressor protein coding sequence;
an interactive fusion protein coding construct comprising a promoter operably linked to a repressible operator repressible by a repressor protein coded by said repressor protein coding sequence, a first interactive protein coding sequence fused in frame with a transcriptional activation domain protein coding sequence and a second interactive protein coding sequence fused in frame with a DNA binding domain protein coding sequence; and
, a reporter construct comprising a DNA binding element bindable to said DNA binding domain protein operably linked to a minimal promoter and a reporter coding sequence.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention provides compositions, methods, and kits for identifying agents that are capable of disrupting protein-protein interaction in a mammalian reverse two-hybrid system. A tetracycline repressor protein was fused with the inhibitory KRAB domain as a suppressor to tightly regulate the reverse two-hybrid system for mammalian cells. Binding of the chimeric protein to the tetracycline operator sequence provided within a promoter entirely suppressed the expression of two interactive proteins. When both an inducer and a candidate protein-protein interaction disrupting agent such as a drug were added in the cell culture together, the reporter gene was either activated by the interaction of protein-pair if the drug was ineffective or remained silent due to the disruption of the protein-protein interaction by the effective drug. The plasmids for the suppressor and the reporter were integrated into chromosomes. Constructs for the expression of interactive proteins were either stably or transiently transfected into the cells. The utility of this system for screening drugs, particularly for enzyme inhibitor drugs, was demonstrated by using two well characterized interactions of the type I receptor for TGFβ with FKPB12 and the EGF receptor with p85. The interaction between TGFβBI and FKPB12 or between EGF receptor and p85 were disrupted by FK506 and kinase inhibitor AG1478, respectively. The mammalian reverse two-hybrid system of the present invention can also be used for high throughput screening of compounds that disrupt protein-protein interactions.
-
Citations
32 Claims
-
1. A mammalian reverse two-hybrid system for identifying a change in protein-protein interaction comprising:
-
a suppressor construct comprising a constitutive promoter operably linked to a repressor protein coding sequence;
an interactive fusion protein coding construct comprising a promoter operably linked to a repressible operator repressible by a repressor protein coded by said repressor protein coding sequence, a first interactive protein coding sequence fused in frame with a transcriptional activation domain protein coding sequence and a second interactive protein coding sequence fused in frame with a DNA binding domain protein coding sequence; and
,a reporter construct comprising a DNA binding element bindable to said DNA binding domain protein operably linked to a minimal promoter and a reporter coding sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30)
-
-
29. A method for identifying a change in protein-protein interaction comprising:
-
(a) stably introducing a suppressor construct and a reporter construct in a cell;
(b)(i) stably introducing a first interactive protein-transcriptional activation domain fusion protein coding construct and a second interactive protein-DNA binding domain fusion protein coding construct into said cell;
or(b)(ii) transiently introducing a first interactive protein-transcriptional activation domain fusion protein coding construct and a second interactive protein-DNA binding domain fusion protein coding construct into said cell;
(c) subjecting a cell resulting from step (b)(i) or (b)(ii) to an inducer and a test protein-protein interaction disrupting agent;
(d) assaying said subjected cell for the non-expression and expression of said reporter coding sequence; and
,(e) identifying said protein-protein interaction disrupting agent based on non-expression of said reporter coding sequence.
-
-
31. A kit for identifying a change in protein-protein interaction comprising:
-
one or more aliquots of a suppressor construct, a first interactive protein-transcriptional activation domain fusion protein coding construct, a second interactive protein-DNA binding domain fusion protein coding construct, and a reporter construct on one or more plasmids; and
,one or more aliquots of a reagent, cell, and apparatus needed for conducting a method for identifying a change in protein-protein interaction. - View Dependent Claims (32)
-
Specification