Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis
First Claim
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1. A method of detecting an interaction between a bait polypeptide and a prey polypeptide comprising:
- introducing a first nucleic acid encoding a first hybrid protein into a host cell, the first nucleic acid having a first exogenously activatable promoter, and the first hybrid protein having a DNA binding region and the bait polypeptide;
introducing a second nucleic acid encoding a second hybrid protein into the host cell, the second nucleic acid having a second exogenously activatable promoter different from the first exogenously activatable promoter, and the second hybrid protein having a transcriptional activation region and the prey polypeptide;
activating the first and second promoters using first and second exogenous activators to induce expression of the first and second hybrid proteins; and
detecting an interaction between the bait polypeptide and the prey polypeptide by activation of a detectable reporter gene in the host cell, wherein the DNA binding region binds near the reporter gene and the transcriptional activation region activates transcription of the reporter gene when brought into proximity to the reporter gene by an interaction between the bait polypeptide and the prey polypeptide;
wherein sensitivity of detecting an interaction may be continuously adjusted by altering the relative or absolute amount of at least one of the first or second hybrid proteins in the host cell and wherein amounts of the first and second hybrid proteins in the host cell are independent of one another.
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Abstract
A method for detecting interactions between first and second interacting molecules a variable sensitivity. This variable sensitivity may be obtained by providing for the overexpression of either a bait hybrid protein containing a DNA binding domain (desensitization) or a prey hybrid protein containing the DNA activation domain for a reporter gene (enhanced sensitivity). The use of exogenous activators of one or the other according to the needs of a particular system is readily accomplished.
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Citations
21 Claims
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1. A method of detecting an interaction between a bait polypeptide and a prey polypeptide comprising:
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introducing a first nucleic acid encoding a first hybrid protein into a host cell, the first nucleic acid having a first exogenously activatable promoter, and the first hybrid protein having a DNA binding region and the bait polypeptide;
introducing a second nucleic acid encoding a second hybrid protein into the host cell, the second nucleic acid having a second exogenously activatable promoter different from the first exogenously activatable promoter, and the second hybrid protein having a transcriptional activation region and the prey polypeptide;
activating the first and second promoters using first and second exogenous activators to induce expression of the first and second hybrid proteins; and
detecting an interaction between the bait polypeptide and the prey polypeptide by activation of a detectable reporter gene in the host cell, wherein the DNA binding region binds near the reporter gene and the transcriptional activation region activates transcription of the reporter gene when brought into proximity to the reporter gene by an interaction between the bait polypeptide and the prey polypeptide;
wherein sensitivity of detecting an interaction may be continuously adjusted by altering the relative or absolute amount of at least one of the first or second hybrid proteins in the host cell and wherein amounts of the first and second hybrid proteins in the host cell are independent of one another. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of detecting an interaction between a bait polypeptide and a prey polypeptide comprising:
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introducing a first nucleic acid encoding a first hybrid protein into a host cell, the first nucleic acid having an estrogen-sensitive promoter, and the first hybrid protein having a GAL4 binding domain and the bait polypeptide;
introducing a second nucleic acid encoding a second hybrid protein into the host cell, the second nucleic acid having a glucocorticoid-sensitive promoter, and the second hybrid protein having a GAL4 transcriptional activation domain and the prey polypeptide;
activating the promoters to induce expression of the first and second hybrid proteins; and
detecting an interaction between the bait polypeptide and the prey polypeptide by activation of a UASg-LacZ reporter gene in the host cell;
wherein sensitivity of detecting an interaction may be continuously adjusted by altering the relative or absolute amount of at least one of the first or second hybrid proteins in the host cell and wherein amounts of the first and second hybrid proteins in the host cell are independent of one another. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21)
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Specification