Methods and assays for screening protein targets
First Claim
1. A method for identifying a protein target as being able to bind a ligand, comprising:
- (a) providing a molecule comprising a methotrexate moiety covalently linked to the ligand, which methotrexate moiety binds to a dihydrofolate reductase;
(b) introducing the molecule into a cell which i) expresses a first fusion protein comprising the dihydrofolate reductase that binds to the methotrexate moiety, ii) expresses a second fusion protein comprising the protein target, wherein either the first or second fusion protein also comprises a transcription activator domain and the other fusion protein comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein and wherein the DNA-binding domain binds upstream of the reporter gene, and wherein the cell is a yeast cell;
(c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and
(d) selecting the cell if it expresses the reporter gene, so as to thereby identify the protein target as being able to bind the ligand.
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Abstract
A method for identifying a protein as being able to bind a ligand comprising, providing a molecule composed of a methotrexate moiety that is covalently bonded to the ligand; introducing the molecule into a cell which a) expresses a first fusion protein comprising a dihydrofolate reductase capable of binding methotrexate, expresses b) a second fusion protein comprising the protein, wherein one of the first and second fusion proteins also comprises a transcription activator domain and the other comprises a DNA-binding domain, and c) has a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein; permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and selecting the cell if it expresses the reporter gene.
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Citations
28 Claims
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1. A method for identifying a protein target as being able to bind a ligand, comprising:
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(a) providing a molecule comprising a methotrexate moiety covalently linked to the ligand, which methotrexate moiety binds to a dihydrofolate reductase; (b) introducing the molecule into a cell which i) expresses a first fusion protein comprising the dihydrofolate reductase that binds to the methotrexate moiety, ii) expresses a second fusion protein comprising the protein target, wherein either the first or second fusion protein also comprises a transcription activator domain and the other fusion protein comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein and wherein the DNA-binding domain binds upstream of the reporter gene, and wherein the cell is a yeast cell; (c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and (d) selecting the cell if it expresses the reporter gene, so as to thereby identify the protein target as being able to bind the ligand. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for identifying a protein target as being able to bind a ligand, comprising:
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(a) providing a molecule comprising a methotrexate moiety covalently linked to the ligand, which methotrexate moiety binds to a dihydrofolate reductase; (b) introducing the molecule into a cell which i) expresses a first fusion protein comprising the dihydrofolate reductase that binds to the methotrexate moiety, ii) expresses a second fusion protein comprising the protein target, wherein either the first or second fusion protein also comprises a transcription activator domain and the other fusion protein comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein and wherein the DNA-binding domain binds upstream of the reporter gene, and wherein the cell is a bacterial cell; (c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and (d) selecting the cell if it expresses the reporter gene, so as to thereby identify the protein target as being able to bind the ligand. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification