Methods and compositions for interaction trap assays
First Claim
1. A method for selecting an interacting pair of test polypeptides, comprising:
- i providing a population of prokaryotic host cells wherein each host cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and a first test polypeptide, (c) a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and second test polypeptide, wherein the first fusion protein is part of a library of at least 107 members, the second fusion protein is part of a library of at least 107 members, or the first and second fusion proteins are both members of a library such that at least 107 unique pairs of test polypeptides could be tested for interaction;
wherein interaction of a first fusion protein and a second fusion protein in a host cell results in a desired level of expression of the reporter gene;
wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell; and
ii isolating host cells with a growth advantage wherein said cells comprise a first fusion protein and a second fusion protein which interact thereby selecting an interacting pair of test polypeptides.
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Abstract
The present invention provides methods and compositions for interaction trap assays for detecting protein-protein, protein-DNA, or protein-RNA interactions. The methods and compositions of the invention may also be used to identify agents which may agonize or antagonize a protein-protein, protein-DNA, or protein-RNA interaction. In certain embodiments, the interaction trap system of the invention is useful for screening libraries with greater than 107 members. In other embodiments, the interaction trap system of the invention is used in conjunction with flow cytometry. The invention further provides a means for simultaneously screening a target protein or nucleic acid sequence for the ability to interact with two or more test proteins or nucleic acids.
6 Citations
272 Claims
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1. A method for selecting an interacting pair of test polypeptides, comprising:
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i providing a population of prokaryotic host cells wherein each host cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and a first test polypeptide, (c) a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and second test polypeptide, wherein the first fusion protein is part of a library of at least 107 members, the second fusion protein is part of a library of at least 107 members, or the first and second fusion proteins are both members of a library such that at least 107 unique pairs of test polypeptides could be tested for interaction;
wherein interaction of a first fusion protein and a second fusion protein in a host cell results in a desired level of expression of the reporter gene;
wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell; and
ii isolating host cells with a growth advantage wherein said cells comprise a first fusion protein and a second fusion protein which interact thereby selecting an interacting pair of test polypeptides. - 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, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 75)
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49. A method for identifying agents which modulate a protein-protein interaction, comprising:
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i providing a population of prokaryotic host cells wherein each host cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and a first test polypeptide, (c) a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and second test polypeptide, wherein the prokaryotic host cell is an imp- or gram positive strain of bacteria;
wherein interaction of a first fusion protein and a second fusion protein in a host cell results in a desired level of expression of the reporter gene;
ii contacting the host cell with at least one test agent; and
iii identifying test agents which modulate expression of the reporter gene in a manner also dependent on the expression of the first and second test polypeptides, thereby identifying agents which modulate a protein-protein interaction. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57)
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58. A method for selecting a polypeptide which differentially interacts with at least two different test polypeptides, comprising:
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i providing a population of prokaryotic host cells wherein each cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a second DNA-binding domain, (c) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a first DNA-binding domain and a first test polypeptide, (d) a second chimeric gene which encodes a second fusion protein, the second fusion protein including a second DNA-binding domain and a second test polypeptide, (e) a third chimeric gene which encodes a third fusion protein, the third fusion protein including an activation tag and third test polypeptide, wherein the third fusion protein is part of a library of at least 107 members;
wherein interaction of the first fusion protein and the third fusion protein in the host cell results in a desired level of expression of the first reporter gene;
wherein interaction of the second fusion protein and the third fusion protein in the host cell results in a desired level of expression of the second reporter gene; and
ii isolating host cells comprising a third fusion protein capable of interacting with the first fusion protein, the second fusion protein, or the first and the second fusion proteins based on a desired level of expression of the first reporter gene, the second reporter gene, or the first and second reporter genes, respectively, thereby selecting a polypeptide which differentially interacts with at least two different test polypeptides. - View Dependent Claims (59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)
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70. A method for selecting a test agent that differentially modulates the interaction of a polypeptide with at least two different test polypeptides, comprising:
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i providing a population of prokaryotic host cells wherein each cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a second DNA-binding domain, (c) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a first DNA-binding domain and a first test polypeptide, (d) a second chimeric gene which encodes a second fusion protein, the second fusion protein including a second DNA-binding domain and a second test polypeptide, (e) a third chimeric gene which encodes a third fusion protein, the third fusion protein including an activation tag and third test polypeptide, wherein the host cell is an imp- or gram positive strain of bacteria;
wherein interaction of the first fusion protein and the third fusion protein in the host cell results in a desired level of expression of the first reporter gene;
wherein interaction of the second fusion protein and the third fusion protein in the host cell results in a desired level of expression of the second reporter gene;
ii contacting the host cell with at least one test agent; and
iii identifying test agents which modulate the expression of the first, second, or first and second reporter genes in a manner also dependent on the expression of the first, second and third test polypeptides, thereby selecting a test agent that differentially modulates the interaction of a polypeptide with at least two different test polypeptides.
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71. A method for detecting an interaction between a test polypeptide and a DNA sequence, comprising
i providing a population of prokaryotic host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein the DBD recognition element is part of a library of at least 107 members, the fusion protein is part of a library of at least 107 members, or the DBD recognition element and the fusion protein are both members of a library such that at least 107 unique pairs of a DBD recognition element and a fusion protein could be tested for interaction; -
wherein interaction between a test polypeptide of a fusion protein and a DBD recognition element in a host cell results in a desired level of expression of the reporter gene;
wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell; and
ii isolating host cells with a growth advantage wherein said cells comprise a fusion protein and a DBD recognition element which interact, thereby detecting an interaction between a test polypeptide and a DNA sequence. - View Dependent Claims (72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101)
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102. A method for identifying agents which modulate an interaction between a test polypeptide and a DNA sequence, comprising
i providing a population of prokaryotic host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein the prokaryotic host cell is an imp− - or gram positive strain of bacteria;
wherein interaction between a test polypeptide of a fusion protein and a DBD recognition element in a host cells results in a desired level of expression of the reporter gene;
ii contacting the host cell with at least one test agent; and
iii identifying agents which modulate expression of the reporter gene in a manner also dependent on the presence of a fusion protein and a DBD recognition element. - View Dependent Claims (103, 104, 105, 106, 107, 108, 109, 110)
- or gram positive strain of bacteria;
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111. A method for selecting a polypeptide that differentially interacts with at least two different DNA sequences, comprising
i providing a population of prokaryotic host cells each of which contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding site (DBD recognition element) for a second DNA-binding domain, (c) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein the fusion protein is part of a library of at least 107 members; -
wherein interaction of a fusion protein with the first DBD recognition element in the host cells results in a desired level of expression of the first reporter gene;
wherein interaction of a fusion protein with the second DBD recognition element in the host cells results in a desired level of expression of the second reporter gene; and
ii isolating host cells comprising a fusion protein that interacts with the first DBD recognition element, the second DBD recognition element, or the first and second DBD recognition elements based on a desired level of expression of the first reporter gene, the second reporter gene, or the first and second reporter genes, respectively, thereby selecting a polypeptide that differentially interacts with at least two different DNA sequences. - View Dependent Claims (112, 113, 114, 115, 116, 117, 118)
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119. A method for selecting a test agent that differentially modulates the interaction of a polypeptide with at least two different DNA sequences, comprising
i providing a population of prokaryotic host cells each of which contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding site (DBD recognition element) for a second DNA-binding domain, (c) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein the prokaryotic host cell is an imp− - or gram positive strain of bacteria;
wherein interaction of a fusion protein with the first DBD recognition element in the host cells results in a desired level of expression of the first reporter gene;
wherein interaction of a fusion protein with the second DBD recognition element in the host cells results in a desired level of expression of the second reporter gene;
ii contacting the host cell with at least one test agent; and
iii identifying test agents which modulate the expression of the first, second, or first and second reporter genes in a manner also dependent on the presence of the fusion protein and the first and second DBD recognition elements, thereby selecting a test agent that differentially modulates the interaction of a polypeptide with at least two different DNA sequences. - View Dependent Claims (120)
- or gram positive strain of bacteria;
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121. A method for detecting an interaction between a test RNA binding domain polypeptide and an RNA sequence, comprising
i providing a population of prokaryotic host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a fusion protein, the fusion protein including a DNA-binding domain and a first RNA binding domain, (c) a second chimeric gene which encodes a fusion protein, the fusion protein including an activation tag and a second RNA binding domain, (d) a third chimeric gene which encodes a hybrid RNA, the hybrid RNA comprising a first RNA sequence that binds one of the first or second RNA binding domains and a second RNA sequence to be tested for interaction with the RNA-binding domain not bound to the first RNA sequence; -
wherein the RNA-binding domain not bound to the first RNA sequence is part of a library of at least 107 members, the second RNA sequence is part of a library of at least 107 members, or the RNA-binding domain not bound to the first RNA sequence and the second RNA sequence are both members of a library such that at least 107 unique pairs of an RNA-binding domain and an RNA sequence could be tested for interaction;
wherein interaction of an RNA-binding domain not bound to the first RNA sequence with the second RNA sequence in a host cell results in a desired level of expression of the reporter gene; and
ii isolating host cells comprising an RNA-binding domain that interacts with the second RNA sequence based on a desired level of expression of the reporter gene thereby detecting an interaction between a test RNA binding domain polypeptide and an RNA sequence.
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122. A kit for selecting a polypeptide that interacts with a test polypeptide, comprising:
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i a first gene construct for encoding a first fusion protein, which first gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a prokaryotic host cell, (b) a DNA sequence that encodes a DNA binding domain and which is operably linked with the transcriptional and translational elements of the first gene construct, and (c) one or more sites for inserting a DNA sequence encoding a first test polypeptide into the first gene construct in such a manner that the first test polypeptide is expressed in-frame as part of a fusion protein containing the DNA binding domain;
ii a second gene construct for encoding a second fusion protein, which second gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a prokaryotic host cell, (b) a DNA sequence that encodes an activation tag and which is operably linked with the transcriptional and translational elements of the second gene construct, and (c) one or more sites for inserting a DNA sequence encoding a second test polypeptide into the second gene construct in such a manner that the second test polypeptide is expressed in-frame as part of a fusion protein containing the activation tag; and
iii a prokaryotic host cell containing at least one reporter gene having one or more binding sites (DBD recognition elements) for the DNA binding domain, and wherein a desired level of expression of the reporter gene is obtained upon interaction of the first and second fusion proteins; and
wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell. - View Dependent Claims (123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135)
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136. A kit for detecting an interaction between a test DNA-binding domain polypeptide and a DNA sequence, comprising:
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i a first gene construct which comprises;
(a) one or more sites for inserting a DNA sequence comprising a transcriptional element which includes at least one binding site (DBD recognition element) for a DNA-binding domain, (b) a translational element operably linked to the transcriptional element, and (c) a DNA sequence for a reporter gene which is operably linked with the transcriptional and translational elements of the first gene construct, and wherein the transcriptional and translational elements direct expression of the reporter gene in a prokaryotic host cell;
ii a second gene construct for encoding a first fusion protein, which second gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a prokaryotic host cell, (b) a DNA sequence that encodes an activation tag and which is operably linked with the transcriptional and translational elements of the second gene construct, and (c) one or more sites for inserting a DNA sequence encoding a first test polypeptide into the second gene construct in such a manner that the first test polypeptide is expressed in-frame as part of a fusion protein containing the activation tag;
iii a prokaryotic host cell, and wherein a desired level of expression of the reporter gene is obtained upon interaction of a test polypeptide with a DBD recognition element; and
wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell. - View Dependent Claims (137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159)
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160. A method for detecting an interaction between a first test polypeptide and a second test polypeptide, comprising:
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i providing a population of host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and a first test polypeptide, (c) a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and second test polypeptide, wherein expression of the reporter gene results in signal detectable by FACS;
wherein interaction of the first fusion protein and second fusion protein in the host cell results in a desired level of expression of the reporter gene; and
ii isolating host cells comprising an interacting pair of fusion proteins based on a desired level of expression of the reporter gene using FACS thereby detecting an interaction between a first test polypeptide and a second test polypeptide. - View Dependent Claims (161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190)
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191. A method for selecting a polypeptide that differentially interacts with at least two different test polypeptides, comprising:
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i providing a population of host cells wherein each cell contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a second DNA-binding domain, (c) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a first DNA-binding domain and a first test polypeptide, (d) a second chimeric gene which encodes a second fusion protein, the second fusion protein including a second DNA-binding domain and a second test polypeptide, (e) a third chimeric gene which encodes a third fusion protein, the third fusion protein including an activation tag and third test polypeptide, wherein expression of the first and second reporter genes results in a signal detectable by FACS;
wherein interaction of the first fusion protein and the third fusion protein in the host cell results in a desired level of expression of the first reporter gene;
wherein interaction of the second fusion protein and the third fusion protein in the host cell results in a desired level of expression of the second reporter gene; and
ii isolating host cells comprising a third fusion protein capable of interacting with the first fusion protein, the second fusion protein, or the first and the second fusion proteins based on a desired level of expression of the first reporter gene, the second reporter gene, or the first and second reporter genes, respectively, using FACS, thereby selecting a polypeptide that differentially interacts with at least two different test polypeptides. - View Dependent Claims (192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204)
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205. A method for detecting an interaction between a test polypeptide and a DNA sequence, comprising
i providing a population of host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein expression of the reporter gene results in signal detectable by FACS; -
wherein interaction between a test polypeptide of a fusion protein and a DBD recognition element in a host cells results in a desired level of expression of the reporter gene; and
ii isolating host cells comprising a fusion protein that interacts with a DBD recognition element based on a desired level of expression of the reporter gene using FACS thereby detecting an interaction between the test polypeptide and the DBD recognition element DNA sequence. - View Dependent Claims (206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231)
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232. A method for selecting a polypeptide that differentially interacts with at least two different DNA sequences, comprising
i providing a population of host cells each of which contains (a) a first reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a first DNA-binding domain, (b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding site (DBD recognition element) for a second DNA-binding domain, (c) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide and an activation tag, wherein expression of the first and second reporter genes results in a signal detectable by FACS; -
wherein interaction of a fusion protein with the first DBD recognition element in the host cells results in a desired level of expression of the first reporter gene;
wherein interaction of a fusion protein with the second DBD recognition element in the host cells results in a desired level of expression of the second reporter gene; and
ii isolating host cells comprising a fusion protein that interacts with the first DBD recognition element, the second DBD recognition element, or the first and second DBD recognition elements based on a desired level of expression of the first reporter gene, the second reporter gene, or the first and second reporter genes, respectively, using FACS, thereby selecting a polypeptide that differentially interacts with at least two different DNA sequences. - View Dependent Claims (233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243)
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244. A method for detecting an interaction between a test RNA binding domain polypeptide and an RNA sequence, comprising
i providing a population of host cells wherein each cell contains (a) a reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain, (b) a first chimeric gene which encodes a fusion protein, the fusion protein including a DNA-binding domain and a first RNA binding domain, (c) a second chimeric gene which encodes a fusion protein, the fusion protein including an activation tag and a second RNA binding domain, (d) a third chimeric gene which encodes a hybrid RNA, the hybrid RNA comprising a first RNA sequence that binds one of the first or second RNA binding domains and a second RNA sequence to be tested for interaction with the RNA-binding domain not bound to the first RNA sequence; -
wherein the expression of the reporter gene produces a signal detectable by FACS;
wherein interaction of an RNA-binding domain not bound to the first RNA sequence with the second RNA sequence in a host cell results in a desired level of expression of the reporter gene; and
ii isolating host cells comprising an RNA-binding domain that interacts with the second RNA sequence based on a desired level of expression of the reporter gene thereby detecting an interaction between a test RNA binding domain polypeptide and an RNA sequence using FACS. - View Dependent Claims (245, 246, 247, 248, 249)
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250. A kit for selecting a polypeptide that interacts with a test polypeptide, comprising:
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i a first gene construct for encoding a first fusion protein, which first gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a host cell, (b) a DNA sequence that encodes a DNA binding domain and which is operably linked with the transcriptional and translational elements of the first gene construct, and (c) one or more sites for inserting a DNA sequence encoding a first test polypeptide into the first gene construct in such a manner that the first test polypeptide is expressed in-frame as part of a fusion protein containing the DNA binding domain;
ii a second gene construct for encoding a second fusion protein, which second gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a host cell, (b) a DNA sequence that encodes an activation tag and which is operably linked with the transcriptional and translational elements of the second gene construct, and (c) one or more sites for inserting a DNA sequence encoding a second test polypeptide into the second gene construct in such a manner that the second test polypeptide is expressed in-frame as part of a fusion protein containing the activation tag;
iii a host cell containing at least one reporter gene having one or more binding sites (DBD recognition elements) for the DNA binding domain;
wherein expression of the reporter gene produces a signal detectable by FACS; and
wherein a desired level of expression of the reporter gene is obtained upon interaction of the first and second fusion proteins and can by analyzed using FACS. - View Dependent Claims (251, 252, 253, 254, 255, 256)
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257. A kit for detecting an interaction between a test DNA-binding domain polypeptide and a DNA sequence, comprising:
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i a first gene construct which comprises;
(a) one or more sites for inserting a DNA sequence comprising a transcriptional element which includes at least one binding site (DBD recognition element) for a DNA-binding domain, (b) a translational element operably linked to the transcriptional element, and (c) a DNA sequence for at least one reporter gene which is operably linked with the transcriptional and translational elements of the first gene construct, and wherein the transcriptional and translational elements direct expression of the reporter gene in a host cell;
ii a second gene construct for encoding a first fusion protein, which second gene construct comprises;
(a) transcriptional and translational elements which direct expression of a protein in a host cell, (b) a DNA sequence that encodes an activation tag and which is operably linked with the transcriptional and translational elements of the second gene construct, and (c) one or more sites for inserting a DNA sequence encoding a first test polypeptide into the second gene construct in such a manner that the first test polypeptide is expressed in-frame as part of a fusion protein containing the activation tag;
iii a host cell;
wherein expression of the reporter gene produces a signal detectable by FACS; and
wherein a desired level of expression of the reporter gene is obtained upon interaction of a test polypeptide with a DBD recognition element and can by analyzed by FACS. - View Dependent Claims (258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272)
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Specification