Identification and characterization of interacting molecules
First Claim
1. A method for detecting formation of complexes including a first test member and a potentially interacting second test member, comprising:
- (a) providing host cells containing;
(i) a first type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said first type of genetic element comprising nucleotide sequences encoding one of said first test member(s), and one of two parts of a functional entity;
(ii) a second type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said second type of genetic element comprising nucleotide sequences encoding one of said second test member(s), and the other of two parts of said functional entity;
wherein selectable and counter-selectable marker(s) associated with the first and second types of genetic elements differ by at least one selectable and at least one counter-selectable marker(s), respectively; and
(iii) a readout system for producing a detectable signal;
wherein interaction between said first and second test members brings said two parts of the functional entity into close proximity, thereby activating said readout system and producing the detectable signal;
(b) culturing the host cells under conditions wherein complexes including said first and second test members, if any, will be formed in the cell;
(c) selecting host cells wherein at least one of said complexes has formed by transferring samples of said host cells to;
(i) at least two different selective media, wherein each of said selective media allows growth of said host cells only in the absence of said counter-selectable marker(s) associated with one of said two types of genetic elements and in the presence of said selectable marker(s) associated with the other of said two types of genetic elements, wherein at least one of said at least two selective media selects for the presence of said first genetic element and the absence of said second genetic element, and at least a second of said at least two selective media selects for the presence of said second genetic element and the absence of said first genetic element; and
(ii) a further selective medium that allows identification of said host cells only on the activation of said readout system; and
(d) detecting formation of at least one of said complexes by identifying host cells wherein;
(i) said readout system is not activated on any of said selective media specified in step (c)(i);
but which (ii) said readout system is activated on said selective medium specified in step (c)(ii).
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Abstract
The present invention relates to an improved method for the identification and optionally the characterization of interacting molecules designed to detect positive clones from the rather large numbers of false positive clones isolated by two-hybrid systems. The method of the invention relies on a novel combination of selection steps used to detect clones that express interacting molecules from false positive clones. The present invention further relates to a kit useful for carrying out the method of the invention. The present invention provides for parallel, high-throughput or automated interaction screens for the reliable identification of interacting molecules.
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Citations
47 Claims
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1. A method for detecting formation of complexes including a first test member and a potentially interacting second test member, comprising:
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(a) providing host cells containing;
(i) a first type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said first type of genetic element comprising nucleotide sequences encoding one of said first test member(s), and one of two parts of a functional entity;
(ii) a second type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said second type of genetic element comprising nucleotide sequences encoding one of said second test member(s), and the other of two parts of said functional entity;
wherein selectable and counter-selectable marker(s) associated with the first and second types of genetic elements differ by at least one selectable and at least one counter-selectable marker(s), respectively; and
(iii) a readout system for producing a detectable signal;
wherein interaction between said first and second test members brings said two parts of the functional entity into close proximity, thereby activating said readout system and producing the detectable signal;
(b) culturing the host cells under conditions wherein complexes including said first and second test members, if any, will be formed in the cell;
(c) selecting host cells wherein at least one of said complexes has formed by transferring samples of said host cells to;
(i) at least two different selective media, wherein each of said selective media allows growth of said host cells only in the absence of said counter-selectable marker(s) associated with one of said two types of genetic elements and in the presence of said selectable marker(s) associated with the other of said two types of genetic elements, wherein at least one of said at least two selective media selects for the presence of said first genetic element and the absence of said second genetic element, and at least a second of said at least two selective media selects for the presence of said second genetic element and the absence of said first genetic element; and
(ii) a further selective medium that allows identification of said host cells only on the activation of said readout system; and
(d) detecting formation of at least one of said complexes by identifying host cells wherein;
(i) said readout system is not activated on any of said selective media specified in step (c)(i);
but which(ii) said readout system is activated on said selective medium specified in step (c)(ii). - 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, 44, 45)
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38. A method for identifying one or more compounds that modulate(s) the formation of complexes including a first test member and a second test member, comprising:
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(a) providing host cells containing;
(i) a first type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said first type of genetic element comprising nucleotide sequences encoding one of said first test member(s), and one of two parts of a functional entity;
(ii) a second type of genetic element associated with at least one each of a selectable and a counter-selectable marker, said second type of genetic element comprising nucleotide sequences encoding one of said second test member(s), and the other of two. parts of said functional entity;
wherein selectable and counter-selectable marker(s) associated with the first and second types of genetic elements differ by at least one selectable and at least one counter-selectable marker(s), respectively; and
(iii) a readout system for producing a detectable signal;
wherein interaction between said first and second test members brings said two parts of the functional entity into close proximity, which activates said readout system, thereby producing the detectable signal;
(b) culturing the host cells under conditions wherein complexes including said first and second test members, if any, will be formed in the cell;
(c) selecting for said complexes by transferring samples of said host cells to;
(i) at least two different selective media, wherein each of said selective media allows growth of said host cells only in the absence of said counter-selectable marker(s) associated with one of said two types of genetic elements and in the presence of said selectable markers associated with the other of said two types of genetic elements, wherein at least one of said at least two selective media selects for the presence of said first genetic element and the absence of said second genetic element, and at least a second of said at least two selective media selects for the presence of said second genetic element and the absence of said first genetic element; and
(ii) a further selective medium that allows identification of said host cells only on the activation of said readout system; and
(d) identifying interaction-positive host cells in which said members do not activate said readout system on any of said selective media specified in step (c)(i);
but which activate the readout system on said selective medium specified in step (c)(ii);
(e) identifying compounds which increase or decrease the amount of activation of said readout system in said interaction-positive host cells identified in step (d). - View Dependent Claims (47)
(a) identifying, using the method of claim 38, one or more compound(s) that modulates the formation of complexes including a first test member and a second test member;
(b) formulating, in a pharmaceutically acceptable excipient, said one or more compound(s) identified in step (a).
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39. A method for detecting interaction between a first test polypeptide and a second test polypeptide, comprising:
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(a) providing an interaction trap system including a host cell which contains;
(i) a reporter gene operably linked to a transcriptional regulatory sequence which includes a DNA-binding domain (DBD) recognition element, (ii) a first genetic element including;
a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and said first test polypeptide; and
coding sequences for at least one each of a selectable marker and a counter-selectable marker,(iii) a second genetic element including;
a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and said second test polypeptide; and
coding sequences for at least one each of a selectable marker and a counter-selectable marker;
wherein interaction of the first fusion protein and the second fusion protein in the host cell results in a measurable change in expression of the reporter gene;
(b) culturing the host cells under conditions wherein interaction between said first and second fusion proteins, if any, will occur in the cell;
(c) selecting host cells wherein a first test polypeptide and a second test polypeptide interact by transferring samples of said host cells to;
(i) at least two different selective media, wherein each of said selective media allows growth of said host cells only in the absence of said counter-selectable marker associated with one of said genetic elements and in the presence of said selectable marker associated with the other of said genetic elements, wherein at least one of said at least two selective media selects for the presence of said first genetic element and the absence of said second genetic element, and at least a second of said at least two selective media selects for the presence of said second genetic element and the absence of said first genetic element; and
(ii) a further selective medium that allows identification of said host cells only on expression of the reporter gene; and
(d) detecting an interaction between a first test polypeptide and a second test polypeptide by identifying host cells that do not activate the reporter gene on any of said selective media specified in step (c)(i);
but which do activate the reporter gene on said further selective medium specified in step (c)(ii).
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40. A kit for detecting formation of complexes, said kit comprising:
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(a) at least two types of genetic elements, each comprising a first genetic information encoding at least one counter-selectable marker, a second genetic information for accepting a sequence encoding one potentially interacting molecule, and a third genetic information encoding one of two parts of a functional entity, wherein each type of genetic element differs by at least one counter-selectable marker, and said functional entity is capable of activating a readout system when said two parts of the functional entity are brought into close proximity; and
(b) instructions to expose host cells containing said at least two types of genetic elements and a readout system that is activated by said functional entity, to at least three environments, wherein at least a first of said environments negatively selects against cells expressing said counter-selectable marker(s) associated with one of said at least two types of genetic elements, and at least a second of said environments negatively selects against cells expressing counter-selectable marker(s) associated with another of said at least two types of genetic elements, and wherein at least a third of said environments allows the identification of host cells in which said readout system is activated; and
to identify interaction-positive host cells in which said members do not activate said readout system in any of said first or second environment, but which activate said readout system in said third environment.- View Dependent Claims (41, 42)
(a) are contained in at least one storage compartment.
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43. A kit for detecting interaction between a first test polypeptide and a second test polypeptide, the kit comprising:
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(a) a first gene construct encoding a first fusion protein, which first gene construct comprises;
(i) a transcriptional regulatory sequence which directs expression of the first fusion protein in a host cell, (ii) a DNA sequence that encodes a DNA-binding domain and which is functionally associated with the transcriptional regulatory sequence of the first gene construct, (iii) a means 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 the first fusion protein containing the DNA binding domain, and (iv) at least one each of coding sequences for a selectable and counter-selectable marker;
(b) a second gene construct for encoding a second fusion protein, which second gene construct comprises;
(i) a transcriptional regulatory sequence which directs expression of the second fusion protein in a host cell, (ii) a DNA sequence that encodes an activation domain, and which is functionally associated with the transcriptional regulatory sequence of the second gene construct, (iii) a means for inserting a DNA sequence encoding a second test polypeptide into the second vector in such a manner that the second test polypeptide is expressed in-frame as part of the second fusion protein containing the activation tag, and (iv) at least one each of coding sequences for a selectable and counter-selectable marker;
(c) a host cell containing a reporter gene having a DNA-binding domain (DBD) recognition element of the first fusion protein, wherein the reporter gene expresses a detectable transcript or protein when the first and second fusion proteins interact; and
(d) optionally at least one storage compartment, planar carrier or computer disc comprising or/and characterizing genetic elements, host cells, storage compartments or carriers identified in any of (a) to (c).
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46. A kit for detecting formation of complexes, said kit comprising:
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(a) a first type and a second type of genetic elements, each comprising a first genetic information, encoding at least one counter-selectable marker, a second genetic information encoding one member of a variegated library of potentially interacting molecules, and a third genetic information encoding one of two parts of a functional entity, wherein the counter-selectable marker(s) associated with one of said two types of genetic elements differs from that counter-selectable markers associated with the other of said two types of genetic elements by at least one counter-selectable marker, wherein each type of genetic element is associated with a different part of the functional entity, and said functional entity is capable of activating a readout system when said two parts of the functional entity are brought into close proximity; and
(b) instructions to expose host cells containing said first and second types of genetic elements to at least two environments, wherein at least a first of said environments negatively selects against cells expressing one or more counter-selectable markers associated with one of said two types of genetic elements, and at least a second of said environments negatively selects against cells expressing one or more counterselectable markers associated with the other of said two types of genetic elements.
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Specification