Interaction trap systems for detecting protein interactions

Abstract

Disclosed herein is a method of determining whether a first protein is capable of physically interacting with a second protein, involving: (a) providing a host cell which contains (i) a reporter gene operably linked to a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including the first protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the second protein covalently bonded to a gene activating moiety and being conformationally-constrained; and (b) measuring expression of the reporter gene as a measure of an interaction between the first and the second proteins. Also disclosed are methods for assaying protein interactions, and identifying antagonists and agonists of protein interactions. Proteins isolated by these methods are also discussed. Finally, populations of eukaryotic cells are disclosed, each cell having a recombinant DNA molecule encoding a conformationally-constrained intracellular peptide.

1 . A method of determining whether a first protein is capable of physically interacting with a second protein, comprising: 
 (a) providing a host cell which contains 
 (i) a reporter gene operably linked to a DNA-binding-protein recognition site;  
 (ii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said first protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site; and  
 (iii) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said second protein covalently bonded to a gene activating moiety and being conformationally-constrained; and  
   (b) measuring expression of said reporter gene as a measure of an interaction between said first and said second proteins.    
     
     
         2 . The method of  claim 1 , wherein said second protein is a peptide of at least 6 amino acids.  
     
     
         3 . The method of  claim 1 , wherein said second protein is a peptide of less than or equal to 60 amino acids in length.  
     
     
         4 . The method of  claim 1 , wherein said second protein comprises a randomly generated or intentionally de signed peptide sequence.  
     
     
         5 . The method of  claim 1 , wherein said second protein is conformationally-constrained because it is covalently bonded to a conformation-constraining protein.  
     
     
         6 . The method of  claim 1 , wherein said second protein comprises one or more loops.  
     
     
         7 . The method of  claim 1 , wherein said first protein is Cdk2.  
     
     
         8 . The method of  claim 1 , wherein said first protein is Ras or an activated Ras.  
     
     
         9 . The method of  claim 5 , wherein said second protein is embedded within said conformation-constraining protein.  
     
     
         10 . The method of  claim 5 , wherein said conformation-constraining protein is thioredoxin.  
     
     
         11 . The method of  claim 5 , wherein said conformation-constraining protein is a thioredoxin-like molecule.  
     
     
         12 . The method of  claim 10 , wherein said second protein is inserted into the active site loop of said thioredoxin protein.  
     
     
         13 . The method of  claim 1 , wherein said second protein is conformationally-constrained by disulfide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of said second protein.  
     
     
         14 . The method of  claim 1 , wherein said host cell is yeast.  
     
     
         15 . The method of  claim 1 , wherein said DNA binding domain is LexA.  
     
     
         16 . The method of  claim 1 , wherein said reporter gene is assayed by a color reaction.  
     
     
         17 . The method of  claim 1 , wherein said reporter gene is assayed by cell viability.  
     
     
         18 . A method of detecting an interacting protein in a population of proteins, comprising: 
 (a) providing a host cell which contains 
 (i) a reporter gene operably linked to a DNA-binding-protein recognition site; and  
 (ii) a fusion gene which expresses a fusion protein, said fusion protein comprising a test protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site;  
   (b) introducing into said host cell a second fusion gene which expresses a second fusion protein, said second fusion protein comprising one of said population of proteins covalently bonded to a gene activating moiety and being conformationally-constrained; and    (c) measuring expression of said reporter gene.    
     
     
         19 . The method of  claim 18 , wherein said population of proteins comprises short peptides of between 1 and 60 amino acids in length.  
     
     
         20 . The method of  claim 18 , wherein said population of proteins is a set of randomly generated or intentionally designed peptide sequences.  
     
     
         21 . The method of  claim 18 , wherein said population of proteins is conformationally-constrained by covalently bonding to a conformation-constraining protein.  
     
     
         22 . The method of  claim 18 , wherein said population of proteins each comprises one or more loops .  
     
     
         23 . The method of  claim 21 , wherein each of said population of proteins is embedded within a conformation-constraining protein.  
     
     
         24 . The method of  claim 21 , wherein said conformation-constraining protein is thioredoxin.  
     
     
         25 . The method of  claim 24 , wherein each of said population of proteins is inserted into the active site loop of said thioredoxin.  
     
     
         26 . The method of  claim 18 , wherein each of said population of proteins is conformationally-constrained by disulfide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of said protein.  
     
     
         27 . The method of  claim 18 , wherein said first protein is Cdk2.  
     
     
         28 . The method of  claim 18 , wherein said first protein is Ras or an activated Ras.  
     
     
         29 . The method of  claim 18 , wherein said host cell is yeast.  
     
     
         30 . The method of  claim 18 , wherein said DNA binding domain is LexA.  
     
     
         31 . The method of  claim 18 , wherein said reporter gene is assayed by a color reaction.  
     
     
         32 . The method of  claim 18 , wherein said reporter gene is assayed by cell viability.  
     
     
         33 . A method of identifying a candidate interactor, comprising: 
 (a) providing a reporter gene operably linked to a DNA-binding-protein recognition site;    (b) providing a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site;    (c) providing a second fusion protein, said second fusion protein comprising a second protein covalently bonded to a gene activating moiety and being conformationally-constrained, said second protein being capable of interacting with said first protein;    (d) contacting said candidate interactor with said first protein and/or said second protein; and    (e) measuring expression of said reporter gene.    
     
     
         34 . The method of  claim 33 , wherein providing said first fusion protein comprises providing a first fusion gene which expresses said first fusion protein and wherein providing said second fusion protein comprises providing a second fusion gene which expresses said second fusion protein.  
     
     
         35 . The method of  claim 33 , wherein said first fusion protein and said second fusion protein are permitted to interact prior to contact with said candidate interactor.  
     
     
         36 . The method of  claim 33 , wherein said first fusion protein and said candidate interactor are permitted to interact prior to contact with said second fusion protein.  
     
     
         37 . The method of  claim 33 , wherein said candidate interactor is conformationally-constrained.  
     
     
         38 . The method of  claim 33 , wherein said candidate interactor comprises one or more loops.  
     
     
         39 . The method of  claim 33 , wherein said candidate interactor is an antagonist and reduces reporter gene expression.  
     
     
         40 . The method of  claim 33 , wherein said candidate interactor is an agonist and increases reporter gene expression.  
     
     
         41 . The method of  claim 33 , wherein said candidate interactor is a member selected from the group consisting of proteins, polynucleotides, and small molecules.  
     
     
         42 . The method of  claim 33 , wherein said candidate interactor is encoded by a member of a cDNA or synthetic DNA library.  
     
     
         43 . The method of  claim 33 , wherein said candidate interactor is a mutated form of said first fusion protein or said second fusion protein.  
     
     
         44 . The method of  claim 34 , wherein said reporter gene, said first fusion gene, and said second fusion gene are included on a single piece of DNA.  
     
     
         45 . The method of  claim 33 , wherein said method further comprises 
 (f) determining whether said second protein interacts with said first protein inside a cell.    
     
     
         46 . The method of  claim 33 , wherein said first protein is Cdk2.  
     
     
         47 . The method of  claim 33 , wherein said first protein is Ras or an activated Ras.  
     
     
         48 . A population of eukaryotic cells, each cell having a recombinant DNA molecule encoding a conformationally-constrained intracellular peptide, there being at least 100 different recombinant molecules in said population, each molecule being in at least one cell of said population.  
     
     
         49 . The population of eukaryotic cells of  claim 48 , wherein said intracellular peptide is conformationally-constrained because it is covalently bonded to a conformation-constraining protein.  
     
     
         50 . The population of  claim 49 , wherein said intracellular peptide is embedded within said conformation-constraining protein.  
     
     
         51 . The population of  claim 49 , wherein said intracellular peptide comprises one or more loops.  
     
     
         52 . The population of eukaryotic cells of  claim 49 , wherein said conformation-constraining protein is thioredoxin.  
     
     
         53 . The population of eukaryotic cells of  claim 48 , wherein said intracellular peptide is conformationally-constrained by disulf ide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of said second protein.  
     
     
         54 . The population of eukaryotic cells of  claim 48 , wherein said cells are yeast cells.  
     
     
         55 . The population of eukaryotic cells of  claim 48 , wherein said recombinant DNA molecule further encodes a gene activating moiety covalently bonded to said intracellular peptide.  
     
     
         56 . The population of eukaryotic cells of  claim 48 , wherein said intracellular peptide physically interacts with a second recombinant protein inside said eukaryotic cells.  
     
     
         57 . A method of assaying an interaction between a first protein and a second protein, comprising: 
 (a) providing a reporter gene operably linked to a DNA-binding-protein recognition site;    (b) providing a first fusion protein comprising said first protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site;    (c) providing a second fusion protein comprising said second protein covalently bonded to a gene activating moiety and being conformationally-constrained;    (d) combining said reporter gene, said first fusion protein, and said second fusion protein; and    (e) measuring expression of said reporter gene.    
     
     
         58 . The method of  claim 57 , wherein providing said first fusion protein comprises providing a first fusion gene which expresses said first fusion protein and wherein providing said second fusion protein comprises providing a second fusion gene which expresses said second fusion protein.  
     
     
         59 . The method of  claim 57 , wherein said second fusion protein comprises one or more loops.  
     
     
         60 . The method of  claim 57 , wherein said method further comprises 
 (f) determining whether said second protein interacts with said first protein inside a cell.    
     
     
         61 . A protein comprising the sequence Leu-Val-Cys-Lys-Ser-Tyr-Arg-Leu-Asp-Trp-Glu-Ala-Gly-Ala-Leu-Phe-Arg-Ser-Leu-Phe (SEQ ID NO: 1).  
     
     
         62 . The protein of  claim 61 , wherein said protein is conformationally-constrained.  
     
     
         63 . A protein comprising the sequence Met-Val-Val-Ala-Ala-Glu-Ala-Val-Arg-Thr-Val-Leu-Leu-Ala-Asp-Gly-Gly-Asp-Val-Thr (SEQ ID NO: 2).  
     
     
         64 . The protein of  claim 63 , wherein said protein is conformationally-constrained.  
     
     
         65 . A protein comprising the sequence Pro-Asn-Trp-Pro -His-Gln-Leu-Arg-Val-Gly-Arg-Val-Leu-Trp-Glu-Arg-Leu-Ser-Phe-Glu (SEQ ID NO: 3).  
     
     
         66 . The protein of  claim 65 , wherein said protein is conformationally-constrained.  
     
     
         67 . A protein comprising the sequence Ser-Val-Arg-Met-Arg-Tyr-Gly-Ile-Asp-Ala-Phe-Phe-Asp-Leu-Gly-Gly-Leu-Leu-His-Gly (SEQ ID NO: 9).  
     
     
         68 . The protein of  claim 67 , wherein said protein is conformationally-constrained.  
     
     
         69 . A protein comprising the sequence Glu-Leu-Arg-His-Arg-Leu-Gly-Arg-Ala-Leu-Ser-Glu-Asp-Met-Val-Arg-Gly-Leu -Ala-Trp-Gly-Pro-Thr-Ser-His-Cys-Ala-Thr-Val-Pro-Gly-Thr-Ser-Asp-Leu-Trp-Arg-Val-Ile-Arg-Phe-Leu (SEQ ID NO: 10).  
     
     
         70 . The protein of  claim 69 , wherein said protein is conformationally-constrained.  
     
     
         71 . A protein comprising the sequence Tyr-Ser-Phe-Val-His-His -Gly-Phe-Phe-Asn-Phe-Arg-Val-Ser-Trp-Arg-Glu-Met-Leu-Ala (SEQ ID NO: 11).  
     
     
         72 . The protein of  claim 71 , wherein said protein is conformationally-constrained.  
     
     
         73 . A protein comprising the sequence Gln-Val-Trp-Ser-Leu-Trp-Ala-Leu-Gly-Trp-Arg-Trp-Leu-Arg-Arg-Tyr-Gly-Trp-Asn-Met (SEQ ID NO: 12).  
     
     
         74 . The protein of  claim 73 , wherein said protein is conformationally-constrained.  
     
     
         75 . A protein comprising the sequence Trp-Arg-Arg-Met-Glu -Leu-Asp-Ala-Glu-Ile-Arg-Trp-Val-Lys-Pro-Ile-Ser-Pro-Leu-Glu (SEQ ID NO: 13).  
     
     
         76 . The protein of  claim 75 , wherein said protein is conformationally-constrained.  
     
     
         77 . A protein comprising the sequence Trp-Ala-Glu-Trp-Cys-Gly-Pro-Val-Cys-Ala-His-Gly-Ser-Arg-Ser-Leu-Thr -Leu-Leu-Thr-Lys-Tyr-His-Val-Ser-Phe-Leu-Gly-Pro-Cys-Lys-Met-Ile-Ala -Pro-Ile-Leu-Asp (SEQ ID NO: 17).  
     
     
         78 . The protein of  claim 77 , wherein said protein is conformationally-constrained.  
     
     
         79 . A protein comprising the sequence Leu-Val-Cys-Lys-Ser-Tyr-Arg-Leu-Asp-Trp-Glu -Ala-Gly-Ala-Leu-Phe-Arg-Ser-Leu-Phe (SEQ ID NO: 18).  
     
     
         80 . The protein of  claim 79 , wherein said protein is conformationally-constrained.  
     
     
         81 . A protein comprising the sequence Tyr-Arg-Trp-Gln-Gln-Gly-Val-Val-Pro-Ser-Asn-Trp-Ala-Ser-Cys-Ser-Phe-Arg-Cys-Gly (SEQ ID NO: 19).  
     
     
         82 . The protein of  claim 81 , wherein said protein is conformationally-constrained.  
     
     
         83 . A protein comprising the sequence Ser-Ser-Phe-Ser-Leu-Trp-Leu-Leu-Met-Val-Lys-Ser-Ile-Lys -Arg-Ala-Ala-Trp-Glu-Leu-Gly-Pro-Ser-Ser-Ala-Trp-Asn-Thr-Ser-Gly-Trp-Ala-Ser-Leu-Ala-Asp-Phe-Tyr (SEQ ID NO: 20).  
     
     
         84 . The protein of  claim 83 , wherein said protein is conformationally-constrained.  
     
     
         85 . A protein comprising the sequence Arg-Val-Lys-Leu-Gly-Tyr-Ser-Phe-Trp-Ala-Gln-Ser-Leu-Leu -Arg-Cys-Ile-Ser-Val-Gly (SEQ ID NO: 21).  
     
     
         86 . The protein of  claim 85 , wherein said protein is conformationally-constrained.  
     
     
         87 . A protein comprising the sequence Gln-Leu-Tyr-Ala-Gly-Cys-Tyr-Leu-Gly-Val-Val-Ile-Ala-Ser-Ser -Leu-Ser-Ile-Arg-Val (SEQ ID NO: 22).  
     
     
         88 . The protein of  claim 87 , wherein said protein is conformationally-constrained.  
     
     
         89 . A protein comprising the sequence Gln-Gln-Arg-Phe-Val-Phe-Ser-Pro-Ser-Trp-Phe-Thr-Cys-Ala-Gly -Thr-Ser-Asp-Phe-Trp-Gly-Pro-Glu-Pro-Leu-Phe-Asp-Trp-Thr-Arg-Asp (SEQ ID NO: 23).  
     
     
         90 . The protein of  claim 89 , wherein said protein is conformationally-constrained.  
     
     
         91 . A protein comprising the sequence Arg-Pro-Leu-Thr-Gly-Arg-Trp-Val-Val-Trp-Gly-Arg-Arg-His-Glu-Glu-Cys-Gly-Leu-Thr (SEQ ID NO: 24).  
     
     
         92 . The protein of  claim 91 , wherein said protein is conformationally-constrained.  
     
     
         93 . A protein comprising the sequence Pro-Val-Cys-Cys-Met-Met-Tyr-Gly-His-Arg-Thr-Ala-Pro-His-Ser-Val-Phe-Asn-Val-Asp (SEQ ID NO: 25).  
     
     
         94 . The protein of  claim 93 , wherein said protein is conformationally-constrained.  
     
     
         95 . A protein comprising the sequence Trp-Ser-Pro-Glu-Leu-Leu-Arg-Ala-Met-Val-Ala-Phe-Arg-Trp -Leu-Leu-Glu-Arg-Arg-Pro (SEQ ID NO: 26).  
     
     
         96 . The protein of  claim 95 , wherein said protein is conformationally-constrained.  
     
     
         97 . Substantially pure DNA encoding the protein of  claim 61 .  
     
     
         98 . Substantially pure DNA encoding the protein of  claim 63 .  
     
     
         99 . Substantially pure DNA encoding the protein of  claim 65 .  
     
     
         100 . Substantially pure DNA encoding the protein of  claim 67 .  
     
     
         101 . Substantially pure DNA encoding the protein of  claim 69 .  
     
     
         102 . Substantially pure DNA encoding the protein of  claim 71 .  
     
     
         103 . Substantially pure DNA encoding the protein of  claim 73 .  
     
     
         104 . Substantially pure DNA encoding the protein of  claim 75 .  
     
     
         105 . Substantially pure DNA encoding the protein of  claim 77 .  
     
     
         106 . Substantially pure DNA encoding the protein of  claim 79 .  
     
     
         107 . Substantially pure DNA encoding the protein of  claim 81 .  
     
     
         108 . Substantially pure DNA encoding the protein of  claim 83 .  
     
     
         109 . Substantially pure DNA encoding the protein of  claim 85 .  
     
     
         110 . Substantially pure DNA encoding the protein of  claim 87 .  
     
     
         111 . Substantially pure DNA encoding the protein of  claim 89 .  
     
     
         112 . Substantially pure DNA encoding the protein of  claim 91 .  
     
     
         113 . Substantially pure DNA encoding the protein of  claim 93 .  
     
     
         114 . Substantially pure DNA encoding the protein of  claim 95 .  
     
     
         115 . A protein isolated by a method comprising: 
 (a) providing a host cell which contains 
 (i) a reporter gene operably linked to a DNA-binding-protein recognition site; and  
 (ii) a fusion gene which expresses a fusion protein, said fusion protein comprising a test protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site;  
   (b) introducing into said host cell a second fusion gene which expresses a second fusion protein, said second fusion protein comprising one of said population of proteins covalently bonded to a gene activating moiety and being conformationally-constrained; and    (c) measuring expression of said reporter gene; and    (d) isolating a protein based on its ability to alter the expression of said reporter gene when present in said second fusion protein.    
     
     
         116 . An interactor protein isolated by a method comprising: 
 (a) providing a reporter gene operably linked to a DNA-binding-protein recognition site;    (b) providing a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said DNA-binding-protein recognition site;    (c) providing a second fusion protein, said second fusion protein comprising a second protein covalently bonded to a gene activating moiety and being conformationally-constrained, said second protein being capable of interacting with said first protein;    (d) contacting a candidate interactor protein with said first protein or said second protein;    (e) measuring expression of said reporter gene; and    (f) isolating an interactor protein based on its ability to alter the expression of said reporter gene when present with said first protein or said second protein.    
     
     
         117 . A method for detecting a protein in a sample, comprising 
 (a) contacting said sample with a conformationally constrained protein which is capable of specifically binding to said protein and forming a complex; and    (b) detecting said complex.    
     
     
         118 . The method of  claim 117 , wherein said detecting step is carried out by an immunoprecipitation, Western blot, or affinity column technique that utilizes said conformationally constrained protein as the complex-forming reagent.  
     
     
         119 . A method of assaying an interaction between a first protein and a second protein, comprising: 
 (a) providing said first protein;    (b) providing a fusion protein comprising said second protein, said second protein being conformationally-constrained;    (c) contacting said first protein with said fusion protein under conditions which allow complex formation;    (d) detecting said complex as an indication of an interaction; and    (e) determining whether said first protein interacts with said fusion protein inside a cell.