Identification and comparison of protein-protein interactions that occur in populations and indentification of inhibitors of these interactors

• US 6,395,478 B1
• Filed: 01/12/1999
• Issued: 05/28/2002
• Est. Priority Date: 06/14/1996
• Status: Expired due to Fees

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Nandabalan, Krishnan, Rothberg, Jonathan Marc
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Wilder, Cynthia B.
• Application Number:
  US09/231,303
• Time in Patent Office: 1,232 Days
• Field of Search: 435/6,5,69.1,252.3,254.21,320.1,69.7,70.1,71.1,254.11 436/501,94 530/350
• US Class Current: 435/6.13
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Methods are described for detecting protein-protein interactions, among two populations of proteins, each having a complexity of at least 1,000. For example, proteins are fused either to the DNA-binding domain of a transcriptional activator or to the activation domain of a transcriptional activator. Two yeast strains, of the opposite mating type and carrying one type each of the fusion proteins are mated together. Productive interactions between the two halves due to protein-protein interactions lead to the reconstitution of the transcriptional activator, which in turn leads to the activation of a reporter gene containing a binding site for the DNA-binding domain. This analysis can be carried out for two or more populations of proteins. The differences in the genes encoding the proteins involved in the protein-protein interactions are characterized, thus leading to the identification of specific protein-protein interactions, and the genes encoding the interacting proteins, relevant to a particular tissue, stage or disease. Furthermore, inhibitors that interfere with these protein-protein interactions are identified by their ability to inactivate a reporter gene. The screening for such inhibitors can be in a multiplexed format where a set of inhibitors will be screened against a library of interactors.

Claims

1. A method of detecting one or more protein-protein interactions comprising:

(a) recombinantly expressing within a population of host cells a first population of first fusion proteins, each said first fusion protein comprising a first protein sequence and a DNA binding domain in which the DNA binding domain is the same in each said first fusion protein, in which said host cells comprise at least one polynucleotide operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain of said first fusion protein such that an interaction of a first fusion protein with a second fusion protein, said second fusion protein comprising a transcriptional regulatory domain which is an activation domain, results in activation of transcription of said at least one polynucleotide by said activation domain, and in which said first population of first fusion proteins has a complexity of at least 10;

(b) negatively selecting to eliminate those host cells expressing said first population of first fusion proteins in which said activation of transcription of said at least one polynucleotide occurs in the absence of a second fusion protein, said negatively selecting being performed by a method comprising incubating said host cells expressing said first population of first fusion proteins in an environment in which substantial death of said host cells occurs if said activation of transcription occurs;

(c) after step (b), recombinantly co-expressing with said population of host cells a second population of second fusion proteins, each said second fusion protein comprising a second protein sequence and an activation domain of a transcriptional regulator, in which the activation domain is the same in each said second fusion protein, and in which said second population of second fusion proteins has a complexity of at least 10; and

(d) detecting said activation of transcription of said at least one polynucleotide, thereby detecting cells in which an interaction between a first protein and a second protein has occurred,

wherein the first and second populations of fusion proteins comprise first and second protein sequences, respectively, that are encoded by DNA sequences which are drawn from different DNA populations.

3. The method described in claim 1 wherein said first population of first fusion proteins has a complexity of at least 100 and said second population of second fusion proteins has a complexity of at least 100.

4. The method described in claim 1 or 2 wherein at least one of said populations of fusion proteins comprise protein sequences that are encoded by cDNA sequences.

5. The method described in claim 4 wherein the cDNA sequences are provided by a cDNA library that has been normalized or subtracted.

6. The method described in claim 1 or 2 wherein at least one of the DNA populations is cDNA library obtained from a cell sample or a tissue sample, wherein said sample originates from a particular species, a particular cell type, a particular age of individual, a particular tissue type, a disease state, a disorder, a stage of a disease state or disorder, or a stage of development.

2. A method of detecting one or more protein-protein interactions comprising

(a) recombinantly expressing in a first population of yeast cells of a first mating type, a first population of first fusion proteins, each first fusion protein comprising a first protein sequence and a DNA binding domain, in which the DNA binding domain is the same in each said first fusion protein;

wherein said first population of yeast cells contains a first polynucleotide operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein, said second fusion protein comprising a transcriptional activation domain results in increased transcription of said first polynucleotide, and in which said first population of first fusion proteins has a complexity of at least 100;

(b) negatively selecting to eliminate those yeast cells expressing said first population of first fusion proteins in which said increased transcription of said first polynucleotide occurs in the absence of said second fusion protein, said negatively selecting being carried out by a method comprising incubating said first population of yeast cells expressing said first population of first fusion proteins in an environment in which substantial death of said first population of yeast cells occurs if said increased transcription occurs;

(c) recombinantly expressing in a second population of yeast cells of a second mating type different from said first mating type, a second population of said second fusion proteins, each second fusion protein comprising a second protein sequence and an activation domain of a transcriptional activator, in which the activation domain is the same in each said second fusion protein, and in which said second population of second fusion proteins has a complexity of at least 100;

(d) mating said first population of yeast cells that has been negatively selected with said second population of yeast cells to form a population of diploid yeast cells, wherein said population of diploid yeast cells contains a second polynucleotide operably linked to a promoter driven by a DNA binding site recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein results in increased transcription of said second polynucleotide, in which the first and second polynucleotide can be the same or different; and

(e) detecting said increased transcription of said first and/or second polynucleotide, thereby detecting colonies in which an interaction between a first protein and a second protein has occurred,

wherein the first and second populations of fusion proteins comprise first and second protein sequences, respectively, that are encoded by DNA sequences which are drawn from different DNA populations.