Methods and compositions for interaction trap assays

• US 20050064477A1
• Filed: 08/10/2004
• Published: 03/24/2005
• Est. Priority Date: 05/16/2000
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Pabo, Carl O., Joung, J. Keith, Miller, Jeffrey
• Granted Patent: US 7,297,491 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2565/201 Two hybrid system
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects

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 interactions. 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.

Claims

1. 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 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 second reporter gene operably linked to a transcriptional regulatory sequence which includes one or more binding sites (DBD recognition elements) for a DNA-binding domain,

(c) a chimeric gene which encodes a fusion protein, the fusion protein including a test polypeptide, a weak DNA-binding domain and an activation tag,

wherein binding of the weak DNA-binding domain of (c) to the binding sites of (a) or (b) does not cause a significant increase in the expression of the first reporter gene or the second reporter gene;

wherein expression of the first reporter gene results in a first detectable signal;

wherein expression of the second reporter gene results in a second detectable signal;

wherein a non-specific interaction between a test polypeptide of the fusion protein and a DBD recognition element of the first and second reporter genes results in an increased level of expression of the first and second reporter genes;

wherein a specific interaction between a test polypeptide of the fusion protein and a DBD recognition element of the first or second reporter gene results in a desired level of expression of either the first or second reporter gene; and

ii isolating host cells comprising a fusion protein that specifically interacts with a DBD recognition element of the first or second reporter gene exhibiting a desired level of expression of the first or second reporter gene, thereby detecting an interaction between the test polypeptide and a DBD recognition element DNA sequence.

2. The method of claim 1, wherein the chimeric gene is a member of a library comprising a plurality of sequences encoding for random test polypeptides or the DNA-binding domain recognition element of one of the reporter genes is a member of a library.

3. The method of claim 1, wherein the weak DNA-binding domain comprises two Cys₂His₂ zinc fingers.

4. The method of claim 1, further comprising isolating a polynucleotide comprising a sequence encoding the test polypeptide.

5. The method of claim 4, further comprising linking the sequence encoding the test polypeptide to a heterologous sequence.