Methods and compositions for interaction trap assays

• US 7,297,491 B2
• Filed: 08/10/2004
• Issued: 11/20/2007
• Est. Priority Date: 05/16/2000
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Pabo, Carl O., Joung, J. Keith, Miller, Jeffrey
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US10/915,233
• Publication Number:
  US 20050064477A1
• Time in Patent Office: 1,197 Days
• Field of Search: 435/6
• US Class Current: 435/6.16
• 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 a binding site for a weak zinc-finger DNA-binding domain,

(b) a second reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site for the weak zinc-finger DNA-binding domain,

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

wherein binding of the weak zinc finger 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 the transcriptional regulatory sequence 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 the transcriptional regulatory sequence 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 the transcriptional regulatory sequence 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 the transcriptional regulatory 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 wherein one or more of the binding sites 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.