Methods and compositions for interaction trap assays

• US 7,029,847 B2
• Filed: 11/14/2001
• Issued: 04/18/2006
• 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): Ponnaluri, Padmashri
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US09/990,762
• Publication Number:
  US 20020119498A1
• Time in Patent Office: 1,616 Days
• Field of Search: 435/6, 435/7.1, 435/455, 435/DIG.1, 435/DIG.2, 435/DIG.3
• 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 interaction. 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 selecting a dimerizing polypeptide, comprising:

providing a population of host cells wherein each host cell contains

(a) a chimeric gene which encodes a fusion protein, including one or more zinc-finger DNA-binding domains, an activation domain that is heterologous to the DNA-binding domain, and a test polypeptide, wherein the chimeric gene is a member of a library comprising a plurality of randomly generated sequences test polypeptides;

(b) a reporter gene operably linked to a transcriptional regulatory sequence which includes two or more binding sites (DBD recognition elements) for the DNA-binding domain of (a), wherein binding of a single copy of the fusion protein to the transcriptional regulatory sequence of the reporter gene does not result in a desired level of expression of the reporter gene;

wherein dimerization of two copies of the fusion protein to each other and binding of the dimerized fusion protein to the transcriptional regulatory sequence of the reporter gene results in a desired level of expression of the reporter gene; and

isolating host cells exhibiting a desired level of expression of the reporter gene thereby selecting a dimerizing polypeptide.

2. The method of claim 1, wherein the host cell further comprises a second reporter gene operably linked to a transcriptional regulatory sequence, the transcriptional regulatory sequence comprising at least one binding site for the DNA binding domain of the fusion protein.

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

4. The method of claim 3, further comprising linking the sequence encoding the dimerizing polypeptide to a heterologous sequence.

5. The method of claim 1, wherein the host cell is a prokaryotic host cell.

6. The method claim 1, wherein the desired level of expression of the reporter gene confers a growth advantage on the host cell.

7. The method of claim 1, wherein the desired level of expression of the reporter gene produces a detectable signal.

8. The method of claim 1, wherein the library comprises at least 10⁷ members.