Interaction trap assay, reagents and uses thereof

• US 6,200,759 B1
• Filed: 04/21/1999
• Issued: 03/13/2001
• Est. Priority Date: 08/23/1996
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Dove, Simon, Joung, Keith J., Hochschild, Ann
• Primary Examiner(s): Schwartzman, Robert A.
• Application Number:
  US09/296,204
• Time in Patent Office: 692 Days
• Field of Search: 435/6, 435/7.1, 435/252.3, 435/252.31, 435/252.33, 435/252.34, 435/252.35, 435/252.5, 435/252.8, 435/253.3, 435/253.5, 536/23.4, 536/23.1
• 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 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

The present invention makes available an interaction trap system (hereinafter “ITS”) which is derived using recombinantly engineered prokaryotic cells.

Claims

1. A method for identifying agents which modulate a target protein-protein interaction, comprising

i. providing an interaction trap system including a prokaryotic host cell which contains:

(a) a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site (DNA binding domain (DBD) recognition element) for a DNA-binding domain,

(b) a first chimeric gene which encodes a first fusion protein, the first fusion protein including a DNA-binding domain and first test polypeptide,

(c) a second chimeric gene which encodes a second fusion protein, the second fusion protein including an activation tag and second test polypeptide,

wherein interaction of the first fusion protein and second fusion protein in the host cell results in a measurable change in expression of the reporter gene;

ii. contacting the host cell with test agents and measuring expression of the reporter gene; and

iii. identifying test agents which modulate expression of the reporter gene in a manner also dependent on expression of the first and second test polypeptides.

2. The method of claim 1 which further comprises comparing the level of expression of the reporter gene to a level of expression in a control interaction trap system in which either one or both of the test polypeptides are absent or altered so as to preclude interaction of the first and second fusion proteins.

3. The method of claim 1, wherein the activation tag is a polymerase interaction domain (PID) which forms active RNA polymerase complexes in the host cell.

4. The method of claim 1, wherein the host cell is selected from the group consisting of bacterial strains of Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia and Shigella.

5. The method of claim 1, wherein the reporter gene encodes a gene product that gives rise to a detectable signal selected from the group consisting of:

color, fluorescence, luminescence, a cell surface tag, cell viability, relief of a cell nutritional requirement, cell growth, and drug resistance.

6. The method of claim 1, wherein the reporter gene encodes a gene product selected from the group consisting of the spectinomycin (spc) gene product, chloramphenicol acetyl transferase, luciferase, p-galactosidase and alkaline phosphatase.

7. The method of claim 1, wherein the coding sequence for one or both of the first and second test polypeptides is provided as a library of different coding sequences.

8. The method of claim 1, wherein the DNA-binding domain is a naturally occurring DNA binding protein.

9. The method of claim 1, wherein the DNA-binding domain is an artificial polypeptide.

10. The method of claim 1, wherein at least one of the first or second fusion proteins further includes an oligomerization motif.

11. The method of claim 1, wherein the test agents are selected from the group consisting of peptides, nucleic acids, carbohydrates, and natural product extract libraries.

12. The method of claim 1, wherein the test agents are small organic molecules.

13. The method of claim 1, wherein test agents which agonize the target protein-protein interaction are identified by increased expression of the reporter gene in the presence of the test agent.

14. The method of claim 1, wherein test agents which antagonize the target protein-protein interaction are identified by decreased expression of the reporter gene in the presence of the test agent.

15. The method of claim 3, wherein the PID includes at least a portion of an RNA polymerase subunit.

16. The method of claim 7, wherein the coding sequence for a test polypeptide is from a eukaryotic cDNA library, a eukaryotic genomic library, a prokaryotic genomic library, or a random or semi-random nucleic acid library.

17. The method of claim 8, wherein the DNA-binding domain includes a DNA binding portion of a transcriptional regulatory protein.

18. The method of claim 11, wherein the natural product extract library includes natural products from animals, plants, fungi or microbes.

19. The method of claim 15, wherein the PID includes at least a portion of an α

or ω

polymerase subunit.

20. A method for identifying agents which modulate a target DNA-protein interaction, comprising:

i. providing a prokaryotic host cell which contains

(a) a reporter gene operably linked to a transcriptional regulatory sequence which includes a binding site (DBD recognition element) for a DNA binding domain,

(b) a chimeric gene which encodes a fusion protein, the fusion protein including a DNA binding domain which binds to the DBD recognition element, and an activation tag;

wherein interaction of the DNA binding domain of the fusion protein with the DBD recognition element in the host cell results in a measurable change in expression of the reporter gene;

ii. contacting the host cell with test agents and measuring expression of the reporter gene; and

iii. identifying test agents which modulate expression of the reporter gene in a manner also dependent on expression of the fusion protein.

21. The method of claim 20, wherein the test agents are selected from the group consisting of peptides, nucleic acids, carbohydrates, and natural product extract libraries.

22. The method of claim 20, wherein the test agents are small organic molecules.

23. The method of claim 20, wherein test agents which agonize the target DNA-protein interaction are identified by increased expression of the reporter gene in the presence of the test agent.

24. The method of claim 20, wherein test agents which antagonize the target DNA-protein interaction are identified by decreased expression of the reporter gene in the presence of the test agent.

25. The method of claim 21, wherein the natural product extract library includes natural products from animals, plants, fungi or microbes.