Methods and compositions for interaction trap assays

• US 7,393,318 B2
• Filed: 03/16/2004
• Issued: 07/01/2008
• Est. Priority Date: 05/16/2000
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Miller, Jeffrey, Pabo, Carl O., Joung, J. Keith
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US10/801,994
• Publication Number:
  US 20040146931A1
• Time in Patent Office: 1,568 Days
• Field of Search: 506/17, 506/16, 506/18, 536/23.4, 536/24.1
• US Class Current: 506/17
• 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 population of host cells, each host cell comprising:

(a) a polynucleotide encoding a fusion protein, the fusion protein comprising

(i) an activation tag; and

(ii) a polypeptide sequence to be assayed for its interaction with a DNA sequence,

(b) a transcriptional regulatory sequence comprising one or more binding sites for a zinc finger DNA-binding domain;

(c) a reporter gene operably linked to the transcriptional regulatory sequence, wherein expression of the reporter gene is modulated when the polypeptide sequence interacts with one or more of the binding sites;

wherein at least 10⁷ unique pairs of the one or more binding sites and fusion proteins that bind to the 10⁷ unique pairs of the one or more binding sites are represented in the population of host cells.

2. The population of host cells according to claim 1, wherein the cells are prokaryotic cells.

3. The population of host cells according to claim 1, wherein expression of the reporter gene confers a growth advantage on a host cell of the population in which host cell the polypeptide sequence interacts with the one or more binding sites.

4. The population of host cells according to claim 3, wherein the growth advantage is relief of a cell nutritional requirement.

5. The population of host cells according to claim 4, wherein the reporter gene is the yeast His3 gene.

6. The population of host cells according to claim 5, wherein the degree of the growth advantage is controllable by exposing the host cell to varying concentrations of 3-aminotriazole.

7. The population of host cells according to claim 3, wherein the degree of the growth advantage conferred by the desired level of expression of the reporter gene is controllable by varying the growth conditions of the host cell.

8. The population of host cells according to claim 7, wherein the reporter gene is a β

-lactamase gene and the degree of the growth advantage is controllable by exposing the host cell to a β

-lactam antibiotic and varying concentrations of a β

-lactamase inhibitor.

9. The population of host cells according to claim 8, wherein the β

-lactam antibiotic is a penicillin.

10. The population of host cells according to claim 8, wherein the β

-lactamase inhibitor is Clavulanic acid.

11. The population of host cells according to claim 3, wherein the reporter gene is a β

-lactamase gene.

12. The population of host cells according to claim 11, wherein the β

-lactamase gene is TEM-1.

13. The population of host cells according to claim 11, wherein the degree of the growth advantage is controllable by exposing the host cell to a β

-lactam antibiotic.

14. The population of host cells according to claim 1, wherein the activation tag comprises an RNA polymerase, an RNA polymerase subunit, a functional fragment of an RNA polymerase, or a functional fragment of an RNA polymerase subunit.

15. The population of host cells according to claim 1, wherein the activation tag covalently or non-covalently interacts with an RNA polymerase, an RNA polymerase subunit, a functional fragment of RNA polymerase, or a functional fragment of an RNA polymerase subunit.

16. The population of host cells according to claim 15, wherein the activation tag comprises a fragment of Gal11P which non-covalently interacts with a fusion polypeptide comprising an RNA polymerase, an RNA polymerase subunit, a functional fragment of RNA polymerase, or a functional fragment of an RNA polymerase subunit.

17. The population of host cells according to claim 16, wherein the fusion polypeptide further comprises Gal4.

18. The population of host cells according to claim 1, wherein the activation tag interacts indirectly with RNA polymerase via at least one intermediary polypeptide, nucleic acid, or small molecule, which functionally links the activation tag to the RNA polymerase.

19. The population of host cells according to claim 1, wherein an intermediary peptide is covalently fused to an RNA polymerase, an RNA polymerase subunit, a functional fragment of RNA polymerase, or a functional fragment of an RNA polymerase subunit.

20. The population of host cells according to claim 1, wherein (a), (b), or (a) and (b), are contained within one or more vectors in the host cell.

21. The population of host cells according to claim 1, wherein the binding site is a member of a library of at least 10⁷ potential binding sites for a zinc finger DNA binding domain.

22. The population of host cells according to claim 1, wherein the binding site is a member of a library of at least 10⁸ potential binding sites for a zinc finger DNA binding domain.

23. The population of host cells according to claim 1, wherein the polypeptide sequence to be assayed is a member of a library of at least 10⁷ polypeptides.

24. The population of host cells according to claim 23, wherein the polypeptide sequence to be assayed is a member of a library of at least 10⁸ polypeptides.

25. A method for detecting an interaction between a polypeptide sequence and a DNA sequence, comprising

providing a population of host cells according to claim 1;

assaying the population of host cells for expression of the reporter gene;

identifying host cells in which expression of the reporter gene is modulated, thereby detecting interaction between the polypeptide sequence and the zinc finger DNA-binding domain in the host cell.

26. The method of claim 25, further comprising the step of identifying the polynucleotide encoding the fusion protein.