Intraction trap assay, reagents and uses thereof

• US 5,925,523 A
• Filed: 08/26/1997
• Issued: 07/20/1999
• 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, J. Keith, Hochschild, Ann
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): SCHWARTZMAN, ROBERT A
• Application Number:
  US08/920,015
• Time in Patent Office: 693 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/7.2, 435/7.32, 435/7.33, 435/7.34, 435/7.35, 435/7.37, 435/29
• 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 detecting interaction between a first test polypeptide and a second test polypeptide, 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 (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;

and

ii. measuring expression of the reporter gene.

2. The method of claim 1 which futher 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 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, Bacillzis, 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, β

-galactosidase and alkaline phosphatase.

7. The method of claim 1, wherein at least one of the first and second test polypeptides are encoded by nucleic acids from a nucleic acid library.

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 at least one of the first and second fusion proteins includes a control polypeptide sequence, the function of which in the fusion protein is sensitive to at least one of stability of the fusion protein, folding of the fusion protein, and functional association of the fusion protein with a polymerase.

12. A method for detecting cleavage of a polypeptide by a proteolytic activity, comprising the method of claim 1, wherein one of either the first or second fusion proteins includes a potential proteolytic cleavage site which, when cleaved, alters expression of the reporter gene, and expression of the reporter gene is detected under conditions wherein the proteolytic activity is active in the host cells.

13. A method for identifying an antibody equivalent, comprising the method of claim 1, wherein the test polypeptide of one of either the first or second fusion proteins includes an epitope of interest and the other is a potential antibody equivalent, expression of the reporter gene being detected under conditions wherein an antibody equivalent binds to the epitope of interest.

14. A drug screening assay comprising the method of claim 1, wherein the expression of the reporter gene is detected in the presence of a test agent applied to the host cell and a test agent which alters the expression of the reporter gene is identified.

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 nucleic acid library is 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 12, wherein the interaction trap system comprises a variegated population of the host cell collectively expressing a library of different potential proteolytic cleavage sites.

19. The method of claim 12, wherein the intreaction trap system comprises a variegated population of the host cell collectively expressing a library of genes including ones encoding potential proteolytic activities.

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

or ω

polymerase subunit.

21. A kit for detecting interaction between a first test polypeptide and a second test polypeptide, the kit comprising:

i. a first gene construct for encoding a first fusion protein, which first gene construct comprises;

(1) transcriptional and translational elements which direct expression of the first fusion protein in a prokaryotic host cell,

(2) a DNA sequence that encodes a DNA-binding domain and which is functionally associated with the transcriptional and translational elements of the first gene construct, and

(3) a means for inserting a DNA sequence encoding a first test polypeptide into the first gene construct in such a manner that the first test polypeptide is expressed in-frame as part of the first fusion protein containing the DNA binding domain;

ii. a second gene construct for encoding a second fusion protein, which second gene construct comprises;

(1) transcriptional and translational elements which direct expression of the second fusion protein in a prokaryotic host cell,

(2) a DNA sequence that encodes an activation tag, and which is functionally associated with the transcriptional and translational elements of the second gene construct, and

(3) a means for inserting a DNA sequence encoding a second test polypeptide into the second vector in such a manner that the second test polypeptide is expressed in-frame as part of the second fusion protein containing the activation tag; and

iii. a prokaryotic host cell containing a reporter gene having a binding site (DBD recognition element) for the DNA-binding domain, wherein the reporter gene expresses a detectable transcript or protein when the first and second fusion proteins interact.

22. The kit of claim 21, wherein the activation tag is a polymerase interaction domain (PID) which forms active RNA polymerase complexes in the host cell.

23. The kit of claim 21, wherein the host cell is selected from the group consisting of bacterial strains of Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia and Shigella.

24. The kit of claim 21, 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.

25. The kit of claim 21, wherein the reporter gene encodes a gene product selected from the group consisting of the spectinomycin (spc) gene product, chloramphenicol acetyl transferase, luciferase, β

-galactosidase and alkaline phosphatase.

26. The kit of claim 21, wherein the DNA-binding domain is a naturally occurring DNA binding protein.

27. The kit of claim 21, wherein the DNA-binding domain is an artificial polypeptide.

28. The kit of claim 21, wherein at least one of the first or second gene constructs further include a coding sequence for including an oligomerization motif in the fusion protein(s).

29. The kit of claim 21, wherein at least one of the first or second gene constructs further comprises a coding sequence for including a control polypeptide sequence in the fusion protein(s), the function of the control sequence in the fusion protein being sensitive to at least one of stability of the fusion protein, folding of the fusion protein, and functional association of the fusion protein with a polymerase.

30. The kit of claim 21, wherein the reporter gene is extrachrosomal.

31. The kit of claim 22, wherein the PID includes at least a portion of an RNA polymerase subunit.

32. The kit of claim 26, wherein the DNA-binding domain includes a DNA binding portion of a transcriptional regulatory protein.

33. The kit of claim 31, wherein the PID includes at least a portion of an α

or ω

polymerase subunit.

34. A method for identifying a nucleic acid encoding a test polypeptide which contacts another test polypeptide, comprising

i. providing an interaction trap system including a variegated population of prokaryotic host cells which each include:

(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 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 a second test polypeptide,

wherein interaction of the first fusion protein and second fusion protein in the host cell results in measurable change of expression of the reporter gene, and one of the first or second chimeric genes is present in the host cell population as a variegated population with respect to nucleic acid sequence encoding test polypeptides;

ii. measuring expression of the reporter gene; and

iii. identifying nucleic acids which encode test polypeptides which increase expression of the reporter gene.

35. A method for detecting interaction between a test polypeptide and a DNA sequence, comprising

i. providing a population of prokaryotic host cells which contain

(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 test polypeptide and an activation tag,

wherein interaction of the test polypeptide of the fusion protein with the DBD recognition element in the host cells results in a measurable change in expression of the reporter gene;

and

ii. measuring expression of the reporter gene,

wherein the host cells comprise a variegated population of test polypeptides.

36. A method for detecting interaction between a test polypeptide and a DNA sequence, comprising

i. providing a population of prokaryotic host cells which contain

(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 test polypeptide and an activation tag,

wherein interaction of the test polypeptide of the fusion protein with the DBD recognition element in the host cells results in a measurable change in expression of the reporter gene;

and

ii. measuring expression of the reporter gene,

wherein the host cells-comprise a variegated population of DBD recognition elements.