Detection systems for registering protein interactions and functional relationships

• US 6,171,792 B1
• Filed: 11/10/1998
• Issued: 01/09/2001
• Est. Priority Date: 11/10/1997
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Mendelsohn, Andrew R., Lok, Walter L., Xu, C. Wilson, Brent, Roger
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): SIU, STEPHEN C
• Application Number:
  US09/189,653
• Time in Patent Office: 791 Days
• Field of Search: 435/240.1, 435/172.3, 435/68, 435/70, 435/71, 435/317, 435/320, 435/5, 435/6, 435/69.1, 435/29, 435/254.21, 435/4, 435/68.1, 435/69.9, 435/70.2, 436/501, 436/63, 436/94, 436/808, 424/93.21
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 14/4738 Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR cell cycle dependent kinases C12N9/12
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/61 containing an enzyme fusion for detection (lacZ, luciferase)
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/81 for yeasts
• 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: C12Q 2565/201 Two hybrid system
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

Disclosed herein are methods for detecting complex protein interactions and protein functional relationships, and reagents for carrying out those methods.

Claims

1. A method for detecting a protein—

protein interaction, comprising;

(a) providing a host cell which contains

(i) a first reporter gene operably linked to a DNA sequence comprising a first protein binding site;

(ii) a second reporter gene operably linked to a DNA sequence comprising a second protein binding site;

(iii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said first protein binding site;

(iv) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising a second protein covalently bonded to a binding moiety which is capable of specifically binding to said second protein binding site; and

(v) a third fusion gene which expresses a third fusion protein, said third fusion protein comprising a third protein covalently bonded to a gene activating moiety;

(b) measuring expression output of said first reporter gene as a measure of said interaction between said first and said third proteins;

(c) measuring expression output of said second reporter gene as a measure of said interaction between said second and said third proteins;

(d) interpreting the expression output results of step (b) and step (c), whereby

(i) increased output of both of said first and said second reporter genes indicates that said third fusion protein interacts with both of said first and said second fusion proteins;

(ii) increased output of said first reporter gene but not said second reporter gene indicates that said third fusion protein interacts with said first fusion protein but not said second fusion protein;

(iii) increased output of said second reporter gene but not said first reporter gene indicates that said third fusion protein interacts with said second fusion protein but not said first fusion protein; and

(iv) no change in output in either of said first or said second reporter genes indicates that said third fusion protein does not interact with either of said first or said second fusion genes;

(e) comparing said expression output results of step (b) and step (c) with the expression output result measured in either

(i) a first comparison host cell which contains

(a) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(b) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said first protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(c) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety;

or

(ii) a second comparison host cell which contains

(a) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(b) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said second protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(c) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety;

or

(iii) both of said first and said second comparison host cells.

2. The method of claim 1, wherein at least one of said first or said second reporter genes may be reduced in expression level.

3. The method of claim 1, wherein one of said first or said second protein binding sites is a tetracycline operator.

4. The method of claim 1, wherein one of said first or said second reporter genes is URA3 or lacZ.

5. The method of claim 1, wherein one of said first or said second reporter genes produces a signal that is received and detected by a second cell.

6. The method of claim 1, wherein said host cell is a yeast cell or a mammalian cell.

7. The method of claim 6, wherein said host cell is a yeast cell.

8. The method of claim 1, wherein said first and said second reporter genes may be expressed simultaneously.

9. The method of claim 1, wherein said first protein and said second proteins are allelic variants.

10. A method for detecting a protein that mediates a change in the state of another protein, comprising:

(a) providing a host cell which contains

(i) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(ii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(iii) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising a second protein which is capable of interacting with said first protein and which is covalently bonded to a gene activating moiety,

wherein at least one of said first or said second proteins may exhibit a change in state;

(b) allowing said first and said second proteins to interact;

(c) measuring expression of said reporter gene as a measure of said interaction between said first and said second proteins;

(d) introducing into said cell a third gene expressing a third protein;

(e) measuring expression of said reporter gene, a change in said reporter gene expression in the presence of said third protein being an indication that said third protein mediates a change in the state of said first or said second protein leading to an alteration in the ability of said first protein and said second protein to interact.

11. The method of claim 10, wherein said change in state is a conformational change.

12. The method of claim 11, wherein said protein exhibiting a conformational change is a Ras protein.

13. The method of claim 10, wherein said host cell is a yeast cell or a mammalian cell.

14. The method of claim 13, wherein said host cell is a yeast cell.

15. The method of claim 10, wherein the expression of each of said first fusion protein and said third protein occurs in response to an extracellular stimulus.

16. The method of claim 10, wherein said reporter gene produces a signal that is received and detected by a second cell.

17. A method for detecting a protein that mediates a change in the state of another protein, comprising:

(a) providing a first host cell which contains

(i) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(ii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said first protein binding site; and

(iii) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising a second protein which is capable of interacting with said first protein and which is covalently bonded to a gene activating moiety,

wherein at least one of said first or said second proteins may exhibit changes in state;

(b) allowing said first and said second proteins to interact;

(c) measuring expression of said reporter gene in said first host cell as a measure of said interaction between said first and said second proteins;

(d) providing a second host cell which contains

(i) said reporter gene operably linked to said DNA sequence comprising said protein binding site;

(ii) said first fusion gene which expresses said first fusion protein; and

(iii) said second fusion gene which expresses said second fusion protein;

(iv) a third gene which expresses a third protein;

(e) allowing said first and said second proteins to interact in the presence of said third protein;

(f) measuring expression of said reporter gene in said second host cell as a measure of said interaction between said first and said second proteins in the presence of said third protein, a change in said reporter gene expression in said second host cell as compared to that measured in said first host cell being an indication that said third protein mediates a change in the state of said first or said second protein resulting in an alteration in the ability of said first protein and said second protein to interact.

18. The method of claim 17, wherein said change in state is a conformational change.

19. The method of claim 18, wherein said protein exhibiting a conformational change is a Ras protein.

20. The method of claim 17, wherein each of said host cells is a yeast cell or a mammalian cell.

21. The method of claim 20, wherein each of said host cells is a yeast cell.

22. The method of claim 17, wherein the expression of each of said first fusion protein and said third protein occurs in response to an extracellular stimulus.

23. The method of claim 17, wherein said reporter gene produces a signal that is received and detected by a second cell.

24. A method for detecting whether a candidate protein interacts with a transcriptional activator, comprising:

(a) providing a host cell which contains

(i) a reporter gene which can be reduced in expression level, said reporter gene being operably linked to a DNA sequence comprising a protein binding site;

(iii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said transcriptional activator covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(v) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said candidate protein covalently bonded to a gene activating moiety;

(b) detecting an increase in expression of said reporter gene as an indication of an interaction between said candidate protein and said transcriptional activator.

25. The method of claim 24, wherein said reporter gene is a URA3 gene.

26. The method of claim 25, wherein said expression level is reduced by 6-azauracil.

27. The method of claim 24, wherein said protein binding site is a tetracycline operator.

28. The method of claim 27, wherein said expression level is reduced by tetracycline or a tetracycline derivative.

29. The method of claim 24, wherein said host cell is a yeast cell or a mammalian cell.

30. The method of claim 29, wherein said host cell is a yeast cell.

31. A method for detecting a protein—

protein interaction, comprising;

(a) providing a host cell which contains

(i) a first reporter gene operably linked to a DNA sequence comprising a first protein binding site;

(ii) a second reporter gene operably linked to a DNA sequence comprising a second protein binding site, wherein one of said first or said second reporter genes produces a signal that is received and detected by a second cell;

(iii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said first protein binding site;

(iv) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising a second protein covalently bonded to a binding moiety which is capable of specifically binding to said second protein binding site; and

(v) a third fusion gene which expresses a third fusion protein, said third fusion protein comprising a third protein covalently bonded to a gene activating moiety;

(b) measuring expression output of said first reporter gene as a measure of said interaction between said first and said third proteins;

(c) measuring expression output of said second reporter gene as a measure of said interaction between said second and said third proteins; and

(d) interpreting the expression output results of step (b) and step (c), whereby

(i) increased output of both of said first and said second reporter genes indicates that said third fusion protein interacts with both of said first and said second fusion proteins;

(ii) increased output of said first reporter gene but not said second reporter gene indicates that said third fusion protein interacts with said first fusion protein but not said second fusion protein;

(iii) increased output of said second reporter gene but not said first reporter gene indicates that said third fusion protein interacts with said second fusion protein but not said first fusion protein; and

(iv) no change in output in either of said first or said second reporter genes indicates that said third fusion protein does not interact with either of said first or said second fusion genes.

32. The method of claim 31, further comprising comparing said expression output results of step (b) and step (c) with the expression output result measured in either

(a) a first comparison host cell which contains

(i) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(ii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said first protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(iii) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety;

or

(b) a second comparison host cell which contains

(i) a reporter gene operably linked to a DNA sequence comprising a protein binding site;

(ii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said second protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and

(iii) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety;

or

(c) both of said first and said second comparison host cells.

33. The method of claim 31, wherein at least one of said first or said second reporter genes may be reduced in expression level.

34. The method of claim 31, wherein one of said first or said second protein binding sites is a tetracycline operator.

35. The method of claim 31, wherein one of said first or said second reporter genes is URA3 or lacZ.

36. The method of claim 31, wherein said host cell is a yeast cell or a mammalian cell.

37. The method of claim 36, wherein said host cell is a yeast cell.

38. The method of claim 31, wherein said first and said second reporter genes may be expressed simultaneously.

39. The method of claim 31, wherein said first protein and said second proteins are allelic variants.