Method to detect protein-protein interactions

• US 5,637,463 A
• Filed: 05/04/1995
• Issued: 06/10/1997
• Est. Priority Date: 05/04/1995
• Status: Expired due to Term

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Osborne, Mark A., Dalton, Stephen, Kochan, Jarema P.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Brusca, John S.
• Application Number:
  US08/434,730
• Time in Patent Office: 768 Days
• Field of Search: 435/6, 435/7.21, 435/7.31, 435/69.1, 435/69.7, 435/172.1, 435/172.3, 435/320.1, 536/23.4, 536/23.5, 536/23.74, 530/350
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

Methods are provided for studying protein-protein interactions which require posttranslational modification of one of the proteins. The interaction is detected by reconstituting the activity of a transcriptional activator. This activity is dependent on the interactions between three different proteins. These include two chimeric proteins, one of which must be posttranslationally modified by the activity of the third protein in order for the chimeric proteins to interact. One of the chimeric proteins contains a transcriptional activation domain fused to a test protein. The second chimeric protein contains a DNA-binding domain of a transcriptional activator fused to the other test protein.

Claims

1. A method for detecting an interaction between a first test protein and a second test protein, comprising:

(A) introducing a first chimeric gene, a second chimeric gene and a third gene into a Saccharomyces cerevisiae or mammalian host cell containing a detectable gene which expresses a detectable protein when the detectable gene is transcribed, wherein;

(i) said first chimeric gene encodes a first hybrid protein, said first hybrid protein comprising;

(a) a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell; and

(b) a first test protein or fragment thereof to be tested for interaction with a second test protein or fragment thereof, wherein the interact loft of said first and second test proteins requires a post-translational interaction of said first test protein with a third test protein;

(ii) said second chimeric gene encodes a second hybrid protein, said second hybrid protein comprising;

(a) a transcriptional activation moiety; and

(b) a second test protein or fragment thereof to be tested for interaction with said first test protein or fragment thereof; and

(iii) said third gene comprising a DNA sequence that encodes a third test protein or fragment thereof to be tested for interaction with said first test protein;

wherein said first chimeric gene, second chimeric gene and third gene also contain an inducible promoter region such that expression of said genes in the host cell is regulated;

(B) subjecting the host cell to conditions such that said first hybrid protein, said second hybrid protein and said third test protein are expressed in sufficient quantity for said third test protein to interact with said first test protein thereby enabling the interaction between said first test protein and said second test protein, and the formation of a complex of the DNA binding moiety of said first hybrid protein and the transcriptional activation moiety of said second hybrid protein which in turn results in the activation and transcription of the detectable gene; and

(C) determining whether the detectable gene has been transcribed, the transcription of the detectable gene being indicative of whether an interaction has occurred between said first and second test proteins.

2. The method of claim 1, wherein the DNA binding moiety and the transcriptional activation moiety are derived from a single transcriptional activator.

3. The method of claim 1, wherein the DNA binding moiety and the transcriptional activation moiety are derived from different proteins.

4. The method of claim 1, wherein said detectable protein is selected from the group consisting of β

-galactosidase, green fluorescent protein luciferase, alkaline phosphatase and chloramphenical acetyl transferase.

5. The method of claim 1, wherein the detectable gene expresses a selectable marker comprising a protein involved in nutrient biosynthesis.

6. The method of claim 1, wherein the inducible promoter is a galactose inducible promoter.

7. A method for detecting an interaction between a first test protein and a second test protein, said interaction dependent upon the post-translational modification of the first test protein by a third test protein, comprising:

(A) introducing into a Saccharomyces cerevisiae or mammalian host cell containing a detectable gene which expresses a detectable protein when the detectable gene is transcribed, the following;

(i) a first chimeric gene encoding a first hybrid protein, said hybrid protein comprising;

(a) a DNA-binding moiety that recognizes a binding site on the detectable gene in the host cell; and

(b) a first test protein or fragment thereof to be tested for interaction with a second test protein or fragment thereof, wherein the interaction of said first and second test proteins requires a post-translational modification of said first test protein by a third test protein;

(ii) a second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, said second hybrid protein comprising;

(a) a transcriptional activation moiety; and

(b) a second test protein or fragment thereof to be tested for interaction with said first test protein or fragment thereof; and

(iii) a third gene comprising a DNA sequence that encodes a third test protein or fragment thereof which post-translationally modifies said first test protein;

wherein said first chimeric gene, second chimeric gene and third gene also contain an inducible promoter region such that expression of said genes in the host cell is regulated;

(B) subjecting the host cell to conditions such that said first hybrid protein, said second hybrid protein and said third test protein are expressed in sufficient quantity for said third test protein to post-translationally modify said first test protein thereby enabling the formation of a complex between said first test protein and said second test protein, and the formation of a complex of the DNA binding moiety of said first hybrid protein and the transcriptional activation moiety of said second hybrid protein which in turn results in the activation and transcription of the detectable gene; and

(C) determining whether the detectable gene has been transcribed, the transcription of the detectable gene being indicative of whether an interaction has occurred between said first and second test proteins.

8. The method of claim 7, wherein said third test protein is a protein kinase.

9. The method of claim 7, wherein said first test protein comprises a subunit of a protein containing an immunoreceptor tyrosine-based activation motif (ITAM).

10. The method of claim 7, wherein said second test protein is a src-homology 2 (SH2) containing protein.

11. The method of claim 8, wherein said third protein is a tyrosine kinase.

12. The method of claim 9, wherein said first test protein is selected from the group consisting of high affinity IgE receptor FceRIγ

and Fcε

RIβ

subunits and T cell receptor ζ

chain.

13. The method of claim 10, wherein said second test protein is selected from the group consisting of Syk and Lyn.

14. The method of claim 11, wherein said third test protein is selected from the group consisting of Lck and Lyn.

15. The method of claim 7, wherein

a) the host cell is Saccharomyces cerevisiae;

b) the host cell contains a detectable gene expressing β

-galactosidase;

c) said first hybrid protein comprises E.coli LexA fused to Fcε

RIγ

CT;

d) said second hybrid protein comprises HSV1Vmw65 fused to the SH2 domains of Syk;

e) said third test protein is Lck; and

f) the inducible promoter region is inducible by galactose.

16. A cDNA consisting of SEQ ID NO:

13.

17. A cDNA consisting of SEQ ID NO:

15 .

18. A protein SH2-A consisting of amino acid science SEQ ID NO:

14.

19. A protein SH2-B consisting of acid SEQ ID NO:

16.

20. A kit for the detection in a Saccharomyces cerevisiae or mammalian host cell of interactions between test proteins wherein said interaction is dependent upon post-translational modification of a first test protein by a third test protein, comprising three or more DNA sequences comprising

a first plasmid encoding a first hybrid protein, said first hybrid protein comprising a DNA-binding moiety fused to a first protein of interest, said first protein to be tested for interaction with a second test protein wherein said first test protein must be post translationally modified by the third test protein for it to interact with said second test protein;

b) a second plasmid encoding a second hybrid protein, said second hybrid protein comprising a transcriptional activation moiety fused to cloning sites for a cDNA library or specific fusion proteins to be tested for interaction with said first test protein;

c) a third plasmid encoding a third test protein having protein modification activity for said first test protein; and

d) a DNA comprising a reporter construct.