System to detect protein-protein interactions

• US 5,468,614 A
• Filed: 02/01/1994
• Issued: 11/21/1995
• Est. Priority Date: 01/24/1990
• Status: Expired due to Term

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Fields, Stanley, Song, Ok-Kyu
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/189,910
• Time in Patent Office: 658 Days
• Field of Search: 435/6, 435/320.1, 536/23.7, 536/23.4, 536/23.5, 935/79, 935/80
• US Class Current: 435/6.13
• 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

Abstract

A method and kit are provided for detecting the interaction between a first test protein and a second test protein, in vivo, using reconstitution of the activity of a transcriptional activator. This reconstitution makes use of chimeric genes which express hybrid proteins. Two types of hybrid proteins are prepared. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to the first test protein. The second hybrid protein contains a transcriptional activation domain fused to the second test protein. If the two test proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a marker gene which contains a binding site for the DNA-binding domain.

Claims

1. A kit for detecting binding between a first test protein and a second test protein or fragments thereof in a host cell, the kit comprising in a container:

(a) a first vector, which comprises;

(i) a first gene, which comprises;

(1) a promoter that functions in a host cell,

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

(3) a means for inserting a DNA sequence encoding a first test protein or a fragment thereof into the first vector in such a manner that the first test protein is capable of being expressed as part of a hybrid protein containing the DNA binding domain,

(ii) a means for replicating the first vector in the host cell, and

(iii) a first marker gene, the expression of which in the host cell permits selection of cells containing the first marker gene from cells that do not contain the first marker gene, and

(b) a second vector, which comprises;

(i) a second gene, which comprises;

(1) a promoter that functions in the host cell,

(2) a DNA sequence that encodes a transcriptional activation domain which is functionally associated with the promoter of the second gene, and

(3) a means for inserting a DNA sequence encoding the second test protein or a fragment thereof into the vector, in such a manner that the second test protein is capable of being expressed as part of a hybrid protein containing the transcriptional activation domain

(ii) a means for replicating the second vector in the host cell, and

(iii) a second marker gene, the expression of which in the host cell permits selection of cells containing the second marker gene from cells that do not contain the second marker gene; and

(c) a host cell containing a detectable gene having a binding site for the DNA-binding domain wherein the detectable gene expresses a detectable protein when the detectable gene is activated by the transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the detectable gene;

the host cell being incapable of expressing a protein having the function of;

(i) the first marker gene;

(ii) the second marker gene;

(iii) the DNA-binding domain; and

(iv) the transcriptional activation domain;

wherein binding of the first test protein and the second test protein in the host cell results in measurably greater expression of the detectable gene than the simultaneous presence of the DNA-binding domain and the transcriptional activation domain in the absence of an interaction between the first test protein and the second test protein.

2. The kit according to claim 1, wherein the DNA-binding domain and transcriptional activation domain are derived from transcriptional activators having separable DNA-binding and transcriptional activation domains.

3. The kit according to claim 2, wherein the DNA binding domain and the transcriptional activation domain are selected from the group consisting of transcriptional activators GAL4, GCN4 and ADR1.

4. The kit according to claim 1, wherein the first hybrid protein or the second hybrid protein is encoded on a library of plasmids containing DNA inserts, derived from the group consisting of genomic DNA, cDNA and synthetically generated DNA.

5. The kit according to claim 1, wherein the first test protein is derived from the group consisting of bacterial protein, viral protein, oncogene-encoded protein, growth factor and enzyme.

6. The kit according to claim 1, wherein the first gene is integrated into the chromosomes of the host cell.

7. The kit according to claim 1, wherein the first gene is integrated into the chromosomes of the host cell and the second gene is introduced into the host cell as part of a plasmid.

8. The kit according to claim 1, wherein the DNA-binding domain and the transcriptional activation domain are from different transcriptional activators.

9. The kit according to claim 1, wherein the first vector and the second vector contain a selectable bacterial marker gene and a replication signal that are functional in bacteria.

10. The kit according to claim 9, wherein one of said vectors is a plasmid and the means for inserting a DNA sequence into the plasmid includes a restriction site.

11. The kit according to claim 1, wherein the host cell is a yeast cell.

12. The kit according to claim 11, wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces pombe.