System to detect protein-protein interactions

• US 5,283,173 A
• Filed: 01/24/1990
• Issued: 02/01/1994
• 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 Moskowitz
• Assistant Examiner(s): Houtteman, Scott W.
• Application Number:
  US07/469,285
• Time in Patent Office: 1,469 Days
• Field of Search: 435/5, 435/6, 435/69.1, 435/172.3, 435/252.3, 435/801, 436/501, 436/63, 436/94, 436/808, 530/350
• 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

Methods 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 method for detecting an interaction between a first test protein and a second test protein, the method comprising:

(a) providing a Saccharomyces cerevisiae host cell containing a detectable gene wherein the detectable gene expresses a detectable protein when the detectable gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the detectable gene;

(b) providing a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising;

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

(ii) a first test protein or fragment thereof that is to be tested for interaction with at least one second test protein or fragment thereof;

(c) providing a second chimeric gene that is capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising;

(i) the transcriptional activation domain; and

(ii) a second test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof;

wherein interaction between the first test protein and the second test protein in the host cell causes the transcriptional activation domain to activate transcription of the detectable gene;

(d) introducing the first chimeric gene and the second chimeric gene into the host cell;

(e) subjecting the host cell to conditions under which the first hybrid protein and the second hybrid protein are expressed in sufficient quantity for the detectable gene to be activated; and

(f) determining whether the detectable gene has been expressed to a degree greater than expression in the absence of an interaction between the first test protein and the second test protein.

2. The method 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 method 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 method 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 method 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 a non-bacterial enzyme and a non-viral enzyme.

6. The method according to claim 1, wherein the chimeric genes are introduced into the host cell in the form of plasmids.

7. The method according to claim 1, wherein the first chimeric gene is integrated into the chromosomes of the host cell.

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

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