Mammalian protein interaction cloning system

• US 6,316,223 B1
• Filed: 07/22/1999
• Issued: 11/13/2001
• Est. Priority Date: 03/30/1998
• Status: Expired due to Term

• Current Assignee: Rigel Pharmaceuticals, Inc.
• Original Assignee: Rigel Pharmaceuticals, Inc.
• Inventors: Luo, Ying, Payan, Donald, Huang, Betty
• Primary Examiner(s): PARK, HANKYEL
• Application Number:
  US09/359,081
• Time in Patent Office: 845 Days
• Field of Search: 435/69.1, 435/70.1
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 2565/201 Two hybrid system

Abstract

The present invention is directed to compositions and methods for a genetic system of detecting protein-protein interactions in a mammalian host cell. Two fusion proteins are made in the host cell. The first fusion protein contains a DNA binding domain which is fused to a so-called bait protein. The second fusion protein consists of a transcriptional activation domain fused to a so-called test protein. The DNA binding domain binds to an operator sequence which controls expression of one or more reporter genes. The transcriptional activation domain is recruited to the promoter through the functional interaction between the bait protein and the test protein. Subsequently the transcriptional activation domain interacts with the basal transcription machinery to activate expression of one or more reporter genes which can be identified and characterized. The individual compositions are useful for analyzing protein-protein interactions between known proteins and to isolate, clone and characterize unknown proteins. The individual compositions can be used to express the fusion proteins either transiently or stably. In addition, the present invention is directed to methods for screening for candidate bioactive agents that modulate the protein-protein interaction between a bait protein and a test protein and thus are useful for the identification of novel therapeutic drugs and the like.

Claims

1. A method for detecting an interaction between a bait protein and a test protein comprising:

a) providing a mammalian host cell comprising;

i) a bait vector comprising;

1) a first selection gene;

2) a bait vector viral origin of replication which requires a bound viral replication protein to effect replication; and

3) a first fusion gene comprising;

A) a first sequence encoding a nucleic acid binding domain; and

B) a second sequence encoding a bait protein;

ii) a test vector comprising;

1) a second selection gene;

2) a test vector viral origin of replication which requires a bound viral replication protein to effect replication; and

3) a second fusion gene comprising;

A) a third sequence encoding a transcriptional activation domain; and

B) a fourth sequence encoding a test protein; and

iii) a reporter vector comprising;

1) a first reporter gene;

2) a gene encoding a viral replication protein that binds to said viral origin of replication; and

3) an operator site, to which said nucleic acid binding domain binds;

b) subjecting the host cell to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein;

c) determining whether a protein-protein interaction between said first fusion protein and said second fusion protein occurred; and

d) isolating said fourth sequence.

5. A method according to claim 1, further comprising using said fourth sequence to isolate nucleic acid encoding a full-length test protein.

6. A method according to claim 5, further comprising expressing said nucleic acid to generate a full-length recombinant test protein or derivative thereof.

7. A method according to claim 6, further comprising determining whether said full-length recombinant test protein or said derivative thereof interacts with said bait protein.

2. A method for detecting an interaction between a bait protein and a test protein comprising:

a) providing a mammalian host cell comprising;

i) a retroviral bait vector comprising a first fusion gene comprising;

1) a first sequence encoding a nucleic acid binding domain; and

2) a second sequence encoding a bait protein;

ii) a retroviral test vector comprising a second fusion gene comprising;

1) a third sequence encoding a transcriptional activation domain; and

2) a fourth sequence encoding a test protein; and

iii) a retroviral reporter vector comprising;

1) a first reporter gene; and

2) an operator site, to which said nucleic acid binding domain binds;

b) subjecting the host cell to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein;

c) determining whether a protein-protein interaction between said first fusion protein and said second fusion protein occurred; and

d) isolating said fourth sequence.

3. A method for detecting an interaction between a bait protein and a test protein comprising:

a) providing a mammalian host cell comprising;

i) a bait vector comprising;

1) a first selection gene;

2) a bait vector viral origin of replication which requires a bound viral replication protein to effect replication; and

3) a first fusion gene comprising;

A) a first sequence encoding a nucleic acid binding domain; and

B) a second sequence encoding a bait protein;

ii) a test vector comprising;

1) a second selection gene;

2) a test vector viral origin of replication which requires a bound viral replication protein to effect replication; and

3) a second fusion gene comprising;

A) a third sequence encoding a transcriptional activation domain; and

B) a fourth sequence encoding a test protein; and

iii) a reporter vector comprising;

1) a first reporter gene;

2) a gene encoding a viral replication protein that binds to said viral origin of replication; and

3) an operator site, to which said nucleic acid binding domain binds;

b) subjecting the host cell to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein;

c) determining whether a protein-protein interaction between said first fusion protein and said second fusion protein occurred; and

d) detennining said fourth sequence.

4. A method for detecting an interaction between a bait protein and a test protein comprising:

a) providing a mannnalian host cell comprising;

i) a retroviral bait vector comprising a first fusion gene comprising;

1) a first sequence encoding a nucleic acid binding domain; and

2) a second sequence encoding a bait protein;

ii) a retroviral test vector comprising a second fusion gene comprising;

1) a third sequence encoding a transcriptional activation domain; and

2) a fourth sequence encoding a test protein; and

iii) a retroviral reporter vector comprising;

1) a first reporter gene; and

2) an operator site, to which said nucleic acid binding domain binds;

b) subjecting the host cell to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein;

c) determining whether a protein-protein interaction between said first fusion protein and said second fusion protein occurred; and

d) determining said fourth sequence.

8. A method for screening for a bioactive agent capable of modulating a protein-protein interaction between a bait protein and a test protein comprising:

a) providing a mammalian host cell comprising a vector composition comprising;

i) a first fusion gene comprising;

1) a first sequence encoding a nucleic acid binding domain; and

2) a second sequence encoding a bait protein;

ii) a second fusion gene comprising;

1) a third sequence encoding a transcriptional activation domain; and

2) a fourth sequence encoding a test protein; and

iii) a reporter gene comprising;

1) a coding region for a reporter protein; and

2) an operator site, to which said nucleic acid binding domain binds;

b) subjecting the host cell to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein;

c) adding a candidate bioactive agent to said host cell; and

d) determining the effect of said candidate bioactive agent on the protein-protein interaction between said bait protein and said test protein.

9. A method according to claim 8, wherein said vector composition further comprises at least one selection gene.

10. A method according to claim 8, wherein said vector composition further comprises at least one origin of replication and at least one gene encoding a replication protein that binds to said origin of replication.

11. A method according to claim 8, further comprising identifying said candidate bioactive agent.