Mammalian protein interaction cloning system

A composition comprising:

• a) a bait vector comprising:
• i) a first selection gene;
• ii) a bait vector viral origin of replication which requires a bound viral replication protein to effect replication;
• and
• iii) a first fusion gene comprising:
• 1) a first sequence encoding a nucleic acid binding domain;
• and
• 2) a cloning sequence comprising at least one cloning site for insertion of a nucleic acid sequence encoding a bait protein;
• and
• b) a test vector comprising:
• i) a second selection gene;
• ii) a test vector viral origin of replication which requires a bound viral replication protein to effect replication;
• and
• iii) a second fusion gene comprising:
• 1) a third sequence encoding a transcriptional activation domain;
• and
• 2) a cloning sequence comprising at least one cloning site for insertion of a nucleic acid sequence encoding a test protein.

A composition comprising:

• a) a retroviral bait vector comprising a first fusion gene comprising:
• i) a first sequence encoding a nucleic acid binding domain;
• and
• ii) a second sequence encoding a bait protein;
• and
• b) a retroviral test vector comprising a second fusion gene comprising:
• i) a third sequence encoding a transcriptional activation domain;
• and
• ii) a fourth sequence encoding a test protein.

A composition comprising:

• a) a retroviral bait vector comprising a first fusion gene comprising:
• i) a first sequence encoding a nucleic acid binding domain;
• and
• ii) a cloning sequence comprising at least one cloning site for insertion of a nucleic acid sequence encoding a bait protein;
• and
• b) a retroviral test vector comprising a second fusion gene comprising:
• i) a third sequence encoding a nucleic acid binding domain;
• and
• ii) a cloning sequence comprising at least one cloning site for insertion of a nucleic acid sequence encoding a test protein.

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

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;
• and
• 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 detectable gene;
• and
• 2) an operator site, to which said nucleic acid binding domain binds;
• and
• 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;
• and
• c) determining whether a protein--protein interaction between said first fusion protein and said second fusion protein occurred.