Two-hybrid double screening system and method

A kit for detecting protein-protein interactions, comprising:

• (a) a first screening assay, comprising a first mammalian cell comprising (i) a reporter gene encoding a reporter polypeptide operably linked to an upstream transcriptional regulatory sequence comprising a DNA binding site for a DNA binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein, the bait fusion protein comprising a DNA binding domain and the bait;
• and (iii) a cDNA library of prey, wherein each prey cDNA encodes a prey fusion protein comprising a transcriptional activation domain and a prey;
• (b) components for conducting a 3-primer PCR;
• and
• (c) a second screening assay, comprising a second mammalian cell, the second mammalian cell comprising (i) a bait nucleotide sequence encoding the bait fusion protein, wherein the bait fusion protein comprises a reporter polypeptide and a bait protein, (ii) a cDNA prey PCR product;
• wherein the first screening identifies a candidate prey cDNA encoding a prey able to interact with the bait, and the 3-primer PCR results in a PCR product comprising the candidate prey cDNA and a membrane-localizing signal, and wherein the second screening identifies a prey cDNA encoding a prey able to interact with the bait.

A method for detecting protein-protein interactions, comprising:

• (a) conducting a first screening assay with a first mammalian cell, wherein the first mammalian cell comprises (i) a reporter gene encoding a reporter polypeptide operably linked to an upstream transcriptional regulatory sequence, wherein the transcriptional regulatory sequence comprises a DNA binding site, (ii) a bait nucleotide sequence encoding a bait fusion protein, wherein the bait fusion protein comprises a DNA binding domain and the bait;
• and (iii) a cDNA library of prey, wherein each prey cDNA encodes a prey fusion protein comprising a transcriptional activation domain and a prey, wherein the first mammalian cell is incubated under conditions conducive to expressing the reporter gene in the presence of an interaction between a bait protein and a prey protein;
• (b) performing a 3-primer PCR resulting in a PCR product comprising the candidate prey cDNA and a membrane-localizing signal;
• and
• (c) conducting a second screening assay with a second mammalian cell, the second mammalian cell comprising (i) a bait nucleotide sequence encoding the bait fusion protein, wherein the bait fusion protein comprises a reporter polypeptide and a bait protein, and (c) the cDNA prey PCR product of step (b), and wherein the second screening identifies a prey cDNA encoding a prey able to interact with the bait.

A method for screening interactions between a bait and a library of prey in a mammalian cell, comprising:

• (a) providing a first mammalian cell comprising (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence containing a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein, the bait fusion protein comprising a DNA binding domain and the bait;
• and (iii) a cDNA library of prey, wherein each prey cDNA encodes a prey fusion protein including a transcriptional activation domain and a prey;
• (b) incubating the mammalian cell for 30 hours or less;
• (c) detecting expression of the reporter gene, if present, wherein the detecting is collecting signal positive cells;
• (d) rescuing cDNA from the recovered positive cells of step (d) with 3-primer PCR, wherein rescued cDNA comprises a cDNA prey PCR product comprising a membrane-localizing signal and a prey;
• (e) providing the cDNA prey PCR product in a second mammalian cell, the second mammalian cell further comprising a bait nucleotide sequence encoding the bait fusion protein, wherein the bait fusion protein comprises a fluorescent polypeptide and a bait protein;
• (f) incubating the second mammalian cell for 30 hours or less;
• (g) detecting membrane-bound signal of the fluorescent polypeptide, if present, wherein detection of a membrane-bound fluorescent signal indicates an interaction between the bait and the prey.

A method for reducing false positives obtained in a conventional yeast two-hybrid screening, comprising:

• (a) performing a conventional yeast two-hybrid screening;
• (b) isolating prey cDNAs from yeast colonies resulted from the yeast two-hybrid screening;
• (c) performing a 3-primers PCR procedure to prepare the prey cDNAs in an expressible form in order to be re-examined in the second screening;
• (d) performing a second screening comprising the steps of:
• (i) providing a mammalian cell comprising:
• (1) a bait nucleotide sequence encoding the bait fusion protein, comprising a fluorescent polypeptide and a bait;
• and (2) a cDNA prey PCR product, comprising a membrane-localizing signal and a prey, resulted from the 3-primers PCR;
• (e) incubating the mammalian cell for 30 hours or less;
• and
• (f) detecting membrane-bound signal of the fluorescent polypeptide, if present, indicating an interaction between the bait and the prey.

A method for detecting an interaction between a bait and a prey in a mammalian cell, comprising:

• (a) providing a mammalian cell containing:
• (i) a bait nucleotide sequence encoding a bait fusion protein, including a fluorescent polypeptide and a bait;
• (ii) a prey nucleotide sequence encoding a prey fusion protein, including a membrane-localizing signal and a prey;
• (b) incubating the cell for 30 hours or less;
• and
• (c) detecting the location of the fluorescent polypeptide signal, if present surrounding the cell membrane, indicating an interaction between the bait and the prey.

A method of identifying an agent that disrupts interaction between a bait and a prey, comprising:

• (a) providing a mammalian cell containing:
• (i) a bait nucleotide sequence encoding a bait fusion protein, including a fluorescent polypeptide and a bait;
• (ii) a prey nucleotide sequence encoding a prey fusion protein, including a membrane-localizing signal and a prey;
• (b) contacting the mammalian cell with a test agent;
• (c) incubating the cell for 30 hours or less;
• and
• (d) detecting a decrease of the fluorescent polypeptide signal surrounding the cell membrane compared to the level of the fluorescent polypeptide signal surrounding the cell membrane in the absent of the test agent, if present, thereby detecting an agent that disrupts an interaction between the bait and the prey.

A method of identifying an agent able to enhance interaction between a bait and a prey, including:

• (a) providing a mammalian cell containing:
• (i) a bait nucleotide sequence encoding a bait fusion protein, including a fluorescent polypeptide and a bait;
• (ii) a prey nucleotide sequence encoding a prey fusion protein, including a membrane-localizing signal and a prey;
• (b) contacting the mammalian cell with a test agent;
• (c) incubating the cell for 30 hours or less;
• and
• (d) detecting a increase of the fluorescent polypeptide signal surrounding the cell membrane compared to the level of the fluorescent polypeptide signal surrounding the cell membrane in the absent of the test agent, if present, thereby detecting an agent able to enhance an interaction between the bait and the prey.