Yeast surface two-hybrid system for quantitative detection of protein-protein interactions

A library of yeast cells, wherein each cell in the library comprises two expression cassettes, wherein the first expression cassette comprises from 5′ to a first promoter, a nucleotide sequence coding for a fusion protein between a bait protein and a yeast cell wall anchor protein wherein the fusion protein comprises a first signal sequence, and a first 3′ untranslated region;

• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• and wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• and wherein (1) the bait protein is the same for all of the cells in the library, and the prey proteins are different for all or at least some of the cells in the library;
• (2) the prey protein is the same for all of the cells in the library, and the bait proteins are different for all or at least some of the cells in the library;
• or (3) the bait and the prey proteins are each different for all or at least some of the cells in the library.

A yeast surface two hybrid (YS2H) system, comprising:

• a yeast cell,
• a vector comprising two expression cassettes, wherein the first expression cassette comprises from 5′ to 3′:
• a first promoter, a nucleotide sequence coding for a fusion protein between a bait protein and a yeast cell wall anchor protein wherein the fusion protein comprises a first signal sequence, and a first 3′ untranslated region;
• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• and wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• and
• reagents or instructions for detecting signals generated upon association of the bait and the prey proteins on the cell surface of said yeast cell.

A method for assessing the interactions between two proteins, comprising:

• providing a yeast cell,
• providing two expression cassettes, wherein the first expression cassette comprises from 5′ to 3′:
• a first promoter, a nucleotide sequence coding for a fusion protein between a bait protein and a yeast cell wall anchor protein wherein the fusion protein comprises a first signal sequence, and a first 3′ untranslated region;
• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• and wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• introducing said two expression cassettes into said yeast cell by transformation;
• expressing said two proteins in said yeast cell to allow interactions between said two proteins;
• and
• detecting signals generated upon association of the bait and the prey proteins on the cell surface of said yeast cell as a basis for assessing the interactions between the two proteins.

A method of identifying a candidate protein which binds to a target protein from a plurality of candidate proteins, comprising:

• providing a library of yeast cells, wherein each cell in the library comprises two expression cassettes, wherein the first expression cassette comprises from 5′ to 3′:
• a first promoter, a nucleotide sequence coding for a fusion protein between said target protein as a bait protein and a yeast cell wall anchor protein wherein said fusion protein comprises a first signal sequence, and a first 3′ untranslated region;
• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for a candidate protein as a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• and wherein the first expression cassettes are identical among all of the cells in the library, and wherein the second expression cassettes are identical among the cells except for the candidate proteins such that a plurality of candidate proteins are represented by said library;
• expressing said proteins to allow association between candidate proteins to said target protein;
• detecting signals generated upon association of candidate proteins to said target protein as a basis for assessing the binding interactions between a candidate protein to said target protein;
• sorting cells in said library based on binding affinities of candidate proteins to said target protein;
• and
• identifying a cell expressing a candidate protein having a selected binding affinity to said target protein.

A method of identifying a candidate protein which binds to a target protein from a plurality of candidate proteins, comprising:

• providing a library of yeast cells, wherein each cell in the library comprises two expression cassettes, wherein the first expression cassette comprises from 5′ to 3′:
• a first promoter, a nucleotide sequence coding for a fusion protein between a candidate protein as a bait protein and a yeast cell wall anchor protein wherein said fusion protein comprises a first signal sequence, and a first 3′ untranslated region;
• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for said target protein as a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• and wherein the second expression cassettes are identical among all of the cells in the library, and wherein the first expression cassettes are identical among the cells except for the candidate proteins such that a plurality of candidate proteins are represented by said library;
• expressing said proteins to allow association between candidate proteins to said target protein;
• detecting signals generated upon association of candidate proteins to said target protein as a basis for assessing the binding interactions between a candidate protein to said target protein;
• sorting cells in said library based on binding affinities of candidate proteins to said target protein;
• and
• identifying a cell expressing a candidate protein having a selected binding affinity to said target protein.

A method of identifying interacting protein pairs, comprising:

• providing a library of yeast cells, wherein each cell in the library comprises two expression cassettes, wherein the first expression cassette comprises from 5′ to 3′:
• a first promoter, a nucleotide sequence coding for a fusion protein between a candidate protein as a bait protein and a yeast cell wall anchor protein wherein said fusion protein comprises a first signal sequence, and a first 3′ untranslated region;
• the second expression cassette comprises from 5′ to 3′:
• a second promoter, a nucleotide sequence coding for a candidate binding partner protein as a prey protein in fusion to a second signal sequence, and a second 3′ untranslated region;
• wherein upon expression of the bait and the prey protein in said yeast cell, said bait protein becomes anchored in the cell wall, and said prey protein is produced as a secretory protein;
• and wherein the first expression cassettes are identical among the cells except for the candidate proteins such that a plurality of candidate proteins are represented by said library, and the second expression cassettes are identical among the cells except for the candidate binding partner proteins such that a plurality of candidate binding partner proteins are represented by said library;
• expressing said proteins to allow association between candidate proteins to said candidate binding partner proteins;
• detecting signals generated upon association of candidate proteins to said candidate binding partner proteins;
• sorting cells in said library based on binding affinities of candidate proteins to said candidate binding partner proteins;
• and
• identifying a cell expressing a candidate protein and a candidate binding partner protein having a selected binding affinity towards each other.