High throughput protein-protein interaction screening in yeast liquid culture
The present invention relates to methods and compositions for the high throughput screening of protein-protein interactions in yeast liquid culture. Protein fusions non-native to yeast may be expressed to replace endogenous sexual agglutination proteins and mediate library-by-library interrogation of protein interactions. The methods and compositions of the invention can be utilized for the characterization of protein interaction networks in high throughput for both binding affinity and specificity, which is crucial for understanding cellular functions, screening therapeutic candidates, and evaluating engineered protein networks.
1. A method for characterizing a plurality of protein-protein interactions, the method comprising:
providing a first quantity of recombinant haploid yeast cells;
providing a second quantity of recombinant haploid yeast cells, wherein at least one of the first quantity of recombinant haploid yeast cells or the second quantity of recombinant haploid yeast cells has been rendered incapable of mating according to any native sexual agglutination process such that the first quantity of recombinant haploid yeast cells and the second quantity of recombinant haploid yeast cells are not capable of mating according to any native sexual agglutination process;
combining the first quantity of recombinant haploid yeast cells and the second quantity of recombinant haploid yeast cells in a liquid medium to produce a culture;
growing the culture for a time and under conditions such that
one or more of a first plurality of exogenous synthetic adhesion proteins is expressed and displayed on the surface of at least a portion of the first quantity of recombinant haploid yeast cells, each synthetic adhesion protein being (1) a fusion protein bound to a cell wall glycosylphosphatidylinositol (GPI) anchored protein residing on the surface of each of the portion of the first quantity of recombinant haploid yeast cells or (2) a glycosylphosphatidylinositol (GPI) anchored fusion protein residing on the surface of each of the portion of the first quantity of recombinant haploid yeast cells,
one or more of a second plurality of exogenous synthetic adhesion proteins is expressed and displayed on the surface of at least a portion of the second quantity of recombinant haploid yeast cells, each synthetic adhesion protein being (1) a fusion protein bound to a cell wall glycosylphosphatidylinositol (GPI) anchored protein residing on the surface of each of the portion of the second quantity of recombinant haploid yeast cells or (2) a glycosylphosphatidylinositol (GPI) anchored fusion protein residing on the surface of each of the portion of the second quantity of recombinant haploid yeast cells, and
one or more interactions between one or more of the first plurality of exogenous synthetic adhesion proteins and one or more of the second plurality of exogenous synthetic adhesion proteins mediates one or more mating events between one or more of the first quantity of recombinant haploid yeast cells and one or more of the second quantity of recombinant haploid yeast cells to produce one or more diploid yeast cells;
determining, based on the number of mating events in the culture, the strength of the interactions between one or more of the first plurality of exogenous proteins and one or more of the second plurality of exogenous proteins; and
identifying the one or more of the first plurality of exogenous proteins and the one or more of the second plurality of exogenous proteins that mediated the one or more mating events.
2. The method of claim 1 , wherein:
the first plurality of exogenous proteins is encoded by a first plurality of nucleic acid constructs within the first quantity of recombinant haploid yeast cells, each yeast cell within the first quantity of recombinant haploid yeast cells comprising one of the first plurality of nucleic acid constructs; and
the second plurality of exogenous proteins is encoded by a second plurality of nucleic acid constructs within the second quantity of recombinant haploid yeast cells, each yeast cell within the second quantity of recombinant haploid yeast cells comprising one of the second plurality of nucleic acid constructs.
3. The method of claim 2 , wherein:
each of the first quantity of recombinant haploid yeast cells comprises a first fluorescent marker; and
each of the second quantity of recombinant haploid yeast cells comprises a second fluorescent marker;
wherein the second fluorescent marker is spectrally distinct from the first fluorescent marker.
4. The method of claim 3 , further comprising determining the number of mating events based on the number of the one or more diploid yeast cells in the culture.
5. The method of claim 4 , wherein the number of mating events is determined by flow cytometry.
6. The method of claim 2 , further comprising determining the number of mating events based on a result of massively parallel sequencing of DNA isolated from the one or more diploid yeast cells.
7. The method of claim 6 , wherein:
each of the first plurality of nucleic acid constructs comprises a first unique primer binding site; and
each of the second plurality of nucleic acid constructs comprises a second unique primer binding site.
8. The method of claim 7 , wherein each of the first plurality of nucleic acid constructs and each of the second plurality of nucleic acid constructs comprises a recombination site.
9. The method of claim 8 , wherein the first quantity of recombinant haploid yeast cells and/or the second quantity of recombinant haploid yeast cells comprises an exogenous recombinase.
10. The method of claim 9 , wherein the exogenous recombinase mediates recombination between the first plurality of nucleic acid constructs and the second plurality of nucleic acid constructs.
11. The method of claim 10 , wherein the result of the massively parallel sequencing of DNA isolated from the one or more diploid yeast cells yields a plurality of sequencing reads, each sequencing read comprising a portion of the first plurality of nucleic acid constructs and a portion of the second plurality of nucleic acid constructs.
12. The method of claim 11 , wherein the strength of the interaction between the one or more of the first plurality of exogenous proteins and the one or more of the second plurality of exogenous proteins is determined by the number of sequencing reads.
13. The method of claim 12 , wherein the strength of the interaction between the one or more of the first plurality of exogenous proteins and the one or more of the second plurality of exogenous proteins is determined by the number of sequencing reads relative to a reference number of sequencing reads corresponding to one or more reference protein-protein interactions.
14. The method of claim 1 , wherein i) the first quantity of recombinant haploid yeast cells lacks a functional Aga2 protein, and/or ii) the second quantity of recombinant haploid yeast cells lacks a functional Sag1 protein.