Protein-protein interaction guided mating of yeast

Patent metadata

Field	Value
Publication number	US-10017758-B1
Application number	US-201715605183-A
Country	US
Kind code	B1
Filing date	May 25, 2017
Priority date	May 25, 2017
Publication date	Jul 10, 2018
Grant date	Jul 10, 2018

Abstract

The invention relates to methods and compositions useful for detection of protein-protein interactions between cell surface proteins.

First claim

What is claimed is:

1. A method of detecting a protein-protein interaction between cell surface proteins, the method comprising:
(a) providing a first plurality of haploid yeast cells of a first mating type, the first plurality of cells comprising a library of first plasmids, wherein each plasmid in the library comprises:
a first selectable marker, a first unique oligonucleotide molecular barcode sequence operatively linked to a first recombination site, and a sequence encoding a unique cell surface protein, such that each unique molecular barcode is associated with a unique cell surface protein;

(b) providing a second plurality of haploid yeast cells of a second mating type, the second plurality of cells comprising a library of second plasmids, wherein each plasmid in the library comprises:
a second selectable marker, a second unique oligonucleotide molecular barcode sequence operatively linked to a second recombination site, and a sequence encoding a unique cell surface protein, such that each unique molecular barcode is associated with a unique cell surface protein,
wherein the unique cell surface proteins of the library of first plasmids and the unique cell surface proteins of the library of second plasmids comprise potential binding pairs;

(c) contacting the first plurality of haploid yeast cells with the second plurality of haploid yeast cells under dilute yeast cell conditions that promote contact between the first plurality of haploid yeast cells and the second plurality of haploid yeast cells when a specific binding interaction between a cell surface protein of the library of first plasmids and a cell surface protein of the library of second plasmids occurs, wherein the specific binding interaction promotes mating of the first and second plurality of haploid yeast cells to produce diploid yeast cells;
(d) recombining within the diploid cells portions of the plasmids to generate a recombined molecular barcode sequence comprising at least a portion of the unique barcode associated with one of the unique cell surface proteins of the library of first plasmids and a portion of the unique molecular barcode associated with one of the unique cell surface proteins of the library of second plasmids; and
(e) sequencing at least a portion of the recombined molecular barcode sequence from the diploid cells to identify a surface protein from the library of first plasmids and a cell surface protein from the library of second plasmids that interact, thus detecting a protein-protein interaction between the cell surface proteins.

2. The method of claim 1 , further comprising using at least one of the selectable markers to select for diploid cells that contain at least one each of the first and second plasmids after contacting the first plurality of haploid yeast cells with the second plurality of haploid yeast cells.

3. The method of claim 1 , wherein the recombined molecular barcode sequence from two or more diploid cells are sequenced to detect two or more different cell surface protein-protein interactions.

4. The method of claim 1 , wherein the specific binding interaction between the first plurality of haploid yeast cells and the second plurality of haploid yeast cells occurs in a dilute mixture that has a spectral absorbance of less than 2.0 O.D. 600 nm .

5. The method of claim 1 , wherein the binding affinity between the first cell surface protein and the second cell surface protein is greater than the mating type binding affinity between a yeast cell from the first mating type and a yeast cell from the second mating type.

6. The method of claim 5 , wherein the binding affinity is at least two-fold, five-fold or ten-fold greater than the mating type affinity.

7. The method of claim 1 , wherein the first selectable marker and the second selectable marker are the same.

8. The method of claim 1 , wherein the first selectable marker and the second selectable marker are different.

9. The method of claim 1 , wherein the recombination is site-specific recombination.

10. The method of claim 1 , wherein the recombination is CRE-Lox recombination.

11. The method of claim 1 , wherein the recombination is FLP-FRT recombination.

12. The method of claim 1 , wherein the recombination is unidirectional integration.

13. The method of claim 12 , wherein the recombination is catalyzed by a serine integrase.

14. The method of claim 13 , wherein the recombination is catalyzed by a PhiC31 integrase.

15. The method of claim 1 , wherein the first and the second unique oligonucleotide molecular barcode sequence is between 5 and 50 nucleotides in length.

16. The method of claim 1 , wherein the plasmids recombine with the genome of the haploid yeast cell or the diploid yeast cell.

17. The method of claim 1 , wherein the cell surface protein is a transmembrane protein.

18. The method of claim 1 , wherein the cell surface protein is an integral membrane protein.

19. The method of claim 1 , wherein the cell surface protein is peripheral membrane protein.

20. The method of claim 1 , wherein the cell surface protein is encoded by a mammalian gene.

21. The method of claim 1 , wherein the cell surface protein is encoded by a human gene.

22. The method of claim 1 , wherein at least one of the haploid yeast cells have a gene coding for a yeast mating factor that is controlled by an inducible promoter.

23. The method of claim 22 , wherein the inducible promoter is Gal1.

24. The method of claim 22 , wherein the mating of the haploid yeast cells is induced by activating the inducible promoter.

25. The method of claim 1 , wherein mating of the haploid yeast cells is detected by a marker that becomes active only in the diploid cells.

26. The method of claim 1 , wherein the diploid cells are detected after recombination between the plasmids.

27. The method of claim 1 , wherein the diploid cells are detected by the presence of two distinct selectable markers.

28. The method of claim 1 , wherein sequencing comprises amplification of the recombined molecular bar code sequence.

29. The method of claim 1 , wherein sequencing comprises amplification of at least a portion of the sequence that encodes the cell surface protein in a first plasmid or at least a portion of the sequence that encodes the cell surface protein in a second plasmid.

30. The method of claim 1 , wherein the cell surface protein is a protein not normally associated with the cell membrane or expressed on the cell surface.

31. The method of claim 30 , wherein the cell surface protein is a hybrid protein comprising at least a portion of a native, soluble protein fused to at least a portion of a native cell surface protein, wherein the at least a portion of the native, soluble protein is expressed on the outer surface of the haploid yeast cell.

32. The method of claim 31 , wherein a linker connects the at least a portion of the native, soluble protein to the at least a portion of the native cell surface protein.

Assignees

• Verily Life Sciences Llc

Inventors

• Peikon Ian
• Tong Gary

Classifications

• C12N15/1055 Primary

  Protein x Protein interaction, e.g. two hybrid selection · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12Q1/025

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title