Fast and exhaustive method for selecting a prey polypeptide interacting with a bait polypeptide of interest: application to the construction of maps of interactors polypeptides

• US 20030134268A1
• Filed: 10/25/2002
• Published: 07/17/2003
• Est. Priority Date: 02/18/1998
• Status: Active Grant

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Rain, Jean-Christophe, Legrain, Pierre, Fromont, Micheline
• Granted Patent: US 6,913,886 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The present invention is directed to a method for selecting a prey polypeptide that is able to interact with a bait polypeptide of interest, to a prey polynucleotide encoding the prey polypeptide as well as to the prey polypeptide itself. The invention also concerns plasmids used for performing the method of the invention as well as prokaryotic or eukaryotic recombinant host organisms containing such plasmids and also a collection of said recombinant host organisms consisting in a DNA library, such as a collection of recombinant haploid Saccharomyces cerevisiae. Finally, the invention is also directed to a technical medium containing the whole information concerning the interactions between metabolically related bait and prey polypeptides and/or polynucleotides coding for bait and prey polypeptides.

Claims

1. A method for selecting a polynucleotide encoding a prey polypeptide, said prey polypeptide being able to interact with a bait polypeptide, comprising the steps of:

a) subjecting a bait polynucleotide encoding the bait polypeptide, to a two-hybrid screening method, wherein said two-hybrid screening method comprises a step of mating at least one first recombinant yeast cell containing the prey polynucleotide to be assayed with a second haploid recombinant yeast cell containing the bait polynucleotide, provided that one haploid yeast cell among the first recombinant yeast cell or the second recombinant yeast cell also contains at least one detectable gene that is activated by a polypeptide including a transcriptional activation domain;

b) selecting the recombinant diploid yeast cell obtained at step a) for which the detectable gene has been expressed to a degree greater than expression in the absence of interaction between the bait polypeptide and the prey polypeptide; and

c) optionally characterizing the prey polynucleotide contained in each diploid yeast cell selected at step b).

2. The method according to claim 1, further comprising repeating at least once steps a) to c) with the previously characterized prey polynucleotide used as the bait polynucleotide.

3. The method according to claim 2, wherein the number of repeats of steps a) to c) with the previously characterized prey polynucleotide used as the bait polynucleotide is in the range from 1 to 10, preferably from 1 to 5 and most preferably 3.

4. The method according to claim 1, wherein the bait polynucleotide used that corresponds to a selected prey belongs to the following group of polynucleotide consisting in:

a) a polynucleotide that is identical to said selected prey polynucleotide;

b) a polynucleotide containing the complete ORF including said selected prey polynucleotide;

c) a polynucleotide which is any polynucleotide fragment comprised in the complete ORF including said selected prey polynucleotide.

5. The method according to claim 1, wherein the mating step is performed on a filter.

6. The method according to claim 5, wherein the mating step is performed during a maximum of 5 hours.

7. The method according to claim 5, wherein the efficiency value of the mating step is up to 50%.

8. The method according to claim 1, wherein the detectable gene is LacZ, HIS3 or both LacZ and HIS3.

9. The method according to claim 1, wherein the prey polynucleotide is contained within the plasmid pACTIIst.

10. The method according to claim 1, wherein the bait polynucleotide is contained within the plasmid pAS2Δ

Δ

.

11. The method according to claim 1, wherein the prey polynucleotide is provided by a DNA library.

12. The method according to claim 11, wherein the DNA library has been prepared from the genome or from the mRNA of a prokaryotic host organism.

13. The method according to claim 11, wherein the DNA library has been prepared from the genome or from the mRNA of a eukaryotic host organism.

14. The method according to claim 13, wherein the DNA library has been prepared from the genomic DNA of Saccharomyces cerevisiae.

15. The method according to claim 14 wherein the DNA library is the FRYL library contained in the collection of recombinant E. coli strain MR32 cells that have been deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.) on Dec. 21, 1995 under the access number I-1651.

16. The method according to claim 1, wherein the prey polynucleotide selected at step b) is classified within one of the following prey polynucleotide classes:

a) a polynucleotide contained in an intergenic region or on the reverse orientation of an ORF contained in the genome of the organism from which the initial DNA library has been prepared (B).

b) a polynucleotide selected several times and contained in different clones of the initial DNA library (A1);

c) a polynucleotide selected only once and having a 5′

end of the coding strand starting close to an initiation codon of an ORF contained in the genome of the organism from which the initial DNA library has been prepared (A2);

d) a polynucleotide selected only once and having a nucleotide length of at least 1000 bases (A3);

e) a polynucleotide selected only once and having characteristics different from the polynucleotides of the a), b), c) and d) classes (A4).

26. A recombinant diploid yeast cell selected by the method according to claim 1.

27. A polynucleotide that has been selected with the method according claim 1.

28. A polypeptide that is encoded by a polynucleotide according to claim 27.

31. A computer useable medium containing computer readable data related to the interactions between at least one bait polypeptide and at least one prey polypeptide encoded at least in part by a prey polynucleotide that has been selected with the method according to claim 1.

17. The recombinant plasmid pACTIIst.

18. The recombinant plasmid pAS2Δ

Δ

.

19. A recombinant host organism containing the recombinant plasmid pACTIIst.

21. The recombinant host organism according to claim 19 or 20, which is chosen among the group consisting in a E. coli and a yeast cell.

22. A collection of recombinant cell clones from a host organism according to claim 19, each different cell clone containing an inserted polynucleotide from a DNA library.

23. The collection of recombinant cell clones according to claim 22, wherein the DNA library is prepared from the genomic DNA of Saccharomyces cerevisiae.

24. The collection of recombinant cell clones according to claim 23, wherein the number of different cell clones is about 15×

10⁶.

25. The collection of recombinant cell clones according to claim 24, which is the FRYL library contained in the collection of recombinant E. coli strain MR32 cells that have been deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.) on Dec. 21, 1995 under the access number I-1651.

20. A recombinant host organism containing the recombinant plasmid pAS2Δ

Δ

.

29. A kit for selecting at least one polypeptide of interest belonging to Saccharomyces cerevisiae, wherein said kit comprises:

a) at least one complete collection of yeast cell clones containing the whole polynucleotide inserts representative of the genome from a prokaryotic or eukaryotic organism having a compact genome;

b) optionally the plasmid pAS2Δ

Δ

;

c) optionally the plasmid pACTIIst;

d) optionally, a haploid yeast cell to be transformed with a plasmid containing a bait polynucleotide of interest; and

e) optionally, the reagents necessary to visualize the expression of at least one detectable gene, such as X-Gal.

30. A kit according to claim 29, wherein the genomic DNA library is the FRYL library.