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 6,531,284 B1
• Filed: 08/14/2000
• Issued: 03/11/2003
• Est. Priority Date: 02/18/1998
• Status: Expired due to Term

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Rain, Jean-Christophe, Legrain, Pierre, Fromont, Micheline
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/637,240
• Time in Patent Office: 939 Days
• Field of Search: 435/6, 435/29, 536/23.1, 536/23.4, 536/23.5
• US Class Current: 435/6.12
• 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 polypepetide as well as to 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 consisitng 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 interaction 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, said method comprising:

(A) providing a yeast cell mixture comprising a first population of yeast cells containing at least one first haploid recombinant yeast cell containing a polynucleotide encoding said bait polypeptide (bait polynucleotide) and a second population of yeast cells containing at least one second haploid recombinant yeast cell containing a polynucleotide encoding said prey polypeptide (prey polynucleotide);

(B) subjecting said polynucleotide encoding said bait polypeptide to a two-hybrid screening method, wherein said two-hybrid screening method comprises a step of mating, without replica plating, said at least one first haploid recombinant yeast cell containing said bait polynucleotide with said second haploid recombinant yeast cell containing said prey polynucleotide, provided that one haploid yeast cell among said first haploid recombinant yeast cell or said second haploid recombinant yeast cell also contains at least one detectable gene that is activated by a polypeptide including a transcriptional activation domain; and

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

2. The method according to claim 1, further comprising characterizing said prey polynucleotide contained in each diploid yeast cell selected at step (C).

3. The method according to claim 1, further comprising repeating at least once steps (B) and (C) with the previously characterized prey polynucleotide used as said bait polynucleotide.

4. The method according to claim 3, wherein the number of repeats of steps (B) and (C) with the previously characterized prey polynucleotide used as said bait polynucleotide is in the range from 1 to 10.

5. The method according to claim 1, further comprising repeating at least once steps (B) and (C) with a bait polynucleotide selected from the group consisting of:

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

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

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

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

7. The method according to claim 6, wherein the efficiency value of the mating step is up to 50%.

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

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

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

Δ

.

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

12. The method according to claim 11, wherein said 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 said 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 said DNA library has been prepared from the genome DNA of Saccharomyces cerevisiae.

15. The method according to claim 14, wherein said DNA library is the library contained in the collection of recombinant E. coli strain MR32 cells deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.) under the accession number I-1651.

16. The method according to claim 1, wherein said prey polynucleotide contained in the recombinant diploid yeast cell selected at step (C) is selected from the group consisting of:

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 category);

b) a polynucleotide contained in different clones of the initial DNA library (A1 category);

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 category);

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

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

17. A method of screening for a prey polypeptide that interacts with a bait polypeptide, said method comprising:

(A) providing a yeast cell mixture comprising a first population of yeast cells containing at least one first haploid recombinant yeast cell and a second population of yeast cells containing at least one second haploid recombinant yeast cell, wherein said least one first haploid recombinant yeast cell comprises a polynucleotide encoding said bait polypeptide (bait polynucleotide), and wherein said at least one second haploid recombinant yeast cell comprises a polynucleotide encoding said prey polypeptide (prey polynucleotide);

(B) mating, without replica plating, said at least one first haploid recombinant yeast cell with said at least one second haploid recombinant yeast cell to produce at least one recombinant diploid yeast cell, provided that said at least one first haploid yeast cell or said at least one second haploid recombinant yeast cell further comprises at least one detectable gene that is activated when said prey polypeptide and said bait polypeptide interact in said at least one recombinant diploid yeast cell; and

(C) selecting said at least one recombinant diploid yeast cell obtained at step (B) for which said at least one detectable gene has been expressed to a degree greater than expression in the absence of interaction between said bait polypeptide and said prey polypeptide.

18. The method according to claim 17 further comprising characterizing said prey polynucleotide contained in said at least one diploid yeast cell selected at step (C).

19. The method according to claim 17, wherein said bait polynucleotide comprises a first polynucleotide encoding a DNA binding domain that recognizes a binding site on said at least one detectable gene and a second polynucleotide, wherein said second polynucleotide is selected from the group consisting of:

a) a polynucleotide that was previously characterized as a prey polynucleotide in a two-hybrid system;

b) a polynucleotide having the complete open reading frame of a polynucleotide that was previously characterized as a prey polynucleotide in a two-hybrid system; and

c) a polynucleotide fragment of the polynucleotide identified in part b).

20. The method according to claim 19, wherein said prey polynucleotide is provided by a DNA library.

21. The method according to claim 20, wherein said DNA library has been prepared from the genome or from the mRNA of a prokaryotic host organism.

22. The method according to claim 20, wherein said DNA library has been prepared from the genome or from the mRNA of a eukaryotic host organism.

23. The method according to claim 22, wherein said DNA library has been prepared from the genome DNA of Saccharomyces cerevisiae.

24. The method according to claim 23, wherein said DNA library is the library contained in the collection of recombinant E. coli strain MR32 cells deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.) under the accession number I-1651.