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 6,187,535 B1
• Filed: 02/18/1998
• Issued: 02/13/2001
• Est. Priority Date: 02/18/1998
• Status: Expired due to Term

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Rain, Jean-Christophe, Fromont, Micheline, LeGrain, Pierre
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/025,151
• Time in Patent Office: 1,091 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 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) 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 the polynucleotide encoding the bait polypeptide to a two-hybrid screening method, wherein said two-hybrid screening method comprises a step of mating for a maximum of 5 hours, without replica plating, said at least one first haploid recombinant yeast cell containing the bait polynucleotide with said second haploid recombinant yeast cell containing the prey polynucleotide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid recombinant yeast cell also contains at least one detectable gene that is activated by a polypeptide including a transcriptional activation domain;

(C) selecting the recombinant diploid yeast cell obtained at step (B) 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

(D) optionally characterizing the prey polynucleotide contained in each diploid yeast cell selected at step (C).

2. The method according to claim 1, further comprising repeating at least once steps (B) to (D) 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 (B) to (D) with the previously characterized prey polynucleotide used as the bait polynucleotide is in the range from 1 to 10.

4. The method according to claim 1, further comprising repeating at least once steps (B) to (D) 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.

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 efficiency value of the mating step is up to 50%.

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

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

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

Δ

.

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

11. The method according to claim 10, wherein the DNA library has been prepared from the genome or from the mRNA of a prokaryotic host organism.

12. The method according to claim 10, wherein the DNA library has been prepared from the genome or from the mRNA of a eukaryotic host organism.

13. The method according to claim 12, wherein the DNA library has been prepared from the genomic DNA of Saccharomyces cerevisiae.

14. The method according to claim 13, wherein the 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.

15. The method according to claim 1, wherein the 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 a), b), c), and d) (A4 category).

31. The method according to claim 1, wherein the mating step is performed on a filter for three to five hours.

16. The recombinant plasmid pACTIIst.

17. The recombinant plasmid pAS2Δ

Δ

.

18. A recombinant host organism comprising the recombinant plasmid pACTIIst.

19. A collection of recombinant cell clones from the recombinant host organism according to claim 18, wherein each recombinant cell clone comprises an inserted polynucleotide from a DNA library.

21. A collection of recombinant cell clones from a host organism according to claim 18, each different cell clone containing an inserted polynucleotide from a DNA library.

22. The collection of recombinant cell clones according to claim 19, wherein the DNA library is prepared from the genomic DNA of Saccharomyces cerevisiae.

23. The collection of recombinant cell clones according to claim 22, wherein the number of different cell clones is about 15×

10⁶.

24. The collection of recombinant cell clones according to claim 22, wherein the DNA library is the library contained in the collections of recombinant E. coli strain MR32 deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.) under the accession number I-1651.

26. The recombinant host organism according to claim 18 or 19, which is selected from the group consisting of E. coli and yeast.

20. A recombinant host organism comprising the recombinant plasmid pAS2Δ

Δ

.

25. 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 recombinant clonal yeast cells containing the whole polynucleotide inserts representative of the genome from a prokaryotic or eukaryotic organism having a genome containing at least 50% of coding sequences; and

b) the plasmid pAS2Δ

Δ

;

or

c) the plasmid pACTIIst; and

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.

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

a) a collection of recombinant cell clones comprising at least one complete yeast genomic DNA library, wherein recombinant cell clones comprise a polynucleotide insert, and wherein the collection of recombinant cell clones comprises polynucleotide inserts representative of the entire Saccharomyces cerevisiae genome; and

b) plasmid pAS2Δ

Δ

or plasmid pACTIIst.

28. A kit according to claim 27, further comprising a haploid yeast cell to be transformed with plasmid pAS2Δ

Δ

or plasmid pACTIIst, wherein the plasmid comprises a bait polynucleotide of interest.

29. A kit according to claim 27, further comprising reagents necessary to visualize the expression of at least one detectable gene.

30. A kit according to claim 27, wherein said yeast genomic 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.

32. 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 haploid recombinant yeast cell containing the polynucleotide encoding the bait polypeptide with a second haploid recombinant yeast cell containing the polynucleotide encoding the prey polypeptide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid 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 in 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 in step b);

wherein the polynucleotide encoding the prey polypeptide is contained within the plasmid pACTIIst.

33. The method according to claim 32, further comprising repeating steps a) to c) with the previously characterized prey polynucleotide used as the bait polynucleotide in the range of from 1 to 3 times.

34. The method according to claim 32, wherein the mating step is performed on a filter for three to five hours, the detectable gene is LacZ, HIS3, or both LacZ and HIS3, and the recombinant diploid yeast cell comprises polynucleotides encoding a DNA binding domain and an activation domain of a transcriptional regulatory protein selected from the group consisting of bacterial LEXA protein and yeast GAL4 gene product.

35. The method according to claim 32, wherein the polynucleotide encoding the prey polypeptide is provided by a DNA 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.

36. 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 haploid recombinant yeast cell containing the polynucleotide encoding the bait polypeptide with a second haploid recombinant yeast cell containing the polynucleotide encoding the prey polypeptide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid 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 in 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 in step b);

wherein the polynucleotide encoding the bait polypeptide is contained within the plasmid pAS2Δ

Δ

.

37. The method according to claim 36, further comprising repeating steps a) or c) with the previously characterized prey polynucleotide used as the bait polynucleotide in the range of from 1 to 3 times.

38. The method according to claim 36, wherein the mating step is performed on a filter for three to five hours, the detectable gene is LacZ, HIS3, or both LacZ and HIS3, and the recombinant diploid yeast cell comprises polynucleotides encoding a DNA binding domain and an activation domain of a transcriptional regulatory protein selected from the group consisting of bacterial LEXA protein and yeast GAL4 gene product.

39. The method according to claim 36, wherein the polynucleotide encoding the prey polypeptide is provided by a DNA 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.

40. 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 haploid recombinant yeast cell containing the polynucleotide encoding the bait polypeptide with a second haploid recombinant yeast cell containing the polynucleotide encoding the prey polypeptide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid 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 in 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 in step b);

wherein the polynucleotide encoding the prey polypeptide is provided by a DNA 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.

41. The method according to claim 40, further comprising repeating steps a) to c) with the previously characterized prey polynucleotide used as the bait polynucleotide in the range of from 1 to 3 times.

42. The method according to claim 40, wherein the mating step is performed on a filter for three to five hours, the detectable gene is LacZ, HIS3, or both LacZ and HIS3, and the recombinant diploid yeast cell comprises polynucleotides encoding a DNA binding domain and an activation domain of a transcriptional regulatory protein selected from the group consisting of bacterial LEXA protein and yeast GAL4 gene product.

43. 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 haploid recombinant yeast cell containing the polynucleotide encoding the bait polypeptide with a second haploid recombinant yeast cell containing the polynucleotide encoding the prey polypeptide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid 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 in 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 in step b);

wherein the polynucleotide encoding the prey polypeptide is contained within the plasmid pACTIIst, and the polynucleotide encoding the bait polypeptide is contained within the plasmid pAS2Δ

Δ

.

44. The method according to claim 43, further comprising repeating steps a) to c) with the previously characterized prey polynucleotide used as the bait polynucleotide in the range of from 1 to 3 times.

45. The method according to claim 43, wherein the mating step is performed on a filter for a maximum of five hours, the detectable gene is LacZ, HIS3, or both LacZ and HIS3, and the recombinant diploid yeast cell comprises polynucleotides encoding a DNA binding domain and an activation domain of a transcriptional regulatory protein selected from the group consisting of bacterial LEXA protein and yeast GAL4 gene product.

46. 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) providing a suspension comprising 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 and a second population of yeast cells containing at least one haploid recombinant yeast cell containing a polynucleotide encoding said prey polypeptide;

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

(C) selecting the recombinant diploid yeast cell obtained in step (B) 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

(D) optionally characterizing the prey polynucleotide contained in each diploid yeast cell selected in step (C);

wherein the mating step is performed on a filter for three to five hours;

the detectable gene is LacZ, HIS3, or both LacZ and HIS3; and

the recombinant diploid yeast cell comprises polynucleotides encoding a DNA binding domain and an activation domain of a transcriptional regulatory protein selected from the group consisting of bacterial LEXA protein and yeast GAL4 gene product.

47. The method according to claim 46, further comprising repeating steps (B) to (D) with the previously characterized prey polynucleotide used as the bait polynucleotide in the range of from 1 to 3 times.

48. The method according to claim 46, wherein the prey polynucleotide encoding the prey polypeplide is provided by a DNA library.

49. The method according to claim 48, wherein said DNA library is obtained by a process comprising cleaving a starting DNA material every four bases to form fragments of 200-1400 nucleotides, and forming a collection of recombinant yeast cell clones, each clone containing an inserted 200-1400 nucleotide fragment from said cleaving step.

50. The method according to claim 49, wherein the collection of recombinant yeast cell clones contains all of the starting DNA material.

51. The method according to claim 46, wherein the mating step is performed on a filter.