Methods for protein interaction determination

• US 20100216649A1
• Filed: 01/25/2008
• Published: 08/26/2010
• Est. Priority Date: 05/09/2003
• Status: Abandoned Application

• Current Assignee: Alex R. Hastie, Lawrence M. Mielnicki, Steven C. Pruitt
• Original Assignee: Alex R. Hastie, Lawrence M. Mielnicki, Steven C. Pruitt
• Inventors: Hastie, Alex R., Pruitt, Steven C., Mielnicki, Lawrence M.
• Application Number:
  US12/011,234
• Publication Number:
  US 20100216649A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection

Abstract

Provided are compositions and methods for identifying pairs of interacting proteins. The pair of plasmids is adapted for use in a modified two hybrid system wherein each plasmid comprises a recombinase recognition site. The method comprises the steps of providing cDNAs encoding test polypeptides, inserting the cDNAs into the first and second plasmids, recombining the first and second plasmids to obtain recombined plasmids, isolating and digesting the recombined plasmids to obtain cDNAs encoding pairs of interacting proteins, and determining the sequence of the digested fragments to determine pairs of interacting proteins.

Claims

1. A method for identifying a plurality of pairs of interacting proteins wherein a pair of interacting proteins comprises a first test protein and a second test protein, wherein the first and second test proteins interact with each other in a cell, the method comprising the steps of:

a) providing a cDNA library;

b) providing a plurality of a first plasmid comprising a coding sequence for a

DNA binding domain of a transcription activator, a first recombinase recognition site, a first selectable marker, a first Type II S restriction site and a first inserted cDNA encoding a first test protein;

c) providing a plurality of a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, a second recombinase recognition site, a second selectable marker and a second Type II S restriction site, and a second inserted cDNA encoding a second test protein, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct;

d) introducing the first and second plasmids from b) and c) into the same cell;

e) inducing the expression of the recombinase to recombine the first and second introduced plasmids;

f) isolating and digesting the recombined plasmids with a Type II S restriction enzyme to obtain a plurality of restriction fragments, and

g) determining the sequence of the plurality of restriction fragments to determine the identity of the plurality of pairs of interacting proteins.

2. The method of claim 1, wherein the first and second inserted cDNAs of steps

b) and c) are inserted by homologously recombining the first and second cDNAs with the first and second plasmids, respectively.

3. The method of claim 1, wherein the Type II S restriction site is selected from the group consisting of BsgI, BpmI, and MmeI sites.

4. The method of claim 1, wherein the recombinase recognition sites are half mutant sites.

5. The method of claim 1 wherein step d) comprises introducing the first and second plasmids into the same cell by mating a first and second yeast cell, wherein the first yeast cell has been transformed with either the first or second plasmid, and wherein the second yeast cell has been transformed with the first or second plasmid with which the first yeast cell was not transformed.

6. The method of claim 1, wherein in step e) the cell into which the first and second plasmids are introduced is selected for by interaction of proteins encoded by the first and second cDNAs, wherein the interaction induces expression of a selectable marker, wherein expression of the selectable marker permits the cell to survive or to be distinguished from cells not expressing the selectable marker.

7. The method of claim 1, wherein step d) is performed by massively parallel pyrosequencing.

8. A plasmid comprising a recombinase recognition site, a cloning site for cloning a cDNA into the plasmid, at least one selectable marker, a Type II S restriction site, and a coding sequence, wherein the coding sequence is selected from the group consisting of:

a) a coding sequence for a DNA binding domain of a transcription activator such that the DNA binding domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the cDNA; and

b) a coding sequence for a transcription activation domain of a transcription activator such that the DNA transcription activation domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the cDNA.

9. The plasmid of claim 8, wherein the recombinase recognition site is recognized by a recombinase selected from the group consisting of Cre recombinase, tamoxefin inducible Cre recombinase, and FLP recombinase.

10. The plasmid of claim 8, wherein the transcription activator is Gal4.

11. The plasmid of claim 8, wherein the Type II S restriction site is selected from the group consisting of BsgI, BpmI, and MmeI sites.

12. The plasmid of claim 8, wherein the recombinase recognition sites are half mutant sites.

13. The recombinase recognition sites of claim 12, wherein the sites are selected from lox71 and lox66 sites.

14. The plasmid of claim 8, wherein the selectable marker is selected from the group consisting of LEU2, URA3, HIS3, TRP1, ADE2 and LYS2.

15. A kit for determining interacting proteins, wherein the kit comprises:

a) a first plasmid comprising a coding sequence for a DNA binding domain of a transcription activator, a cloning site for cloning a first cDNA into the first plasmid such that the DNA binding domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the first cDNA, a first recombinase recognition site, a first selectable marker, and a first Type II S restriction site; and

b) a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, the cloning site for cloning a second cDNA into the second plasmid such that the transcription activation domain of the transcription activator can be expressed as a fusion protein with the protein encoded by the second cDNA, a second recombinase recognition site, a second selectable marker, and a second Type II S restriction site, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct.

16. The first and second plasmids of claim 15, wherein the first and second recombinase recognition sites are recognized by a recombinase selected from the group consisting of Cre recombinase, tamoxefin inducible Cre recombinase, and FLP recombinase.

17. The first and second plasmids of claim 15, wherein the transcription activator is Gal4.

18. The first and second plasmids of claim 16, wherein the first and second Type II S restriction enzymes are selected from the group consisting of BsgI, BpmI, and MmeI.

19. The first and second plasmids of claim 16, wherein the first and second recombinase recognition sites are half mutant sites.

20. The first and second plasmids of claim 16, wherein the first plasmid has either the first recombinase recognition site lox71 or lox66, and wherein the second plasmid has a second recombinase recognition site selected from lox71 or lox66, wherein the second recombinase recognition site is the site the first plasmid does not have.