Methods and assays for screening protein targets

• US 7,419,780 B2
• Filed: 11/10/2003
• Issued: 09/02/2008
• Est. Priority Date: 01/24/2000
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Cornish, Virginia W.
• Primary Examiner(s): Robinson; Hope A
• Application Number:
  US10/705,644
• Publication Number:
  US 20040106154A1
• Time in Patent Office: 1,758 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 49/0006 Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
• CPC Class Codes: C07J 41/0066 the 17-beta position being substituted by a carbon atom forming part of an amide group
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 5/06139 with the first amino acid being heterocyclic
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

A method for identifying a protein as being able to bind a ligand comprising, providing a molecule composed of a methotrexate moiety that is covalently bonded to the ligand; introducing the molecule into a cell which a) expresses a first fusion protein comprising a dihydrofolate reductase capable of binding methotrexate, expresses b) a second fusion protein comprising the protein, wherein one of the first and second fusion proteins also comprises a transcription activator domain and the other comprises a DNA-binding domain, and c) has a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein; permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and selecting the cell if it expresses the reporter gene.

Claims

1. A method for identifying a protein target as being able to bind a ligand, comprising:

(a) providing a molecule comprising a methotrexate moiety covalently linked to the ligand, which methotrexate moiety binds to a dihydrofolate reductase;

(b) introducing the molecule into a cell which i) expresses a first fusion protein comprising the dihydrofolate reductase that binds to the methotrexate moiety, ii) expresses a second fusion protein comprising the protein target, wherein either the first or second fusion protein also comprises a transcription activator domain and the other fusion protein comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein and wherein the DNA-binding domain binds upstream of the reporter gene, and wherein the cell is a yeast cell;

(c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and

(d) selecting the cell if it expresses the reporter gene, so as to thereby identify the protein target as being able to bind the ligand.

2. The method of claim 1, wherein the protein target is encoded by genomic DNA or a cDNA.

3. The method of claim 1, wherein the first fusion protein is (dihydrofolate reductase)-(DNA-binding domain).

4. The method of claim 1, wherein the first fusion protein is (dihydrofolate reductase)-(LexA).

5. The method of claim 1, wherein the first fusion protein is (dihydrofolate reductase)-(transcription activation domain).

6. The method of claim 1, wherein the first fusion protein is (dihydrofolate reductase)-(B42).

7. The method of claim 1, wherein the second fusion protein comprises a DNA-binding domain.

8. The method of claim 1, wherein the second fusion protein comprises LexA.

9. The method of claim 1, wherein the second fusion protein comprises a transcription activation domain.

10. The method of claim 1, wherein the second fusion protein comprises B42.

11. The method of claim 1, wherein the cell is Sacchromyces cerevisiae.

12. The method of claim 1, wherein the reporter gene is lacZ, Gal4 or Ura-3.

13. The method of claim 1, wherein the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is LacZ.

14. The method of claim 1, wherein the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is Gal4.

15. A method for identifying a protein target as being able to bind a ligand, comprising:

(a) providing a molecule comprising a methotrexate moiety covalently linked to the ligand, which methotrexate moiety binds to a dihydrofolate reductase;

(b) introducing the molecule into a cell which i) expresses a first fusion protein comprising the dihydrofolate reductase that binds to the methotrexate moiety, ii) expresses a second fusion protein comprising the protein target, wherein either the first or second fusion protein also comprises a transcription activator domain and the other fusion protein comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein and wherein the DNA-binding domain binds upstream of the reporter gene, and wherein the cell is a bacterial cell;

(c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and

(d) selecting the cell if it expresses the reporter gene, so as to thereby identify the protein target as being able to bind the ligand.

16. The method of claim 15, wherein the protein target is encoded by genomic DNA or a cDNA.

17. The method of claim 15, wherein the first fusion protein is (dihydrofolate reductase)-(DNA-binding domain).

18. The method of claim 15, wherein the first fusion protein is (dihydrofolate reductase)-(LexA).

19. The method of claim 15, wherein the first fusion protein is (dihydrofolate reductase)-(transcription activation domain).

20. The method of claim 15, wherein the first fusion protein is (dihydrofolate reductase)-(42).

21. The method of claim 15, wherein the second fusion protein comprises a DNA-binding domain.

22. The method of claim 15, wherein the second fusion protein comprises LexA.

23. The method of claim 15, wherein the second fusion protein comprises a transcription activation domain.

24. The method of claim 15, wherein the second fusion protein comprises B42.

25. The method of claim 15, wherein the cell is Escherichia coli.

26. The method of claim 15, wherein the reporter gene is lacZ, Gal4 or Ura-3.

27. The method of claim 15, wherein the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is LacZ.

28. The method of claim 15, wherein the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is Gal4.