High throughput assay using fusion proteins

• US 5,783,398 A
• Filed: 09/04/1996
• Issued: 07/21/1998
• Est. Priority Date: 09/15/1995
• Status: Expired due to Fees

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Merck & Co., Inc.
• Inventors: Marcy, Alice, Wisniewski, Douglas, Salowe, Scott P.
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): Lathrop, Brian
• Application Number:
  US08/707,792
• Time in Patent Office: 685 Days
• Field of Search: 435/7.1, 435/7.5, 435/7.72, 435/7.8, 435/69.7, 435/71.2, 435/320.1, 435/849, 435/969, 435/973, 530/324, 530/344, 530/350, 530/415, 530/812, 530/816, 530/827, 536/23.4, 536/24.1, 930/280
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/9446 Antibacterials
• CPC Class Codes: G01N 33/9493 Immunosupressants

Abstract

This application describes a high throughput assay for screening for compounds capable of binding to a fusion protein which consists of a target protein and an FK506-binding protein. The method for preparing the DNA encoding for the fusion protein and for expressing that DNA is also described in the application. The invention also discloses the recombinant DNA and protein sequences for several fusion proteins.

Claims

1. A method of screening for compounds capable of binding to a fusion protein, which is defined as an FK506-binding protein linked to a target protein through a peptide linker, which -comprises the steps of:

a) mixing a test compound, a tagged- ligand for the target protein, which is selected from a biotinylated ligand or an epitope-tagged ligand, the fusion protein, a radiolabeled ligand selected from ³ H!- or ¹²⁵ I!-labeled F506 analog and a coated microscintillation plate selected from a streptavidin-coated or anti-antibody coated or protein A-coated microscintillation plates;

b) incubating the mixture from between about 1 hour to about 24 hours;

c) measuring the plate-bound counts attributable to the binding of the tagged ligand to, the fusion protein in the presence of the test compound using scintillation counting; and

d) determining the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay run in the absence of the test compound.

2. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 1, wherein the target protein comprises a single or multiple signal transduction domain.

3. The method for screening for compounds capable of binding to a fusion protein, as recited in claim 2, wherein the single or multiple signal transduction domain is selected from the group consisting of:

SH1, SH2, SH3 and PH domains.

4. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 3, wherein the target protein is a single or multiple SH2 domain.

5. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 4, wherein the radiolabeled ligand is ³ H!-dihydroFK506.

6. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 5, wherein the FK506-binding protein is a 12 kDA human FK506-binding protein.

7. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 6, wherein the target protein is a single or multiple SH2 domain selected from the group consisting of:

ZAP;

SH2, SYK;

SH2 and LCK;

SH2.

8. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 7, wherein the target protein is the SH2 domain, ZAP:

SH2.

9. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 7, wherein the target protein is the SH2 domain, SYK:

SH2.

10. The method of screening for compounds capable of binding to a fusion protein, as recited in claim 7, wherein the target protein is the SH2 domain, LCK:

SH2.

11. A process for preparing a recombinant DNA expression vector encoding for a fusion protein, which is defied as an FK506-binding protein linked to a target protein through a peptide linker, comprising the steps of:

a) removing the stop codon on DNA encoding for the FK506-binding protein;

b) synthesizing a modified DNA fragment on the DNA encoding for the FK506-binding protein which encodes for a peptide linker;

c) digesting an expression vector at cloning sites;

d) cloning the modified DNA fragment encoding for the FK506-binding protein with a peptide linker into the digested expression vector to generate a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker; and

e) cloning DNA -encoding for a target protein into a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker to produce the recombinant DNA expression vector encoding for the fusion protein.

12. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 11, wherein the target protein is a single or multiple signal transduction domain.

13. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 12, wherein the single or multiple signal transduction domain is selected from the group consisting of:

SH1, SH2, SH3 and PH domains.

14. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 13, wherein the single or multiple signal transduction domain is an SH2 domain.

15. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 14, wherein the single or multiple signal transduction domain is an SH2 domain selected from the group consisting of ZAP:

SH2, SYK;

SH2 and LCK;

SH2.

16. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 15, wherein the FK506-binding protein is a 12 kDa FK506 binding protein.

17. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 16, wherein the target protein is ZAP:

SH2.

18. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 16, wherein the target protein is SYK:

SH2.

19. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in claim 16, wherein the target protein is LCK:

SH2.

20. Isolated DNA encoding for a fusion protein comprising the sequence:

(SEQ. ID. NO.

     1).

21. Isolated DNA encoding for a fusion protein comprising the sequence:

(SEQ. ID. NO.

     2).

22. Isolated DNA encoding for a fusion protein comprising the sequence:

(SEQ. ID. NO.

     3).

23. A FKBP-ZAP:

SH2 fusion protein comprising the sequence;

(SEQ. ID. NO.

     4).

24. A FKBP-SYK:

SH2 fusion protein comprising the sequence;

(SEQ. ID. NO.

     5).

25. A FKBP-LCK:

SH2 fusion protein comprising the sequence;

(SEQ. ID. NO.

     6).

26. A process for expressing recombinant DNA encoding for a fusion protein, which is defined as a-FK506-binding protein linked to a target protein through a peptide linker in an expression vector comprising the steps of;

a) transforming a host cell with the fusion protein expression vector;

b) inducing expression of the fusion protein in the host cell;

c) recovering the fusion protein from the host cell; and

d) purifying the fusion protein.

27. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 26, wherein the target protein is a single or multiple signal transduction domain.

28. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 27, wherein the single or multiple signal transduction domain is selected from the group consisting of:

SH1, SH2, SH3 and PH domains.

29. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 28, wherein the single or multiple signal transduction domain is a single or multiple SH2 domain.

30. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 29, wherein the single or multiple SH2 domain is selected from a group consisting of ZAP:

SH2, SYK;

SH2 and LCK;

SH2.

31. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 30, wherein the FK506-binding protein is human 12kDa FK506-binding protein.

32. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 31, wherein the host cell is from bacteria, yeast, blue green algae, plant cells, insect cells, or animal cells.

33. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 32, wherein the host cell is an E. coli strain selected from a group consisting of BL21 (DE3), Nova Blue (DE3), and JM109 (DE3).

34. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 33, wherein the single or multiple SH2 domain is ZAP:

SH2.

35. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 33, wherein the single or multiple SH2 domain is SYK:

SH2.

36. The process for expressing recombinant DNA encoding a fusion protein, as recited in claim 33, wherein the single or multiple SH2 domain is LCK:

SH2.

37. The process for purifying an isolated FKBP-SH2 fusion protein comprising the steps of:

a) preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support;

b) preparing a freeze/thaw extract from cells expressing the fusion protein;

c) loading the extract onto the affinity matrix and washing off unbound protein; and

d) eluting the desired fusion protein with phenyl phosphate.

38. A recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector, wherein the DNA encodes for the FKBP-ZAP:

SH2 fusion protein and has the DNA sequence (SEQ. ID. NO.1).

39. A recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector, wherein the DNA encodes for the FKBP-SYK:

SH2 fusion protein and has the DNA sequence (SEQ. ID. NO.

     2).

40. A recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector, wherein the DNA encodes for the FKBP-LCK:

SH2 fusion protein and has the DNA sequence (SEQ. ID. NO.3).

41. A recombinant host cell containing the recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector wherein the recombinant host cell is selected from the group consisting of:

E. coli BL21 (DE3), E. coli Nova Blue (DE3), and E. coli JM109 (DE3).

42. The recombinant host cell containing the recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector as recited in claim 41, wherein the recombinant host cell is E. coli BL21 (DE3).