Use of templated self assembly to create novel multifunctional species

• US 20050064509A1
• Filed: 09/23/2003
• Published: 03/24/2005
• Est. Priority Date: 09/23/2003
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Waldo, Geoff, Pavlik, Peter, Bradbury, Andrew
• Application Number:
  US10/670,167
• Publication Number:
  US 20050064509A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides self-assembling bifunctional polypeptides, kits comprising the self assembling bifunctional polypeptides, methods for assembling the bifunctional polypeptides, and methods for screening for the presence of an antigen using the bifunctional polypeptides.

Claims

1. A bifunctional polypeptide comprising a binding ligand linked to a first member of a coil-coil binding pair and a reporter molecule linked to the second member of a coil-coil binding pair, wherein binding between the first coil domain and the second coil domain joins the binding ligand to the reporter molecule.

2. The bifunctional polypeptide of claim 1, wherein the coil-coil binding pair is an E coil and a K coil.

3. The bifunctional polypeptide of claim 1, wherein the coil-coil binding pair is an A coil and a B coil.

4. The bifunctional polypeptide of claim 1, wherein the first and the second members of the coil-coil binding pair are each at least 35 amino acids in length.

5. The bifunctional polypeptide of claim 1, wherein the binding ligand is an antibody selected from the group consisting of an scFv, an Fab fragment, an isolated V_H, and an isolate V_L.

6. The bifunctional polypeptide of claim 1, wherein the binding ligand is a peptide.

7. The bifunctional polypeptide of claim 1, wherein the binding ligand is a receptor.

8. The bifunctional polypeptide of claim 1, wherein the reporter molecule is a fluorescent protein or chromophoric protein.

9. The bifunctional polypeptide of claim 8, wherein the fluorescent protein is green fluorescent protein.

10. The bifunctional polypeptide of claim 8, wherein the fluorescent protein is red fluorescent protein.

11. The bifunctional polypeptide of claim 1, wherein the reporter molecule is a fluorescent dye.

12. The bifunctional polypeptide of claim 1, wherein the reporter molecule is an enzyme.

13. The bifunctional polypeptide of claim 12, wherein the enzyme is horseradish peroxidase.

14. The bifunctional polypeptide of claim 12, wherein the enzyme is alkaline phosphatase.

15. The bifunctional polypeptide of claim 1, wherein the reporter molecule is a biotin binding protein.

16. The bifunctional polypeptide of claim 1, wherein the reporter molecule has luminescent activity.

17. The bifunctional polypeptide of claim 16, wherein the reporter molecule is luciferase.

18. A multifunctional polypeptide comprising:

a first member of a coil-coil binding pair linked to a binding ligand; and

a second member of the coil-coil binding pair linked to a polypeptide that undergoes spontaneous multimerization to form a self-assembled complex;

wherein binding between the first member of the coil-coil binding pair and the second member of the coil-coil pair joins the binding ligand and the self-assembled complex.

19. The multifunctional polypeptide of claim 18, further comprising a reporter molecule that is individually linked to a first member of the coil-coil binding pair, wherein the coil-coil binding interaction joins the reporter molecule to the multifunctional polypeptide.

20. The multifunctional polypeptide of claim 18, further comprising a second binding ligand that is individually linked to a first member of a coil-coil binding pair, wherein the second binding ligand binds to an epitope different from the first binding ligand, and wherein the coil-coil binding interaction joins the self-assembled complex to the first and second binding ligands.

21. The multifunctional polypeptide of claim 18, further comprising a second polypeptide individually linked to a second member of the coil-coil binding pair, wherein the second polypeptide undergoes spontaneous multimerization with the first polypeptide to form the self-assembled complex, and wherein the coil-coil binding interaction joins the self-assembled complex to the binding ligand.

22. The multifunctional polypeptide of claim 18, wherein the polypeptide is a soluble ferritin subunit.

23. The multifunctional polypeptide of claim 18, wherein the polypeptide is a viral coat protein.

24. The multifunctional polypeptide of claim 18, wherein the coil-coil binding pair is an E coil and a K coil.

25. The multifunctional polypeptide of claim 18, wherein the coil-coil binding pair is an A coil and a B coil.

26. The multifunctional polypeptide of claim 18, wherein the first and the second members of the coil-coil binding pair are each at least 35 amino acids in length.

27. The multifunctional polypeptide of claim 18, wherein the binding ligand is an antibody selected from the group consisting of a single chain Fv, an Fab, an isolated V_H, and an isolated V_L.

28. The multifunctional polypeptide of claim 18, wherein the binding ligand is a peptide.

29. The multifunctional polypeptide of claim 18, wherein the binding ligand is a fluorobody.

30. The multifunctional polypeptide of claim 18, wherein the binding ligand is a receptor.

31. The multifunctional polypeptide of claim 18, wherein the reporter molecule is a fluorescent protein.

32. The multifunctional polypeptide of claim 18, wherein the fluorescent protein is green fluorescent protein.

33. The multifunctional polypeptide of claim 18, wherein the fluorescent protein is red fluorescent protein.

34. The multifunctional polypeptide of claim 18, wherein the reporter molecule is an enzyme.

35. The multifunctional polypeptide of claim 18, wherein the reporter molecule is a biotin binding protein.

36. The multifunctional polypeptide of claim 18, wherein the reporter molecule is luciferase.

37. A kit comprising:

a binding ligand linked to a first member of a coil-coil binding pair; and

a reporter molecule linked to a second member of a coil-coil binding pair.

38. The kit of claim 37, wherein the binding ligand is an antibody.

39. The kit of claim 37, wherein the binding ligand is a peptide.

40. The kit of claim 37, wherein the binding ligand is a fluorobody.

41. The kit of claim 37, wherein the reporter molecule is a fluorescent protein.

42. The kit of claim 37, wherein the reporter molecule is an enzyme.

43. The kit of claim 37, wherein the reporter molecule is a biotin binding protein.

44. The kit of claim 37, wherein the reporter molecule is luciferase.

45. A kit comprising:

a first subunit that is a first member of a coil-coil binding pair linked to a binding ligand; and

a second subunit that is a second member of the coil-coil binding pair linked to a polypeptide that undergoes spontaneous multimerization.

46. The kit of claim 45, further comprising a third subunit that is a first member of the coil-coil binding pair linked to a reporter molecule.

47. The kit of claim 45, wherein the polypeptide that undergoes spontaneous multimerization is soluble ferritin.

48. The kit of claim 45, wherein the polypeptide that undergoes spontaneous multimerization is a viral coat protein.

49. The kit of claim 45, wherein the binding ligand is an antibody selected from the group consisting of an scFV, an Fab, a V_H region and a V_L region.

50. The kit of claim 45, wherein the binding ligand is a fluorobody.

51. The kit of claim 45, wherein the binding ligand is a peptide.

52. The kit of claim 45, wherein the binding ligand is a receptor.

53. The kit of claim 45, wherein the reporter molecule is a polypeptide selected from the group consisting of a fluorescent protein and an enzyme.

54. The kit of claim 53, wherein the reporter molecule is a fluorescent protein or an enzyme.

55. A method of making a multifunctional polypeptide, the method comprising:

providing a binding ligand linked to a first member of a coil-coil binding pair;

providing a molecule linked to a second member of a coil-coil binding pair, wherein the molecule is a reporter molecule or a spontaneously multimerizing polypeptide; and

incubating the binding pair under conditions in which the first binding pair member specifically binds to the second binding pair member, thereby assembling the bifunctional polypeptide.

56. A method of screening for the presence of an antigen, the method comprising:

incubating a sample comprising the antigen with a bifunctional polypeptide comprising a binding ligand linked to a first member of a coil-coil binding pair and a reporter molecule linked to the second member of the coil-coil binding pair, wherein the binding ligand is joined to the reporter polypeptide by the binding interaction between the binding pair members;

wherein the antigen and the bifunctional polypeptide are incubated under conditions in which the antigen specifically binds to the binding ligand; and

detecting activity of the reporter molecule, thereby detecting the presence of the antigen.

57. A method of screening for the presence of an antigen, the method comprising:

incubating a sample comprising the antigen with a binding ligand linked to a first member of a coil-coil binding pair under conditions in which the antigen specifically binds to the binding ligand and subsequently incubating the sample with a reporter molecule linked to the second member of the coil-coil binding pair, wherein the binding ligand becomes joined to the reporter molecule by the binding interaction between the binding pair members; and

detecting activity of the reporter polypeptide, thereby detecting the presence of the antigen.

58. A method of screening for the presence of an antigen, the method comprising:

(a) incubating a sample comprising the antigen with a bifunctional polypeptide comprising;

(i) a binding ligand linked to a first member of a coil-coil binding pair; and

(ii) a polypeptide that undergoes spontaneous multimerization linked to the second member of a coil-coil binding pair, wherein binding between the first coil domain and the second coil domain joins the binding ligand and the spontaneously multimerizing polypeptide,

wherein the antigen and the bifunctional polypeptide are incubated under conditions in which the antigen specifically binds to the binding ligand; and

(b) detecting the presence of the bifunctional polypeptide, thereby detecting the presence of the antigen.

59. A method of screening for the presence of an antigen, the method comprising:

incubating a sample comprising the antigen with a binding ligand linked to a first member of a coil-coil binding pair under conditions in which the antigen specifically binds to the binding ligand and subsequently incubating the sample with a second polypeptide linked to a second member of the coil-coil binding pair, wherein the second polypeptide undergoes spontaneous multimerization;

wherein the binding ligand is joined to the second polypeptide by the binding interaction between the coil-coil binding pair members to form a multifunctional polypeptide; and

detecting the presence of the multifunctional polypeptide, thereby detecting the presence of the antigen.