Traitement en cours

Veuillez attendre...

Paramétrages

Paramétrages

Aller à Demande

1. WO2020118198 - SYSTÈMES DE CRIBLAGE D'INTERACTIONS PROTÉINE-PROTÉINE

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

CLAIMS

WHAT IS CLAIMED IS:

1. A system for protein-protein interaction screening comprising:

a) a first nucleic acid encoding a bait polypeptide coupled to a first fragment of a protease polypeptide, a protease cleavage site, and a transcription regulating polypeptide;

b) a second nucleic acid encoding a prey polypeptide coupled to a second fragment of a protease polypeptide; and

c) a third nucleic acid comprising a reporter element, wherein said reporter element

comprises a regulatory element, a first reporter gene and a second reporter gene, wherein said regulatory element is configured to be bound by said transcription regulating polypeptide, wherein said second reporter gene encodes an RNA sequence that is unique to said prey polypeptide.

2. The system of claim 1, wherein said first fragment of a protease polypeptide and said second fragment of a protease polypeptide form an active protease when said bait polypeptide interacts with said prey polypeptide, whereupon said protease cleavage site is cleaved by said active protease, said transcription regulating polypeptide is released from said bait polypeptide, binds to said regulatory element, and initiates transcription of said first reporter gene and said second reporter gene.

3. The system of claim 1, wherein said first fragment of a protease polypeptide is a C-terminal fragment of a protease polypeptide and said second fragment of a protease polypeptide is an N-terminal fragment of a protease polypeptide.

4. The system of claim 1, wherein said first fragment of a protease polypeptide is a N-terminal fragment of a protease polypeptide and said second fragment of a protease polypeptide is a C- terminal fragment of a protease polypeptide.

5. The system of any one of claims 1 to 4, wherein said first fragment of a protease polypeptide and said second fragment of a protease polypeptide are derived from Tobacco Etch Virus nuclear -inclusion-a endopeptidase (TEV protease).

6. The system of any one of claims 1 to 5, wherein said transcription regulating polypeptide comprises a synthetic transcription factor.

7. The system of claim 6, wherein said synthetic transcription factor comprises a fusion of a Gal4 DNA binding domain and-VPR activation domain.

8. The system of claim 7, wherein said transcription regulating polypeptide comprises a transcriptional repressor.

9. The system of any one of claims 1 to 8, wherein said second nucleic acid encodes a second transcription regulating polypeptide coupled to said prey polypeptide, wherein said second transcription regulating polypeptide is configured to be cleaved by said active protease.

10. The system of any one of claims 1 to 9, wherein said bait polypeptide or said prey

polypeptide is a membrane bound signaling polypeptide.

11. The system of claim 10, wherein said membrane-bound bait polypeptide or said prey

polypeptide comprises a G protein coupled receptor, a receptor tyrosine kinase, or an ion channel.

12. The system of claim 11, wherein said membrane-bound signaling polypeptide or said prey polypeptide comprises a G protein coupled receptor.

13. The system of any one of claims 1 to 9, wherein said bait polypeptide or said prey

polypeptide is an intracellular signaling polypeptide.

14. The system of claim 13, wherein said intracellular bait polypeptide or said prey polypeptide comprises a nuclear hormone receptor.

15. The system of any one of claims 1 to 14, wherein said bait polypeptide or said prey

polypeptide comprises an intracellular molecule that potentially interacts with said bait polypeptide.

16. The system of any one of claims 1 to 14, wherein said regulatory element comprises an

inducible regulatory element.

17. The system of claim 16, wherein said inducible regulatory element comprises a Gal4

Upstream activation Sequence (Gal4 UAS).

18. The system of any one of claims 1 to 17, wherein said first reporter gene encodes a

fluorescent protein, a luciferase protein, a beta-galactosidase, a beta-glucuronidase, a chloramphenicol acetyltransferase, a secreted placental alkaline phosphatase.

19. The system of any one of claims 1 to 18, wherein said first reporter gene encodes a

fluorescent protein or a luciferase protein.

20. The system of any one of claims 1 to 19, wherein said first nucleic acid comprises a sequence encoding a promoter-less fluorescent protein.

21. The system of claim 20, wherein said fluorescent protein is a green fluorescent protein.

22. The system of any one of claims 1 wherein said first nucleic acid comprises a sequence that directs site specific integration into a genome.

23. The system of claim 22, wherein said first nucleic acid comprises an attB sequence that directs site specific integration into a genome.

24. The system of any one of claims 1 to 23, wherein said second nucleic acid comprises a

sequence encoding a selectable surface marker.

25. The system of claim 24, wherein said selectable surface marker is a hemagglutinin

polypeptide.

26. A cell comprising said first nucleic acid, said second nucleic acid, and/or said third nucleic acid of any one of claims 1 to 25.

27. The cell of claim 26, wherein said first nucleic acid, said second nucleic acid, and/or said third nucleic acid are integrated at an integration site for a transposable element.

28. The cell of claim 26, wherein said first nucleic acid, said second nucleic acid, and/or said third nucleic acid are integrated at a predetermined genomic location.

29. A method for testing a test substances effect on a protein-protein interaction comprising contacting a cell or a populations of cells according to any one of claims 26 to 28 to said test substance.

30. The method of claim 29, wherein the test substance is a chemical.

31. A system for protein-protein interaction screening comprising:

a) a first nucleic acid encoding a bait polypeptide coupled to a first fragment of a ubiquitin polypeptide;

b) a second nucleic acid encoding a prey polypeptide coupled to a second fragment of a ubiquitin polypeptide and a transcription regulating polypeptide; and

c) a third nucleic acid comprising a reporter element, wherein said reporter element

comprises a regulatory element, a first reporter gene and a second reporter gene, wherein said regulatory element is configured to be bound by said transcription regulating polypeptide, wherein said second reporter gene encodes an RNA sequence that is unique to said prey polypeptide.

32. The system of claim 31, wherein said first fragment of a ubiquitin polypeptide and said second fragment of a ubiquitin polypeptide form a cleavable ubiquitin molecule when said bait polypeptide interacts with said prey polypeptide, whereupon said cleavable ubiquitin

molecule is cleaved by a deubiquitinating enzyme, said transcription regulating polypeptide is released from said bait polypeptide, and initiates transcription of said first reporter gene and said second reporter gene.

33. The system of claim 31, wherein said first fragment of a ubiquitin polypeptide is a C-terminal fragment of a ubiquitin polypeptide and said second fragment of a ubiquitin polypeptide is an N-terminal fragment of a ubiquitin polypeptide.

34. The system of claim 31, wherein said first fragment of a ubiquitin polypeptide is an N- terminal fragment of a ubiquitin polypeptide and said second fragment of a ubiquitin polypeptide is a C-terminal fragment of a ubiquitin polypeptide.

35. The system of any one of claims 31 to 34, wherein said transcription regulating polypeptide comprises a synthetic transcription factor.

36. The system of claim 35, wherein said synthetic transcription factor comprises a fusion of a Gal4 DNA binding domain and-VPR activation domain.

37. The system of any one of claims 31 to 34, wherein said transcription regulating polypeptide comprises a transcriptional repressor.

38. The system of any one of claims 31 to 37, wherein said bait polypeptide or said prey

polypeptide is a membrane bound signaling polypeptide.

39. The system of claim 38, wherein said membrane-bound signaling polypeptide comprises a G protein-coupled receptor, a receptor tyrosine kinase, or an ion channel.

40. The system of claim 39, wherein said membrane-bound signaling polypeptide comprises a G protein-coupled receptor.

41. The system of any one of claims 31 to 36, wherein said bait polypeptide or said prey

polypeptide is an intracellular signaling polypeptide.

42. The system of claim 41, wherein said intracellular bait polypeptide or said prey polypeptide comprises a nuclear hormone receptor.

43. The system of any one of claims 31 to 42, wherein said bait polypeptide or said prey

polypeptide comprises an intracellular molecule that potentially interacts with said signaling polypeptide.

44. The system of any one of claims 31 to 43, wherein said regulatory element comprises an inducible regulatory element.

45. The system of claim 44, wherein said inducible regulatory element comprises a Gal4

Upstream activation Sequence (Gal4 UAS).

46. The system of any one of claims 31 to 45, wherein said first reporter gene encodes a

fluorescent protein, a luciferase protein, a beta-galactosidase, a beta-glucuronidase, a chloramphenicol acetyltransferase, or a secreted placental alkaline phosphatase.

47. The system of any one of claims 31 to 45, wherein said first reporter gene encodes a

fluorescent protein or a luciferase protein.

48. The system of any one of claims 31 to 47, wherein said first nucleic acid comprises a

sequence encoding a promoterless fluorescent protein.

49. The system of claim 48, wherein said fluorescent protein is a green fluorescent protein.

50. The system of any one of claims 31 to 49, wherein said first nucleic acid comprises a

sequence that directs site specific integration into a genome.

51. The system of claim 50, wherein said first nucleic acid comprises an attB sequence that

directs site specific integration into a genome.

52. The system of any one of claims 31 to 51, wherein said second nucleic acid comprises a

sequence encoding a selectable surface marker.

53. The system of claim 52, wherein said selectable surface marker is a hemagglutinin

polypeptide.

54. A cell comprising said first nucleic acid, said second nucleic acid, or said third nucleic acid of any one of claims 31 to 53.

55. The cell of claim 54, wherein said first nucleic acid, said second nucleic acid, or said third nucleic acid are integrated at an integration site for a transposable element.

56. The cell of claim 54, wherein said first nucleic acid, said second nucleic acid, or said third nucleic acid are integrated at a predetermined genomic location.

57. The cell of claim 54, wherein said first nucleic acid, said second nucleic acid, or said third nucleic acid are integrated as a single copy at a genomic location.

58. A method for testing a test substances effect on a protein-protein interaction comprising

contacting a cell or a populations of cells according to any one of claims 54 to 57 to said test substance.

59. The method of claim 58, wherein the test substance is a chemical.