Modified green fluorescent proteins

• US 5,777,079 A
• Filed: 11/20/1996
• Issued: 07/07/1998
• Est. Priority Date: 11/10/1994
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tsien, Roger Y., Heim, Roger
• Primary Examiner(s): Wax, Robert A..
• Assistant Examiner(s): Slobodyansky, Elizabeth
• Application Number:
  US08/753,143
• Time in Patent Office: 594 Days
• Field of Search: 435/91.1, 435/91.2, 435/172.1, 435/69.1, 435/69.7, 435/189, 530/350, 530/855
• US Class Current: 530/350
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: Y10S 530/855 Proteins from animals other than mammals or birds

Abstract

Modifications in the sequence of Aequorea wild-type GFP provide products having markedly different excitation and emission spectra from corresponding products from wild-type GFP. In one class of modifications, the product derived from the modified GFP exhibits an alteration in the ratio of two main excitation peaks observed with the product derived from wild-type GFP. In another class, the product derived from the modified GFP fluoresces at a shorter wavelength than the corresponding product from wild-type GFP. In yet another class of modifications, the product derived from the modified GFP exhibits only a single excitation peak and enhanced emission relative to the product derived from wild-type GFP.

Claims

1. A composition of matters comprising:

a fluorescent modified form of an Aequorea wild-type GPP polypeptide,

characterized in that upon oxidation and cyclization of amino acid residues in said fluorescent modified form corresponding to positions 65 to 67 of wild-type GFP polypeptide sequence (SEQ ID NO;

2) said fluorescent modified form exhibits a different excitation and/or emission spectrum from a corresponding product of said wild-type GFP polypeptide sequence,

with the proviso that when said fluorescent modified form comprises a mutation at S65, said mutation at S65 is selected from the group consisting of S65A, S65C, S65T, S65L, S65V, and S65I.

2. The composition according to claim 1,

wherein said fluorescent modified form exhibits and alteration in the ration of two main excitation peaks relative to said wild-type GFP polypeptide sequence.

3. The composition according to claim 2,

wherein said fluorescent modified form exhibits increased fluorescence at a shorter-wavelength peak of the two main excitation peaks than said wild-type GFP polypeptide sequence.

4. The composition according to claim 3,

wherein said fluorescent modified form comprises a replacement of Ser at a position corresponding to position 202 in said wild-type GFP polypeptide sequence by Phe and a replacement of Thr at a position corresponding to position 203 by Ile.

5. The composition according to claim 2,

wherein said fluorescent modified form exhibits increased fluorescence at a longer-wavelength peak of the two main excitation peaks than said wild-type GFP polypeptide sequence.

6. The composition according to claim 5,

wherein said fluorescent modified form comprises a replacement of Ile at a position corresponding to position 167 of said wild-type GFP polypeptide sequence by Val or Thr.

7. The composition according to claim 5,

wherein said fluorescent modified form comprises a replacement of Ser at a position corresponding to position 65 of said wild-type GFP sequence by Thr, a replacement of Met at position 153 with Ala, and a replacement of Lys at position 238 with Glu.

8. The composition according to claim 1,

wherein said fluorescent modified form fluoresces at a shorter wavelength than said wild-type GFP polypeptide sequence.

9. The composition according to claim 8,

wherein said fluorescent modified form comprises a replacement of Tyr at a position corresponding to position 66 of said wild-type GFP polypeptide sequence by Phe, His or Trp.

10. The composition according to claim 8,

wherein said fluorescent modified form comprises a replacement of Tyr at a position corresponding to position 66 of said wild-type GFP polypeptide sequence by His and a replacement of Tyr at position 145 with Phe.

11. The composition according to claim 8,

wherein said fluorescent modified form comprises a replacement of Tyr at a position corresponding to position 66 of said wild-type GFP polypeptide sequence by Trp, a replacement of Asn at position 146 by Ile, a replacement of Met at position 153 by Thr, a replacement of Val at position 163 by Ala, and a replacement of Asn at position 212 by Lys.

12. The composition according to claim 8,

wherein said fluorescent modified form comprises a replacement of Tyr at a position corresponding to position 66 of said wild-type GFP polypeptide sequence by Trp, a replacement of Ile at position 123 by Val, a replacement of Tyr at position 145 by His, a replacement of His at position 148 by Arg, a replacement of Met at position 153 by Thr, a replacement of Val at position 163 by Ala, and a replacement of Asn at position 212 by Lys.

13. The composition according to claim 1,

wherein said fluorescent modified form exhibits enhanced emission relative to said wild-type GFP polypeptide sequence.

14. The composition according to claim 13,

wherein said fluorescent modified comprises a replacement of Ser at a position corresponding to position 65 of said wild-type GFP polypeptide sequence by an amino acid selected from the group consisting of Ala, Cys, Thr, Leu, Val and Ile.

15. The composition according to claim 14,

wherein said amino acid is Cys of Thr.

16. A functional mutant fluorescent protein, comprising:

a protein with an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO;

2) by at least one amino acid substitution that is at position 65, wherein said at least one substitution is either S65A, S65C, S65T, S65L, S65V, or S651,

wherein said functional mutant fluorescent protein has an excitation or emission different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.

17. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65A.

18. The functional mutant fluorescent protein of claim 17,

wherein said functional mutant fluorescent protein comprises a fusion protein.

19. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65C.

20. The functional mutant fluorescent protein of claim 19,

wherein said functional mutant fluorescent protein comprises a fusion protein.

21. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65T.

22. The functional mutant fluorescent protein of claim 21,

wherein said functional mutant fluorescent protein comprises a fusion protein.

23. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65L.

24. The functional mutant fluorescent protein of claim 23,

wherein said functional mutant fluorescent protein comprises a fusion protein.

25. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65V.

26. The functional mutant fluorescent protein of claim 25,

wherein said functional mutant fluorescent protein comprises a fusion protein.

27. The functional mutant fluorescent protein of claim 16,

wherein said at least one amino acid substitution that is at position 65 is S65I.

28. The functional mutant fluorescent protein of claim 27,

wherein said functional mutant fluorescent protein comprises a fusion protein.

29. The functional mutant fluorescent protein of claim 16,

wherein said functional mutant fluorescent protein consists of mutations S65T, M153A, and K238E.

30. The functional mutant fluorescent protein of claim 29,

wherein said functional mutant fluorescent protein comprises a fusion protein.

31. The functional mutant fluorescent protein of claim 16,

wherein said functional mutant fluorescent protein further comprises an amino acid sequence which targets said protein to the specific cellular locations.

32. A functional mutant fluorescent protein, comprising:

a protein with an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO;

2) by at least one amino acid substitution that is at position 66, wherein said at least one substitution is either Y66H or Y66W, further wherein said functional mutant fluorescent protein has an excitation or emission different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.

33. The functional mutant fluorescent protein of claim 32,

wherein said at least one amino acid substitution that is at position 66 is Y66W.

34. The functional mutant fluorescent protein of claim 33,

wherein said functional mutant fluorescent protein comprises a fusion protein.

35. The functional mutant fluorescent protein of claim 32,

wherein said at least one amino acid substitution that is at position 66 is Y66H.

36. The functional mutant fluorescent protein of claim 35,

wherein said functional mutant fluorescent protein comprises a fusion protein.

37. The functional mutant fluorescent protein of claim 32,

wherein said functional mutant fluorescent protein consists of mutations Y66H and Y145F.

38. The functional mutant fluorescent protein of claim 37,

wherein said functional mutant fluorescent protein comprises a fusion protein.

39. The functional mutant fluorescent protein of claim 32,

wherein said functional mutant fluorescent protein consists of mutations Y66W, N146I, M153T, V163A and N212K.

40. The functional mutant fluorescent protein of claim 39,

wherein said functional mutant fluorescent protein comprises a fusion protein.

41. The functional mutant fluorescent protein of claim 32,

wherein said functional mutant fluorescent protein consists of mutations Y66W, I123V, Y145H, H148R, M153T, V163A, and N212K.

42. The functional mutant fluorescent protein of claim 41,

wherein said functional mutant fluorescent protein comprises a fusion protein.

43. A functional mutant fluorescent protein, comprising:

a protein with an amino acid sequence that differs from an amino acid sequence of an Aequorea wild type green fluorescent protein (SEQ ID NO;

2) by at least one amino acid substitution in the region consisting of positions 65 and 66,

wherein said functional mutant fluorescent protein has an excitation or emission different from an excitation spectrum or emission spectrum of said Aequorea wild type green fluorescent protein.

44. The functional mutant fluorescent protein of claim 43,

wherein said functional mutant fluorescent protein exhibits an alteration in the ratio of two main excitation peaks relative to Aequorea wild type green fluorescent protein.

45. The functional mutant fluorescent protein of claim 44,

wherein said functional mutant fluorescent protein exhibits increased fluorescence at the shorter-wavelength peak of said two main excitation peaks.

46. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 202.

47. The functional mutant fluorescent protein of claim 46,

wherein said amino acid substitution that is at position 202 is S202F.

48. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 203.

49. The functional mutant fluorescent protein of claim 48,

wherein said amino acid substitution that is at position 203 is T203I.

50. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 167.

51. The functional mutant fluorescent protein of claim 50,

wherein said amino acid substitution that is at position 167 is I167 V or I167T.

52. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 153.

53. The functional mutant fluorescent protein of claim 52,

wherein said amino acid substitution that is at position 153 is M153T or M153A.

54. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 238.

55. The functional mutant fluorescent protein of claim 54,

wherein said amino acid substitution that is at position 238 is K238E.

56. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 145.

57. The functional mutant fluorescent protein of claim 56,

wherein said amino acid substitution that is at position 145 is Y145H or Y145F.

58. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 146.

59. The functional mutant fluorescent protein of claim 58,

wherein said amino acid substitution that is at position 146 is N146I.

60. The functional mutant fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 163 or 148.

61. The functional mutant fluorescent protein of claim 60,

wherein said amino acid substitution that is at position 163 is V163A and said amino acid substitution at position 148 is H148R.

62. The functional fluorescent protein of claim 43,

further comprising an amino acid substitution that is at position 212 or 123.

63. The functional fluorescent protein of claim 62,

wherein said amino acid substitution that is at position 212 is N212K and said amino acid substitution at position 123 is I123V.

64. The functional fluorescent protein of claim 43,

wherein said functional fluorescent protein comprises a fusion protein.