FACS-optimized mutants of the green fluorescent protein (GFP)

• US 5,804,387 A
• Filed: 01/31/1997
• Issued: 09/08/1998
• Est. Priority Date: 02/01/1996
• Status: Expired due to Term

• Current Assignee: Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Falkow, Stanley, Valdivia, Raphael H., Cormack, Brendan P.
• Primary Examiner(s): HUTSON, RICHARD G
• Assistant Examiner(s): Stole, Einar
• Application Number:
  US08/791,332
• Time in Patent Office: 585 Days
• Field of Search: 435/325, 435/410, 435/243, 435/252.3, 435/254.11, 435/69.1, 435/6, 435/7.21, 530/350, 536/23.1, 935/22
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays

Abstract

Three classes of GFP mutants having single excitation maxima around 488 nm are brighter than lid-type GFP following 488 nm excitation. GFPmut1 has a double substitution: F64L, S65T; GFPmut2 has a triple substitution: S65A, V68L, S72A; and GFPmut3 is characterized by the double substitution S65G, S72A. The excitation maxima of the three mutants are at 488 nm, 481 nm and 501 nm respectively. The fluorescence intensities following excitation at 488 nm are an order of magnitude higher than that of wild-type GFP excited at 488 nm in E. coli. The expression of GFP is observable minutes after induction.

Claims

1. A nucleic acid comprising a sequence encoding a mutant Aequorea victoria green fluorescent protein brighter than a wild-type Aequorea victoria green fluorescent protein upon 488 nm excitation, wherein a set of mutation positions of said mutant protein comprises position 72.

2. The nucleic acid of claim 1 wherein said set of mutation positions further comprises position 65.

3. The nucleic acid of claim 1 wherein said set of mutation positions further comprises position 68.

4. The nucleic acid of claim 1 wherein said set of mutation positions further comprises positions 65 and 68.

5. The nucleic acid of claim 1 wherein a set of mutations of said nucleic acid comprises a S72A mutation.

6. The nucleic acid of claim 5 wherein said set of mutations further comprises a S65A mutation.

7. The nucleic acid of claim 5 wherein said set of mutations further comprises a V68L mutation.

8. The nucleic acid of claim 5 wherein said set of mutations further comprises a S65A mutation and a V68L mutation.

9. The nucleic acid of claim 8 wherein said set of mutations further comprises a S65G mutation.

10. The nucleic acid of claim 1 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ ID NO:

2.

11. The nucleic acid of claim 1 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ ID NO:

3.

12. The nucleic acid of claim 1 wherein said mutant protein is more soluble than said wild-type protein at 37°

C.

13. A host cell comprising a nucleic acid including a sequence encoding a mutant Aequorea victoria green fluorescent protein brighter than a wild-type Aequorea victoria green fluorescent protein upon 488 nm excitation, wherein a set of mutation positions of said mutant protein comprises position 72.

14. The host cell of claim 13 wherein said set of mutation positions further comprises position 65.

15. The host cell of claim 13 wherein said set of mutation positions further comprises position 68.

16. The host cell of claim 13 wherein said set of mutation positions further comprises positions 65 and 68.

17. The host cell of claim 13 wherein a set of mutations of said mutant protein comprises a S72A mutation.

18. The host cell of claim 17 wherein said set of mutations further comprises a S65A mutation.

19. The host cell of claim 17 wherein said set of mutations further comprises a V68L mutation.

20. The host cell of claim 17 wherein said set of mutations further comprises a S65A mutation and a V68L mutation.

21. The host cell of claim 20 wherein said set of mutations further comprises a S65G mutation.

22. The host cell of claim 13 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ ID NO:

2.

23. The host cell of claim 13 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ. ID NO:

3.

24. The host cell of claim 13 wherein said mutant protein is more soluble than said wild-type protein at 37°

C.

25. The host cell of claim 13 wherein a chromophore formation time of said mutant protein is less than one hour in said host cell.

26. The host cell of claim 13 wherein said host cell is a bacterial cell.

27. The host cell of claim 13 wherein said host cell is a mammalian cell.

28. A nucleic acid comprising a sequence encoding a mutant Aequorea victoria green fluorescent protein brighter than a wild-type Aequorea victoria green fluorescent protein upon 488 nm excitation, wherein a set of mutations of said mutant protein comprises a F64L and a S65T mutation.

29. The nucleic acid of claim 28 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ ID NO:

1.

30. The nucleic acid of claim 28 wherein said mutant protein is more soluble than said wild-type protein at 37°

C.

31. A host cell comprising a nucleic acid including a sequence encoding a mutant Aequorea victoria green fluorescent protein brighter than a wild-type Aequorea victoria green fluorescent protein upon 488 nm excitation, wherein a set of mutations of said mutant protein comprises a F64L and a S65T mutation.

32. The host cell of claim 31 wherein an amino acid sequence containing a chromophore of said mutant protein comprises the amino acid sequence of SEQ ID NO:

1.

33. The host cell of claim 31 wherein said mutant protein is more soluble than said wild-type protein at 37°

C.

34. The host cell of claim 31 wherein a chromophore formation time of said mutant protein is less than one hour in said host cell.

35. The host cell of claim 31 wherein said host cell is a bacterial cell.

36. The host cell of claim 31 wherein said host cell is a mammalian cell.

37. A method of analyzing a cell comprising the steps of:

a) expressing a nucleic acid encoding a mutant green fluorescent protein, wherein a set of mutations of said mutant protein comprises a subset selected from a group consisting of a (F64L,S65T) subset, a (S65A,V68L,S72A) subset, and a (S65G, S72A) subset; and

b) measuring a fluorescence signal from said mutant protein.

38. The method of claim 37 further comprising the step of sorting said cell according to said signal.

39. The method of claim 38 wherein said step of sorting comprises sorting said cell by fluorescence activated cell sorting.

40. The method of claim 37 further comprising the step of illuminating said cell at an excitation wavelength substantially equal to 490 nm.

41. The method of claim 37 comprising the step of measuring said signal at an emission wavelength substantially equal to 510 nm.

42. The method of claim 37 wherein:

a) said nucleic acid comprises a gene of interest encoding a protein of interest, wherein said protein of interest is distinct from said mutant protein; and

b) said signal indicates a presence of said gene of interest in said cell.

43. The method of claim 37 wherein:

a) said cell further comprises a protein of interest fused to said mutant protein; and

b) said method further comprises the step of identifying an intracellular location of said mutant protein, thereby identifying an intracellular location of said protein of interest.

44. The method of claim 37 wherein:

a) said nucleic acid comprises a regulatory element operatively connected to a coding portion encoding said mutant protein; and

b) said method further comprising the step of exposing said regulatory element to an environmental stimulus, whereby said signal measures an effect of said stimulus on an activity of said regulatory element.

45. The method of claim 44 wherein said regulatory element comprises a promoter.

46. The method of claim 44 wherein said stimulus comprises a compound of interest.

47. The method of claim 44 wherein said stimulus is selected from a group consisting of a temperature, an acidity, and a species-specific factor.

48. The method of claim 37 comprising the steps of:

a) exposing a library of promoters to a first environmental stimulus, wherein a promoter of said library is operatively connected to a coding portion encoding said mutant protein; and

b) generating a first pattern characterizing an effect of said first stimulus on said library.

49. The method of claim 48 further comprising comparing said first pattern with a second pattern, said second pattern characterizing an effect of a second environmental stimulus on said library.

50. The method of claim 37 further comprising the steps of:

a) expressing a nucleic acid encoding an additional green fluorescent protein spectrally distinguishable from said mutant protein; and

b) measuring an additional fluorescence signal from said additional protein.