Circularly permuted fluorescent protein indicators

• US 20020157120A1
• Filed: 10/23/2001
• Published: 10/24/2002
• Est. Priority Date: 05/21/1999
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Howard Hughes Medical Institute
• Inventors: Tsien, Roger Y., Baird, Geoffrey
• Granted Patent: US 7,060,793 B2
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

Polynucleotides encoding fluorescent indicators, which contain a sensor polypeptide inserted within a fluorescent moiety, are provided, as are polypeptides encoded by such polynucleotides. Also provided are circularly permuted fluorescent polypeptides and polynucleotides encoding the circularly permuted fluorescent polypeptides. In addition, methods of using the fluorescent indicators and the circularly permuted fluorescent polypeptides are provided.

Claims

1. An isolated nucleic acid sequence encoding a circularly permuted fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and

two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end, and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.

2. The nucleic acid of claim 1, wherein the fluorescent protein is an Aequorea-related fluorescent protein moiety.

3. The nucleic acid sequence of claim 2, wherein the Aequorea-related protein moiety is GFP, CFP, or YFP.

4. The nucleic acid sequence of claim 2, wherein the Aequorea-related protein moiety is citrine (SEQ ID NO:

65).

5. The nucleic acid of claim 1, wherein the linker moiety encodes GGTGEL (SEQ ID NO:

1), GGTGGS (SEQ ID NO;

63) or FKTRHN (SEQ ID NO;

2).

6. The nucleic acid of claim 2, wherein the amino-terminal end or the carboxy terminal end of the indicator has an amino acid corresponding to residues 128-148, residues 155-160, residues 168-176, or residues 227-229 of the fluorescent protein.

7. The nucleic acid of claim 2, wherein the amino-terminal end is selected from the group consisting E142, Y143, Y145, H148, D155, H169, E172, D173, A227 and I229, and the carboxy-terminal end is selected from the group consisting of N144, N146, N144, N149, K162, K156, N170, I171, D173, E172, A227, and I229, of the fluorescent protein, respectively.

8. The nucleic acid of claim 1, further comprising a polynucleotide encoding a sensor polypeptide.

9. The nucleic acid of claim 8, wherein the sensor polypeptide changes conformation upon interaction with a response-inducing member.

10. The nucleic acid of claim 8, wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmodulin-related protein, recoverin, a nucleoside diphosphate or triphosphate binding protein, an inositol-1,4,5-triphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a ligand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety.

11. The nucleic acid of claim 8, wherein the sensor polypeptide is calmodulin or a calmodulin-related protein moiety.

12. The nucleic acid of claim 8, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineurin, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromodulin, Spectrin, MARCKS, F52, β

-Adducin, HSP90a, HIV-1 gp160, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, and Model Peptide CBP2.

13. The nucleic acid of claim 1, wherein the circularly permuted fluorescent protein further comprises a localization sequence.

14. An expression vector containing the nucleic acid sequence of claim 1.

15. A transgenic non-human animal comprising a nucleic acid sequence according to claim 1.

18. The cell of claim 17, wherein the cell is a prokaryote.

19. The cell of claim 18, wherein the cell is E. coli.

20. The cell of claim 17, wherein the cell is a eukaryotic cell.

21. The cell of claim 20, wherein the cell is a yeast cell.

22. The cell of claim 20, wherein the cell is a mammalian cell.

24. The polypeptide of claim 23, wherein the fluorescent protein is an Aequorea-related fluorescent protein moiety.

25. The polypeptide of claim 24, wherein the Aequorea-related protein moiety is a GFP (SEQ ID NO:

3), CFP, or YFP

26. The polypeptide of claim 24, wherein the Aequorea-related protein moiety is a citrine (SEQ ID NO:

65).

27. The polypeptide of claim 23, wherein the linker moiety is GGTGEL (SEQ ID NO:

1) GGTGGS (SEQ ID NO;

63) or FKTRHN (SEQ ID NO;

2).

28. The polypeptide of claim 24, wherein the amino-terminal end or the carboxy terminal end of the indicator has an amino acid of the fluorescent protein, the amino acid corresponding to residues 128-148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein.

29. The polypeptide of claim 24, wherein the amino-terminal end is selected from the group consisting E142, Y143, Y145, H148, D155, H169, E172, D173, A227 and I229, and the carboxy-terminal end is selected from the group consisting of N144, N146, N144, N149, K162, K156, N170, I171, D173, E172, A227, and I229, of the fluorescent protein, respectively.

30. The polypeptide of claim 23, further comprising a sensor polypeptide.

31. The polypeptide of claim 30, wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmodulin-related protein, recoverin, a nucleoside diphosphate or triphosphate binding protein, an inositol-1,4,5-triphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a ligand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety.

32. The polypeptide of claim 30, wherein the sensor polypeptide is calmodulin or a calmodulin-related protein moiety.

33. The polypeptide of claim 30, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineurin, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromodulin, Spectrin, MARCKS, F52, β

-Adducin, HSP90a, HIV-1 gp160, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, and Model Peptide CBP2.

34. The polypeptide of claim 23, wherein the circularly permuted fluorescent protein further comprises a localization sequence.

36. The circularly permuted fluorescent protein moiety of claim 35, wherein the fluorescent protein is citrine (SEQ ID NO:

65).

38. The method of claim 37, wherein the polynucleotide encoding the fluorescent protein comprises SEQ ID NO:

64.

39. A method of producing a circularly permuted fluorescent protein comprising expressing the nucleic acid sequence produced by the method of claim 39.

40. The circularly permuted fluorescent protein produced by the method of claim 39.

41. The circularly permuted fluorescent protein of claim 40, which comprises citrine (SEQ ID NO:

65).

16. An expression vector comprising expression control sequences operatively linked to a circularly permuted fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and

two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.

17. A host cell transfected with an expression vector comprising an expression control sequence operatively linked to a sequence coding for the expression of a circularly permuted fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and

two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.

23. An isolated polypeptide comprising a circularly permuted fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and

two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.

35. A circularly permuted fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and

two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein; and

a sensor polypeptide which is response to a chemical, biological, electrical or physiological parameter.

37. A method of producing a circularly permuted fluorescent nucleic acid sequence, comprising:

linking a nucleic acid sequence encoding a linker moiety to the 5′

nucleotide of a polynucleotide encoding a fluorescent protein;

circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence; and

cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide.