Circularly permuted fluorescent protein indicators

• US 7,060,793 B2
• Filed: 10/23/2001
• Issued: 06/13/2006
• Est. Priority Date: 05/21/1999
• 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: Baird, Geoffrey, Tsien, Roger Y.
• Primary Examiner(s): MURPHY, JOSEPH F
• Application Number:
  US09/999,745
• Publication Number:
  US 20020157120A1
• Time in Patent Office: 1,694 Days
• Field of Search: 530/350, 530/402, 435/69.7, 435/320.1, 435/325
• US Class Current: 530/350
• 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 polypeptide comprising a circularly permuted Aequorea-related 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 Aequorea-related 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 Aequorea-related fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein,

wherein the amino acid residues of said fluorescent protein are numbered in conformance with native Aquorea green fluorescent protein (SEQ ID NO;

     3) and wherein the first serine residue following methionine-1 in said fluorescent protein is designated amino acid residue 2, and wherein the amino-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is selected from the group consisting of E142, Y143, Y145, H148, D155, H169, E172, D173, A227 and I229, and the carboxy-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is selected from the group consisting of N144, N146, N149, K162, K156, N170, I171, D173, E172, A227, and I229, and

wherein said circularly permuted fluorescent protein moiety comprises an optically active polypeptide capable of emitting light.

2. The polypeptide of claim 1, wherein the Aequorea-related fluorescent protein moiety is a GFP (SEQ ID NO:

3), CFP, or YFP.

3. The polypeptide of claim 1, wherein the Aequorea-related fluorescent protein moiety is a citrine (SEQ ID NO:

65).

4. The polypeptide of claim 1, wherein the linker moiety is GGTGEL (SEQ ID NO:

1), GGTGGS (SEQ ID NO;

63), or FKTRHN (SEQ ID NO;

2).

5. The polypeptide of claim 1, wherein said linker moiety is inserted at a site corresponding to a residue selected from residues 128–

148, residues 155–

160, residues 168–

176 or residues 227–

229 of the fluorescent protein.

6. The polypeptide of claim 1, wherein the amino-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is Y145 and the carboxy-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is selected from the group consisting of N144, N146, N149, K162, K156, N170, I171, D173, E172, A227, and I229, of the fluorescent protein, respectively.

7. The polypeptide of claim 1, further comprising a sensor polypeptide wherein the sensor polypeptide is calmodulin, a zif zinc finger domain, or citrine.

8. The polypeptide of claim 7, wherein the sensor polypeptide is a zif zinc-finger domain.

9. The polypeptide of claim 7, wherein the sensor polypeptide is calmodulin.

10. The polypeptide of claim 7, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, and Calspermin.

11. The polypeptide of claim 1, wherein the circularly permuted Aequorea-related fluorescent protein further comprises a localization sequence.

12. A circularly permuted Aequorea-related fluorescent protein moiety comprising:

a linker moiety linking the amino-terminal and carboxy-terminal amino acids of citrine (SEQ ID NO;

65), 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,

wherein the amino acid residues of said citrine (SEQ ID NO;

     65) are numbered in conformance with native Aquorea green fluorescent protein (SEQ ID NO;

     3) and wherein the first serine residue following methionine-1 in said fluorescent protein is designated amino acid residue 2, and wherein the amino-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is selected from the group consisting of E142, Y143, Y145, H148, D155, H169, E172, D173, A227 and I229, and the carboxy-terminal end of the circularly permuted Aequorea-related fluorescent protein moiety is selected from the group consisting of N144, N146, N149, K162, K156, N170, I171, D173, E172, A227, and I229,

wherein said circularly permuted fluorescent protein moiety comprises an optically active polypeptide capable of emitting light; and

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

13. A circularly permuted fluorescent protein encoded by a nucleic acid sequence produced by the method of producing a circularly permuted-fluorescent-protein-encoding nucleic acid sequence comprising:

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

nucleotide of a polynucleotide encoding a circularly-permuted Aequorea-related fluorescent protein moiety of claim 12;

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.