Oxidation reduction sensitive green fluorescent protein variants

• US 20040171112A1
• Filed: 03/08/2004
• Published: 09/02/2004
• Est. Priority Date: 03/12/2001
• Status: Active Grant

• Current Assignee: Oregon State University (Oregon University System)
• Original Assignee: Oregon State University (Oregon University System)
• Inventors: Remington, S. James, Hanson, George T.
• Granted Patent: US 7,015,310 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineering to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

Claims

1. A mutant Aequorea victoria green fluorescent protein (GFP), with a fluorescence spectrum that is sensitive to redox status, wherein at least one of the residues at position 147 or 149, and at least one of the residues at position 202 or 204, is mutated from the wild-type amino acid to cysteine.

2. The mutant GFP of claim 1, selected from the group consisting of GFPs in which:

residue 147 and residue 202 are cysteine;

residue 147 and residue 204 are cysteine;

residue 149 and residue 202 are cysteine;

residue 149 and residue 204 are cysteine; and

residues 147, 149, 202 and 204 are each cysteine.

3. The mutant GFP of claim 1, further comprising a mutation at position 65.

4. The mutant GFP of claim 3, wherein the mutation at position 65 is S65T.

5. The mutant GFP of claim 1, wherein the fluorescence spectrum is also pH sensitive.

6. The mutant GFP of claim 1, further comprising a mutation at position 48.

7. The mutant GFP of claim 6, wherein the mutation at position 48 is C48S.

8. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S65T, N149C, and S202C.

9. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S65T, N149C, and Q204C.

10. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S147C, and Q204C.

11. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S147C, and S202C.

12. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S65T, S147C, N149C, S202C, and Q204C.

13. The mutant GFP of claim 1, wherein said mutant protein comprises the following mutations compared to wild-type GFP:

C48S, S147C, N149C, S202C, and Q204C.

14. An isolated nucleic acid molecule encoding the mutant GFP of claim 1.

15. An isolated nucleic acid molecule encoding the mutant GFP of any one of claims 2-13

16. The isolated nucleic acid molecule of claim 14, comprising an expression control sequence.

17. A recombinant nucleic acid comprising the nucleic acid molecule of claim 14 functionally linked to a promoter.

18. A host cell comprising a nucleic acid according to claim 14.

19. The host cell of claim 18 wherein the host cell is a bacterial cell, a plant cell, or an animal cell.

20. The host cell of claim 18 wherein said host cell is a mammalian cell.

21. A method of analyzing an oxidation-reduction condition of or in a cell comprising:

expressing the mutant GFP of claim 1 in the cell; and

measuring a fluorescence signal from the mutant GFP.

23. The method of claim 21, wherein the mutant GFP is expressed as a fusion protein

24. The method of claim 21, further comprising analyzing a pH condition of or in the cell using the mutant GFP.

25. The mutant GFP of claim 1, comprising an amino acid sequence as shown in SEQ ID NO:

3, SEQ ID NO;

5, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, or SEQ ID NO;

13.