Cytosolically-active peroxidases as reporters for microscopy

1. An imaging method, comprising:

providing a sample containing a cell that expresses a Class I heme peroxidase, or a fusion protein comprising (a) the Class I heme peroxidase, and (b) a protein or a cellular localization signal peptide, and

contacting the sample with a substrate of the Class I heme peroxidase to allow conversion of the substrate into a product via an oxidation reaction catalyzed by the Class I heme peroxidase, wherein the product releases a signal detectable by a microscope.

2. The method of claim 1 , further comprising detecting the signal under a microscope.

3. The method of claim 1 wherein the substrate is 3-methyl-2-benzothiazolinone hydrazone, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), a phenol, or an aniline.

4. The method of claim 3 , wherein the phenol is selected from the group consisting of guaiacol, pyrogallol, Amplex UltraRed, dihydrofluorescin, p-cresol, dopamine, 3-methylphenol, 4-methoxyphenol, 4-hydroxybenzaldehyde, 5-aminosalicylic acid, and 4-chloro-1-naphthol.

5. The method of claim 3 , wherein the aniline is selected from the group consisting of diaminobenzidine (DAB), 3-amino-9-ethylcarbazole, o-phenylenediamine, 3,3′,5,5′-tetramethylbenzidine, o-diansidine, 5-aminosalicylic acid, Luminol, 4-aminophthalhydrazide, N-(6-Aminohexyl)-N-ethylisoluminol, N-(4-Aminobutyl)-N-ethylisoluminol, 3-methylaniline, 4-methylaniline, and 4-methoxyaniline.

6. The method of claim 1 wherein the Class I heme peroxidase is an ascorbate peroxidase (APX), a yeast cytochrome c peroxidase (CCP), or a bacterial catalase-peroxidase (BCP).

7. The method of claim 1 wherein the Class I heme peroxidase is monomeric.

8. The method of claim 7 , wherein the Class I heme peroxidase is a mutated APX, which, as compared to its wild-type counterpart, contains

(i) one or more mutations at positions corresponding to K14, E17, K18, K20, R21, R24, A28, E106, E112, 1185, E228, and D229 in SEQ ID NO:1; or

(ii) one or more mutations at positions corresponding to W41, G69, D133, T135, and K136 in SEQ ID NO:1.

9. The method of claim 8 , wherein the mutant APX is:

(a) single mutant K14D, single mutant A28K, single mutant E112K, single mutant E228K, single mutant D229K, double mutant K14D/E112K, double mutant K14D/E228K, double mutant K14D/D229K, double mutant A28K/E112K, double mutant E112K/E228K, double mutant E112K/D229K, triple mutant E17N/K20A/R21L, triple mutant A28K/E112K/D229K, triple mutant K14D/E112K/D229K, triple mutant K14D/E112K/E228K, triple mutant A28K/E112K/E228K, or triple mutant K14D/W41F/E112K,

(b) single mutant W41F, single mutant G69F, single mutant G174F, double mutant W41F/G69F, triple mutant D133A/T135F/K136F, quadruple mutant W41F/D133A/T135F/K136F, quadruple mutant G69F/D133A/T135F/K136F, or quintuple mutant W41F/G69F/D133A/T135F/K136F, or

(c) a combination of (a) and (b).

10. The method of claim 6 , wherein the Class I heme peroxidase is a mutated yeast CCP, which, as compared to its wild-type counterpart, comprises one or more amino acid substitutions at positions corresponding to W51, S81, D146, D148, K149, and G186 in SEQ ID NO:2.

11. The method of claim 6 , wherein the Class I heme peroxidase is a mutated BCP, which, as compared to its wild-type counterpart, comprises one or more amino acid residue substitutions at positions corresponding to W107, D137, N231, E223, and G316 in SEQ ID NO:3.

12. The method of claim 1 wherein the fusion protein comprises the Class I heme peroxidase and the protein.

13. The method of claim 1 , wherein the cellular localization signal peptide is an ER-targeting signal peptide, a Golgi-targeting signal peptide, a mitochondria-targeting signal peptide, a nuclear localization signal peptide, or a nuclear export signal peptide.

14. The method of claim 13 , wherein the cellular localization signal peptide comprises an amino acid sequence selected from the group consisting of:

DPVVVLGLCLSCLLLLSLWKQSYGGG,

(SEQ ID NO: 4)

MLATRVFSLVGKRAISTSVCVRAH,

(SEQ ID NO: 5)

LQLPPLERLTLD,

(SEQ ID NO: 6)

and

KDEL.

(SEQ ID NO: 7)

15. The method of claim 1 wherein the Class I heme peroxidase or the fusion protein comprises a protein tag.

16. The method of claim 1 wherein the cell that expresses the Class I heme peroxidase or the fusion protein is a mammalian cell, a bacterial cell, or a yeast cell.

17. The method of claim 1 wherein the sample is a tissue sample.

18. The method of claim 2 wherein in the detecting step, the sample contains either a live cell or a fixed cell.