JP5772892B2 - Method for producing coelenteramide or an analog thereof - Google Patents

Description

The present invention relates to a method for producing coelenteramide or an analog thereof, a method for producing green fluorescent protein (gFP), and the like.

One of the proteins involved in bioluminescence is a calcium-binding photoprotein. This photoprotein specifically binds to Ca ²⁺ and emits light instantaneously. The calcium-binding photoprotein is a complex containing a protein having an oxygenation function and a peroxide of luciferin, which is a luminescent substrate. In the calcium-binding photoprotein, a protein having an oxygen addition function is called an apoprotein. Luciferin peroxide is coelenterazine peroxide (2-hydrope).
roxycoelenterazine). Specific examples of such calcium-binding proteins include aequorin, clytin-I, clytin-II (clyt).
in-II), mitrocomin, and obelin are known.

Among these, aequorin is a photoprotein isolated from Aequorea aequorea, which is a luminescent jellyfish (Non-patent Document 1: Shimomura, In: Bioluminescence, Chemic).
al principles and methods (2006) pp90-158, World Scientific Pub. Co .; Non-Patent Document 2: Shimomura et al., (1962) J. Cell. Comp. Physiol. 59, pp223-240). This aequorin is a noncovalent complex of apoprotein apoaequorin (21.4 kDa) and coelenterazine hydroperoxide (Non-patent Document 3: Head et al., (2000) Nature,
405 372-376). Apoaequorin is a simple polypeptide composed of 189 amino acid residues and has three EF hand motifs characteristic of the Ca ²⁺ binding site (Non-patent Document 4: Ino)
uye et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158). In the presence of Ca ²⁺
Aequorin emits blue light by intramolecular reaction (λ _max = ˜460 nm) and decomposes into apoaequorin, coelenteramide, and CO ₂ (Non-patent Document 5: Shimomura & Johnson (1972) B
iochemistry 11, 1602-1608 .; Non-Patent Document 6: Shimomura & Johnson (1973) Tetrahedro
n Lett. 2963-2966). The complex of Ca ²⁺ -binding apoaequorin and coelenteramide obtained by this degradation is known as blue fluorescent protein (BFP) (Non-patent Document 7: Shimomura &
Johnson (1975) Nature 256, 236-238).

The fluorescence emission spectrum of BFP is the same as the bioluminescence spectrum of aequorin (Non-patent document 8: Shimomura, Biochem. J. 306 (1995) 537-543; Non-patent document 9: Inouye, F
EBS Lett. 577 (2004) 105-110). Recombinant apoaequorin prepared from E. coli is EDTA
And incubating with coelenterazine and oxygen in the presence of a reducing reagent,
Recombinant aequorin can be regenerated (Non-patent Document 10: Inouye et al., (1986) Bi
ochemistry 25: 8425-8429; Non-Patent Document 11: Inouye et al., (1989) J. Biochem., 1
05, 473-477). This recombinant aequorin is highly purified (Non-patent Document 12: Shi
momura & Inouye (1999) Protein Express. Purif. 16, 91-95). The luminescent properties of recombinant aequorin are consistent with those of natural aequorin (Non-patent Document 13: Shimomura et al.
al., (1990) Biochem. J. 270 309-312). Crystal structures of aequorin and semi-synthetic aequorin were analyzed (Non-patent document 3: Head et al., Nature, 405 (2000) 372-376; Non-patent document 1).
4: Toma et al., (2005) Protein Science 14: 409-416), the binding properties of Mg ²⁺ to the EF hand motif of aequorin have also been investigated by NMR analysis (Non-patent Document 15: Oha).
shi et al., (2005) J. Biochem. 138: 613-620).

Recently, BFP was quantitatively prepared from purified recombinant aequorin (Non-patent Document 9: Inouy).
e, FEBS Lett. 577 (2004) 105-110; Non-Patent Document 16: Inouye & Sasaki, FEBS Lett. 5
80 (2006) 1977-1982), it became clear that this BFP has substantial luminescence activity and catalyzes the oxidation of coelenterazine like luciferase. The emission intensity of this BFP is Ca ^2
+ Approximately 10 times the emission intensity of bound apoaequorin (Non-patent document 9: Inouye, FEBS Lett. 57
7 (2004) 105-110). That is, this BFP is a novel bifunctional protein having fluorescence activity and luciferase activity. Furthermore, BFP has a maximum fluorescence emission peak of about 470 nm by EDTA treatment.
Is converted to a green fluorescent protein (gFP) having gFP is a non-covalent complex of apoaequorin and coelenteramide, and aequorin can be regenerated by incubation of coelenterazine at 25 ° C. in the absence of a reducing reagent (Non-patent Document 9: Inouye, FEBS Let
t. 577 (2004) 105-110). Semi-synthetic aequorin can also be prepared by incubating various coelenterazine analogs with BFP or gFP in the presence of EDTA and dithiothreitol (DTT) (Non-patent Document 16: Inouye et al. & Sasaki, FEBS Lett . 580 (2006)
1977-1982). Furthermore, the luminescence activity of BFP as luciferase is activated by adding imidazole at a concentration of 30 to 300 mM using coelenterazine and its analog as a substrate (Non-patent Document 17: Inouye & Sasaki (2007) Biochem. Biophys. Res. Commun. 354: 650-65
Five). However, in the development of applications of BFP and gFP, the protein catalytic region or amino residue for oxygen addition to coelenterazine, which is important basic information, has not been solved. In solving these problems and developing applications, it is necessary to easily prepare tens of milligrams of BFP and gFP. That is, it is essential to establish a method for preparing high-purity gFP from apoaequorin and coelenteramide and converting it to BFP.

Here, as a method for producing coelenteramide as a raw material for producing BFP and gFP, the following formula
A compound represented by the following formula
A method for producing coelenteramide is known, which comprises reacting with p-hydroxyphenylacetic acid represented by the formula (Non-Patent Document 6: Shimomura & Johnson, Tetrahedron Lett. (1973)).
2963-2966). The yield of coelenteramide in this method is about 50%.

Shimomura, In: Bioluminescence, Chemical principles and methods (2006) pp90-158, World Scientific Pub. Co. Shimomura et al., (1962) J. Cell. Comp. Physiol. 59, pp223-240 Head et al., (2000) Nature, 405, 372-376 Inouye et al., (1985) Proc. Natl. Acad. Sci. USA. 82, 3154-3158 Shimomura & Johnson (1972) Biochemistry 11, 1602-1608 Shimomura & Johnson (1973) Tetrahedron Lett. 2963-2966 Shimomura & Johnson (1975) Nature 256, 236-238 Shimomura, (1995) Biochem. J. 306, 537-543 Inouye, (2004) FEBS Lett. 577, 105-110 Inouye et al., (1986) Biochemistry 25, 8425-8429 Inouye et al., (1989) J. Biochem., 105 473-477 Shimomura & Inouye (1999) Protein Express. Purif. 16, 91-95 Shimomura et al., (1990) Biochem. J. 270, 309-312 Toma et al., (2005) Protein Science 14, 409-416 Ohashi et al., (2005) J. Biochem. 138, 613-620 Inouye & Sasaki, (2006) FEBS Lett. 580, 1977-1982 Inouye & Sasaki, (2007) Biochem. Biophys. Res. Commun. 354, 650-655

In the above situation, there has been a demand for a method for producing high-yield coelenteramide or an analog thereof.

As a result of intensive studies to solve the above problems, the present inventors have obtained coelenteramide,
The present invention was completed by establishing a new synthesis route for synthesizing from O-methylcoelenteramine via di-O-methylcoelenteramide.
That is, this invention provides the manufacturing method of coelenteramide or its analog shown below.

(1) The following general formula (1)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ² is amino;
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group. )
The
The following general formula (2)
A compound represented by the formula:
X is a leaving group,
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
Including reacting with
The following general formula (3)
(Wherein R ¹ , R ³ , and R ⁴ are as defined above).
Manufacturing method.
(2) In general formula (1),
The method according to (1) above, wherein R ¹ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl.
(3) In general formula (1),
R ³ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, heptyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexyl The method according to (1) or (2) above, which is methyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl.
(4) In general formula (2),
X is chlorine, bromine, iodine, methanesulfonyloxy, benzenesulfonyloxy, toluenesulfonyloxy, or (4-R ⁴ O) C ₆ H ₄ CCOO— (wherein R ⁴ is as described above). The method according to any one of (1) to (3) above.
(5) In the general formula (2),
R ⁴ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl, according to any one of (1) to (4) above. Method.
(6) The compound represented by the general formula (1) is the following compound:
The method according to any one of (1) to (5) above, which is a compound selected from the group consisting of:
(7) The compound represented by the general formula (2) is the following compound:
The method according to any one of (1) to (6) above, which is a compound selected from the group consisting of:
(8) The following formula
A compound represented by the following formula
Comprising reacting with a compound represented by the formula:
The manufacturing method of the compound represented by these.
(9) The following general formula (3)
A compound represented by
(Where
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group;
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
(10) The following formula
A compound represented by
(11) The following general formula (3)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group;
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
Further removing the group represented by R ¹ and the group represented by R ⁴ ,
The following general formula (4)
(Wherein R ³ is as defined above).
Manufacturing method.
(12) In the general formula (3),
The method according to (11) above, wherein R ¹ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl.
(13) In general formula (3),
R ³ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, heptyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexyl The method according to (11) or (12) above, which is methyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl.
(14) In the general formula (3),
R ⁴ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl, according to any one of (11) to (13) above. Method.
(15) The compound represented by the general formula (3) is the following compound:
The method according to any one of (11) to (14) above, which is a compound selected from the group consisting of:
(16) The following formula
Including elimination of methyl from the compound represented by formula
The manufacturing method of the compound represented by these.
(17) In the presence of a chelating agent for removing calcium ions or divalent or trivalent ions replaceable with calcium ions,
The following general formula (4)
A compound represented by the formula:
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group. )
A method for producing green fluorescent protein (gFP), which comprises reacting apoprotein with a calcium-binding photoprotein apoprotein.
(18) In the presence of a chelating agent for removing calcium ions or divalent or trivalent ions replaceable with calcium ions, the following formula:
A method for producing a green fluorescent protein (gFP), which comprises reacting a compound represented by the formula with apoaequorin.
(19) The method according to (17) or (18) above, wherein the reaction is carried out in the presence of a reducing agent.
(20) The following general formula (1)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ² is amino;
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group. )
The
The following general formula (2)
A compound represented by the formula:
X is a leaving group,
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
In response to
The following general formula (3)
(Wherein R ¹ , R ³ , and R ⁴ are as defined above).
Generating
By removing the group represented by R ¹ and the group represented by R ⁴ from the compound represented by the general formula (3) ,
The following general formula (4)
Compound represented by
( Wherein R ³ is as described above.)
Generating
A blue fluorescent protein comprising reacting a compound represented by the general formula ( 4 ) with an apoprotein of a calcium-binding photoprotein in the presence of calcium ion or a divalent or trivalent ion replaceable with calcium ion ( BFP) manufacturing method.

According to a method for producing coelenteramide or an analog thereof according to an aspect of the present invention, coelenteramide or an analog thereof can be produced in a high yield as compared with a conventional method. According to another aspect of the present invention, gFP can be easily prepared directly from apoaequorin.

It is a figure which shows the SDS-PAGE analysis result of the protein in each step of apoaequorin purification. (a) Each step of purification of apoaequorin using a nickel chelate column. Lane M: Molecular weight marker (TEFCO): β-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (69.0 kDa), glutamate dehydrogenase (55.0 kDa), lactate dehydrogenase (36.5 kDa), carbonic acid Dehydrase (29.0 kDa) and trypsin inhibitor (20.1 kDa). Lane 1: 12,000 g of supernatant (20 μl) obtained from the crude extract. Lane 2: Fraction (20 μl) obtained by washing the nickel chelate column with 50 mM Tris-HCl (pH 7.6). Lane 3: Fraction (20 μl) eluted from the nickel chelate column with 50 mM Tris-HCl (pH 7.6) containing 0.1 M imidazole. (b) Purification of apoaequorin by treatment with 0.1M acetic acid. Lane M: Molecular weight marker (TEFCO). Lane 1: purified apoaequorin (5 μg). It is a figure which shows the fluorescence spectrum of the apoaequorin origin reconstituted syn-gFP obtained by incubating using coelenteramide under reducing conditions. Purified recombinant apoaequorin (50 μg) for 16 hours at 4 ° C. with coelenteramide (1.2 μg dissolved in 1 μl methanol) in 1 ml 50 mM Tris-HCl (pH 7.6) containing 1 mM DTT in the presence of 10 mM EDTA Fluorescence spectrum excited at 335 nm after incubation. It is the figure which compared the fluorescence spectrum of syn-gFP and gFP. It is a figure which shows the light emission pattern of syn-BFP and BFP which used coelenterazine as a substrate. It is a figure which shows an example of the manufacturing method of gFP of this invention. It is a figure which shows the scheme for manufacturing BFP, gFP, aequorin, etc. from coelenteramide manufactured with the method of this invention.

Hereinafter, the present invention will be described in detail.
1. In the present invention, coelenteramide or an analog thereof is produced from O-methylcoelenteramine or an analog thereof via di-O-methylcoelenteramide or an analog thereof. In some embodiments of the method for producing coelenteramide or an analog thereof of the present invention, the yield before purification of coelenteramide or an analog thereof from O-methylcoelenteramine or an analog thereof is higher than that of a conventional production method, For example, it is 55% or more, 60%, 65% or more, or 70% or more.

1.1. Production of di-O-methylcoelenteramide or an analog thereof The present invention provides di-O-methylcoelenteramide or an analog thereof (a compound represented by the general formula (3)). That is, the present invention provides the following general formula (3)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ³ is alkyl having 1 to 7 carbon atoms, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbon atoms, which may be substituted with an aliphatic cyclic group,
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
I will provide a.

In general formula (3), examples of the alkyl having 1 to 3 carbon atoms represented by R ¹ include methyl,
Examples include ethyl, propyl, or isopropyl. Examples of the arylalkyl having 7 to 10 carbon atoms represented by R ¹ include benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, and 4-phenylbutyl. In some aspects of the invention,
R ¹ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl. Preferably R ¹ is methyl, ethyl, isopropyl or benzyl.

In general formula (3), examples of the alkyl having 1 to 7 carbon atoms which may be substituted with an aliphatic cyclic group represented by R ³ include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s -Butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, heptyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, or cyclohexylmethyl. Examples of the aliphatic cyclic group represented by R ³ include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of the arylalkyl having 7 to 10 carbon atoms represented by R ³ include benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, and 4-phenylbutyl. In some embodiments of the invention, R ³ is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, isopentyl, neopentyl, t-pentyl, hexyl, heptyl, cyclopropylmethyl. , Cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl. Preferably R ³ is cyclohexylmethyl,
Cyclopentylmethyl or benzyl.

In the general formula (3), examples of the alkyl having 1 to 3 carbon atoms represented by R ⁴ include methyl,
Examples include ethyl, propyl, or isopropyl. Examples of the arylalkyl having 7 to 10 carbon atoms represented by R ⁴ include benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, and 4-phenylbutyl. In some aspects of the invention,
R ⁴ is methyl, ethyl, propyl, isopropyl, benzyl, α-methylbenzyl, phenethyl, 3-phenylpropyl, or 4-phenylbutyl. Preferably R ⁴ is methyl, ethyl, isopropyl or benzyl.

In some embodiments of the present invention, the di-O-methylcoelenteramide represented by the general formula (3) or an analog thereof is, for example, a compound selected from the group consisting of the following compounds.

In another aspect of the present invention, the di-O-methylcoelenteramide analog represented by the general formula (3) is, for example, a compound selected from the group consisting of the following compounds.

In still another aspect of the present invention, the di-O-methylcoelenteramide analog represented by the general formula (3) is, for example, a compound selected from the group consisting of the following compounds.

In still another aspect of the present invention, the di-O-methylcoelenteramide analog represented by the general formula (3) is, for example, a compound selected from the group consisting of the following compounds.

In still another aspect of the present invention, the di-O-methylcoelenteramide analog represented by the general formula (3) is, for example, a compound selected from the group consisting of the following compounds.

In some aspects of the invention, di-O-methylcoelenteramide or an analog thereof is preferably a compound selected from the group consisting of the compounds shown below.

In another aspect of the present invention, the di-O-methylcoelenteramide analog is preferably a compound selected from the group consisting of the compounds shown below.

In yet another aspect of the invention, the di-O-methylcoelenteramide analog is preferably
It is a compound selected from the group consisting of the compounds shown below.

In yet another aspect of the invention, the di-O-methylcoelenteramide analog is preferably
It is a compound selected from the group consisting of the compounds shown below.

In yet another aspect of the invention, the di-O-methylcoelenteramide analog is preferably
It is a compound selected from the group consisting of the compounds shown below.

In one embodiment of the present invention, the di-O-methylcoelenteramide or an analog thereof is di-O-methylcoelenteramide shown below.

Moreover, this invention provides the manufacturing method of the said di-O-methylcoelenteramide or its analog. More specifically, the present invention relates to O-methylcoelenteramine or an analog thereof (general formula (
1), 4-methoxyphenylacetyl halide or its analog (
There is provided a process for producing the above di-O-methylcoelenteramide or an analog thereof (compound represented by the general formula (3)), which comprises reacting with the compound represented by the general formula (2). That is, the present invention provides the following general formula (1)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ² is amino;
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group. )
The
The following general formula (2)
A compound represented by the formula:
X is a leaving group,
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
Including reacting with
The following general formula (3)
A compound represented by the formula:
R ¹ , R ³ , and R ⁴ are as described above. )
A manufacturing method is provided.

In the general formula (1) or the general formula (2), R ¹ , R ³ , and R ⁴ are as described in the general formula (3).

In the general formula (2), examples of the leaving group represented by X include halogen (for example, chlorine, bromine, or iodine), sulfonic acid reactive residues (for example, methanesulfonyloxy, benzenesulfonyloxy, or Toluenesulfonyloxy), or acyloxy (for example, (4-R ⁴ O) C ₆ H ₄ CH ₂ COO— (wherein R ⁴ is as defined above)) that forms an acid anhydride. Of these, halogen is preferable, and chlorine is more preferable.

Examples of O-methylcoelenteramine represented by the general formula (1) or an analog thereof include compounds selected from the group consisting of the following compounds.

O-methylcoelenteramine or an analog thereof is preferably a compound selected from the group consisting of the following compounds.

In one embodiment of the present invention, O-methylcoelenteramine or an analog thereof is the following compound.

O-methylcoelenteramine represented by the general formula (1) or an analog thereof can be produced by a known production method, or a commercially available product can be obtained. For example, Kishi
et al., Tetrahedron Lett., 13, 2747-2748 (1972), or Adamczyk et al., Org. Prep.
In the method described in Proced. Int., 33, 477-485 (2001) or a method analogous thereto, the general formula (1)
The O-methylcoelenteramine represented by these or its analog can be manufactured.

Moreover, as 4-methoxyphenyl acetyl halide represented by the said General formula (2) or its analog, the compound selected from the group which consists of the following compound can be mentioned, for example.

4-methoxyphenylacetyl halide or an analog thereof is preferably a compound selected from the group consisting of the following compounds.

In one embodiment of the present invention, 4-methoxyphenylacetyl halide or an analog thereof is
It is 4-methoxyphenyl acetyl chloride shown below.

4-methoxyphenylacetyl halide represented by the general formula (2) or an analog thereof is
It can be produced by a known production method, or a commercially available product can be obtained. Specifically, each compound is heated under reflux with an excess of thionyl chloride acting on the corresponding carboxylic acid, and then concentrated under reduced pressure, or a catalytic amount of N, N-
In the presence of dimethylformamide (DMF), the corresponding carboxylic acid can be reacted with oxalyl dichloride and then concentrated under reduced pressure. 4
-Methoxyphenylacetyl chloride can be purchased from Aldrich, and 4-benzyloxyphenylacetyl chloride and 4-methoxyphenylacetic anhydride can be purchased from Tokyo Chemical Industry.

Di-O-methylcoelenteramide represented by the general formula (3) or an analog thereof is represented by the general formula (1
O-methylcoelenteramine or its analog represented by the formula (4) and 4-methoxyphenylacetyl halide or its analog represented by the general formula (2), for example, in an organic solvent, in the presence of a base, or basic It can manufacture by making it react in an organic solvent. More specifically, di-O-methylcoelenteramide can be produced using the method described in the examples below or a method analogous thereto.

Here, as the organic solvent or basic organic solvent used in the method for producing di-O-methylcoelenteramide of the present invention or an analog thereof, various solvents can be used, but aqueous solvents or organic solvents other than alcohols can be used. Or a basic organic solvent is particularly preferable. The organic solvent or the basic organic solvent is, for example, pyridine, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, acetone, toluene, dioxane, ether or the like, and these can be used alone or as a mixture.

The base used in the method for producing di-O-methylcoelenteramide or an analog thereof of the present invention is not particularly limited, and various bases can be used. For example, pyridine, triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, lutidine, collidine and the like can be used alone or in combination.

Moreover, in the manufacturing method of di-O-methylcoelenteramide or its analog of this invention, although reaction temperature and reaction time are not specifically limited, For example, -20 degreeC-200 degreeC, 1 hour-
72 hours, preferably 0 ° C. to 100 ° C., 6 hours to 36 hours, more preferably room temperature to
It is 12 to 24 hours at 50 ° C.

One embodiment of the present invention is the following formula:
O-methylcoelenteramine represented by the formula
Comprising reacting with 4-methoxyphenylacetyl chloride represented by the following formula:
The manufacturing method of di-O-methylcoelenteramide represented by these is provided.

1.2. Production of coelenteramide or an analog thereof The present invention further eliminates a group represented by R ¹ and a group represented by R ⁴ from di-O-methylcoelenteramide or an analog thereof (a compound represented by the general formula (3)). A method for producing coelenteramide or an analog thereof (compound represented by the general formula (4)). That is, the present invention provides the following general formula (3)
A compound represented by the formula:
R ¹ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons;
R ³ is alkyl having 1 to 7 carbon atoms, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbon atoms, which may be substituted with an aliphatic cyclic group,
R ⁴ is alkyl having 1 to 3 carbons or arylalkyl having 7 to 10 carbons. )
The elimination of the group represented by R ¹ and the group represented by R ⁴ ,
The following general formula (4)
(Wherein R ³ is as described above.)
A manufacturing method is provided.

In the method for producing coelenteramide or an analog thereof according to the present invention, the di-O-methylcoelenteramide represented by the general formula (3) or an analog thereof is as described above. J-O
Examples of -methylcoelenteramide or an analog thereof include, for example, compounds produced by the above-described method for producing di-O-methylcoelenteramide or an analog thereof.

In the method for producing coelenteramide or an analog thereof according to the present invention, elimination of the group represented by R ¹ and the group represented by R ⁴ can be carried out using an ordinary method, and the method of elimination is not particularly limited.
For example, elimination of methyl can be performed using the method described in the examples or a method analogous thereto.

Here, the solvent used in the method for producing coelenteramide or an analog thereof of the present invention,
Various solvents can be used, and nonpolar solvents are particularly preferred. Examples of the solvent include dichloromethane, chloroform, tetrahydrofuran, toluene, dioxane, ether, and the like, and these can be used alone or in combination.

Further, the reaction temperature and reaction time in the production of the coelenteramide of the present invention or an analog thereof are not particularly limited, but are, for example, −100 ° C. to 200 ° C., 1 hour to 72 hours, preferably −20 ° C. to 100 ° C. 6 hours to 36 hours, more preferably 0 ° C to 50 ° C,
Time to 24 hours.

Examples of coelenteramide represented by the general formula (4) produced by the method of the present invention or an analog thereof include compounds selected from the group consisting of the following compounds.

Coelenteramide or an analog thereof is preferably a compound selected from the group consisting of the following compounds.

In one embodiment of the present invention, the coelenteramide or an analog thereof is coelenteramide shown below.

One embodiment of the present invention is the following formula:
Demethylating di-O-methylcoelenteramide represented by
Following formula
The manufacturing method of coelenteramide represented by these is provided.

2. Fluorescent protein As shown in FIG. 6, green fluorescent protein (gFP) is coelenteramide or its coexistent in the presence of calcium ions such as EDTA or a chelating agent for removing divalent or trivalent ions that can be substituted for calcium ions. It can be produced by reacting an analog (a compound represented by the general formula (4)) with an apoprotein such as apoaequorin. In addition, blue fluorescent protein (BFP) can be produced by adding calcium ions to gFP.

As shown in FIG. 6, BFP can also be obtained by reacting coelenteramide or an analog thereof with apoprotein such as apoaequorin in the presence of calcium ions or divalent or trivalent ions that can be substituted for calcium ions. Can be manufactured. In addition, by reacting coelenterazine or its analog with gFP in the presence of a chelating agent for removing calcium ions such as EDTA or divalent or trivalent ions that can be substituted with calcium ions,
Calcium-binding photoproteins such as aequorin can also be produced.

2.1. Green fluorescent protein (gFP)
2.1.1. Method for Producing Green Fluorescent Protein (gFP) The green fluorescent protein (gFP) produced in the method for producing the green fluorescent protein (gFP) of the present invention is coordinated with coelenteramide or its analog to the apoprotein of the calcium-binding photoprotein. It is a complex. gFP can generate fluorescence upon excitation of light.

In the present invention, gFP is produced from coelenteramide or an analog thereof as follows. That is, coelenteramide or an analog thereof (for example, a compound represented by the general formula (4)) in the presence of a chelating agent for removing calcium ions or divalent or trivalent ions replaceable with calcium ions, GFP is produced by reacting with apoprotein, a calcium-binding photoprotein.

In the present invention, coelenteramide used to produce gFP or an analog thereof is, for example,
It is a compound represented by the general formula (4). The compound represented by the general formula (4) is as described above. As coelenteramide or its analog, the compound manufactured by the manufacturing method of the coelenteramide or its analog of the said this invention can be mentioned, for example.

The chelating agent used for producing gFP in the present invention is not particularly limited as long as it binds strongly to calcium ions or divalent or trivalent ions that can be substituted for calcium ions. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N, N, N ′, N′-tetraacetic acid (EGTA)
, Trans-1,2-diaminocyclohexane N, N, N ′, N′-tetraacetic acid (CyDT
A) or N- (2-hydroxyethyl) iminodiacetic acid (HIDA). Here, divalent or trivalent ions that can be substituted for calcium ions are divalent or trivalent ions that cause a luminescent reaction when reacted with a calcium-binding photoprotein instead of calcium ions. is there. That is, it acts on calcium-binding photoproteins in the same manner as calcium ions. Examples of calcium ions or divalent or trivalent ions that can be substituted for calcium ions include calcium ions (Ca ²⁺ ), magnesium ions (Mg ²⁺ ), strontium ions (Sr ²⁺ ), and barium ions (Ba ²⁺ ). ), Lead ion (Pb ²⁺ ), cobalt ion (Co ²⁺ ), nickel ion (Ni ²⁺ ), cadmium ion (Cd ²⁺ ), yttrium ion (Y ³⁺ ), lanthanum ion (La ³⁺ ), Examples include samarium ion (Sm ³⁺ ), europium ion (Eu ³⁺ ), dysprosium ion (Dy ³⁺ ), thulium ion (Tm ³⁺ ), and yttrium ion (Yb ³⁺ ). Of these, divalent metal ions are preferred. More preferred are divalent metal ions other than transition metals, such as Ca ²⁺ , Sr ²⁺ , or Pb ²⁺ .

The amount of the chelating agent used for the production of gFP is not particularly limited as long as it does not affect gFP regeneration. For example, 3 mol or more is preferable because 1 mol of ion apoaequorin is shown to bind 3 mol of calcium ions.

The apoprotein of the calcium-binding photoprotein used for producing gFP in the present invention is, for example, apoaequorin, apocritin-I, apocritin-II, apooverin, apomytrocomin, apomineopsin or apovelvoin . In one embodiment of the present invention, the apoprotein is apoaequorin, apocrytin-I, apocrytin-II, apoovelin, or apomytrocomine, for example, apoaequorin. The apoprotein may be collected from nature or may be produced by genetic engineering. Furthermore, as long as the apoprotein can form gFP, its amino acid sequence may be mutated from a natural one by a gene recombination technique.

The nucleotide sequence and amino acid sequence of the apoprotein (natural apoprotein) of the photoprotein collected from nature are as follows. That is, the base sequence of natural apoaequorin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence of natural apocrytin-I is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The base sequence of natural apocritin-II is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. The base sequence of natural apomitrocomine is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The base sequence of natural type natural apooverin is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The base sequence of natural apovelvoin is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12.

The apoprotein mutated by the recombinant technique is, for example, a protein selected from the group consisting of the following (a) to (c).

(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of a natural apoprotein, and having an apoprotein activity or function of a calcium-binding photoprotein ,
(B) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of a natural apoprotein and having an apoprotein activity or function of a calcium-binding photoprotein, and (c) a natural apoprotein It consists of an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the protein base sequence, and has an apoprotein activity or function of a calcium-binding photoprotein protein.

The above-mentioned “natural type apoprotein” is, for example, apoaequorin, apocrytin-I, apocrytin-II, apoovelin, apomytrocomin, apomineopsin, or apovelvoin. In one embodiment of the present invention, the apoprotein is apoaequorin, apocrytin-I, apocritin-II, apoovelin, or apomytrocomine, and preferably apoaequorin. The amino acid sequences or base sequences of these natural apoproteins are as described above.

The above-mentioned “apoprotein activity or function of calcium-binding photoprotein” means, for example, the activity or function of apoprotein binding to the coelenterazine peroxide or the peroxide of coelenterazine analog to form a calcium-binding photoprotein. Specifically, “a protein binds to a peroxide of coelenterazine or a peroxide of coelenterazine to form a calcium-binding photoprotein” specifically, (1) the protein binds to a peroxide of coelenterazine or a peroxide of coelenterazine analog. (2) A photoprotein containing a protein and a peroxide of coelenterazine or a coelenterazine analog by contacting the coelenterazine or a derivative thereof in the presence of oxygen. It also means forming a (complex). Here, “contact” means that a protein and coelenterazine or an analog thereof are present in the same reaction system, for example, adding a protein to a container containing coelenterazine or an analog thereof, For example, adding coelenterazine or an analog thereof to the container accommodated, or mixing the protein with coelenterazine or an analog thereof. In addition, “coelenterazine analog” is similar to coelenterazine as an apoprotein,
It refers to a compound that can constitute a calcium-binding photoprotein such as aequorin. Coelenterazine or its analogs include, for example, coelenterazine, h-coelenterazine, f-coelenterazine, cl-coelenterazine, n-coelenterazine, cp-coelenterazine, ch-coelenterazine, hch-coelenterazine, fch-coelenterazine, e-coelenterazine, ef
-Coelenterazine, ech-coelenterazine, or hcp-coelenterazine. Methods for obtaining these coelenterazines or their analogs will be described later.

The range of “1 to plural” in the above “amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added” is, for example, 1 to 20, 1 to 15, 1 to 10 1-9
1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to
Two and one. Generally, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously. Such a region is described in "Sambrook J. et al., Molecular Cloning: A Laborato
ry Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) "," Ausbel
FM et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wil
ey and Sons (1987-1997) "," Nuc. Acids. Res., 10, 6487 (1982) "," Proc. Natl. Acad
Sci. USA, 79, 6409 (1982) "," Gene, 34, 315 (1985) "," Nuc. Acids. Res., 13, 443
1 (1985) ", or" Proc. Natl. Acad. Sci. USA, 82, 488 (1985) "or the like.

The range of “90% or more” in the “amino acid sequence having 90% or more identity” is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95%.
Or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2%
99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7%
Above, it is 99.8% or more, or 99.9% or more. In general, the larger the numerical value of identity, the better. The identity of amino acid sequences and base sequences is determined by BLAST (for example, Altz
shul SF et al., J. Mol. Biol. 215, 403 (1990), etc.). When using BLAST, the default parameters of each program are used.

The above-mentioned “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide consisting of a base sequence complementary to the base sequence of the natural apoprotein or a polynucleotide encoding the amino acid sequence of the natural apoprotein. A polynucleotide (for example, a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of the probe as a probe (for example,
DNA). Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which a polynucleotide derived from colony or plaque was immobilized, and then 0.1 to 2 Double-concentration SSC (Saline-sodium citrate) solution (composition of single-concentration SSC solution is 150 mmol / L sodium chloride, 15 mmol
/ L consisting of sodium citrate) and a polynucleotide that can be identified by washing the filter under 65 ° C conditions.

Hybridization was performed using Sambrook J. et al., Molecular Cloning: A Laboratory.
Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel FM
et al., Current Protocols in Molecular Biology, Supplement 1-38, John Wiley and
Sons (1987-1997) or Glover DM and Hames BD, DNA Cloning 1: Core Techniq
It can be carried out according to the methods described in experimental documents such as ues, A practical Approach, Second Edition, Oxford University Press (1995).

The “stringent conditions” referred to herein may be low stringent conditions, medium stringent conditions, or high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide, 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 ×
Denhardt's solution, 0.5% (w / v) SDS, 50% (v / v) formamide at 42 ° C. “High stringent conditions” include, for example, 5 × SSC, 5 × Denhardt's solution, 0.5 (w / v)% SDS, 50% (v
/ v) Formamide at 50 ° C. The tighter the conditions, the higher the complementarity required for duplex formation. Specifically, for example, it can be expected that a polynucleotide (eg, DNA) having high homology as the temperature is increased can be efficiently obtained under these conditions. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

In addition, when using a commercially available kit for hybridization, for example, Alkphos Direct
Labeling Reagents (manufactured by Amersham Pharmacia) can be used. In this case, follow the protocol attached to the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. , Hybridized DNA can be detected.

As other polynucleotides that can be hybridized, when calculated using the default parameters by an analysis program such as BLAST, the polynucleotide encoding the amino acid sequence of the apoprotein is about 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more, 99.5% Above, DNA with 99.7% or more, 99.8% or more, 99.9% or more identity
Can give. The identity of amino acid sequences and base sequences can be determined using the method described above.

In some embodiments of the invention, the apoprotein has all cysteine residues replaced with serine residues. gFP loses chemiluminescence activity when the free SH group of a cysteine residue in an apoprotein is oxidized to form an SS bond. Therefore, an apoprotein in which a cysteine residue is substituted with a serine residue and cannot generate an SS bond hardly loses chemiluminescence activity and does not generate an SS bond, so that the activity continues.

The amount of coelenteramide or its analog used for the production of gFP is not particularly limited, but for example, 1 mol to 5 mol, preferably 1 mol to 2 mol, more preferably 1 mol to 1.2 mol with respect to 1 mol of apoprotein. is there.

In the production of gFP, the reaction between coelenteramide or its analog and apoprotein is preferably carried out in the presence of a reducing agent. Examples of the reducing agent used here include dithiothreitol (DTT) and mercaptoethanol. The amount of reducing agent used for the production of gFP is not particularly limited as long as it does not affect gFP regeneration.
Since there are three cysteine residues, the concentration is preferably such that SS bond formation is prevented. For example, the final concentration is 1 mM dithiothreitol or 0.1% mercapethanol.

Although the reaction temperature and reaction time in manufacture of gFP are not specifically limited, For example, 0 degreeC-42
0.1 ° C to 2 hours at 4 ° C, 0.1 to 2 hours at 4 ° C to 37 ° C, or 0.1 to 24 hours at 4 ° C to 15 ° C
It's time.

The gFP thus obtained may be further subjected to purification. The purification of gFP can be performed according to a normal separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in appropriate combination. be able to.

Some embodiments of the present invention have the following formula in the presence of a chelating agent for removing calcium ions or divalent or trivalent ions replaceable with calcium ions:
A green fluorescent protein (gFP) comprising reacting a compound represented by apoaequorin with
). In one embodiment of the invention, as shown in FIG. 5, in the presence of EDTA,
GFP is produced by reacting coelenteramide with apoaequorin.

2.1.2. Use of green fluorescent protein (gFP) (1) Use as a reporter protein
gFP can be used as a reporter protein for measuring transcriptional activity of a promoter and the like. For example, a vector is constructed in which a polynucleotide encoding an apoprotein is fused to a target promoter or other expression control sequence (for example, an enhancer). The vector is introduced into a host cell, and coelenteramide or an analog thereof is contacted in the presence of calcium ion or a chelating agent for removing a divalent or trivalent ion that can replace the calcium ion. Thus, the activity of the target promoter or other expression control sequence can be measured by generating gFP and detecting fluorescence derived from gFP.

(2) Use as a detection marker
gFP can be used as a fluorescent detection marker. The detection marker is, for example,
It can be used for detection of a target substance in an immunoassay or a hybridization assay. gFP can be used by binding to a target substance (protein or nucleic acid) by a commonly used method such as a chemical modification method. A detection method using such a detection marker can be performed by a normal method. The detection marker of the present invention is, for example,
It is expressed as a fusion protein of an apoprotein and a target substance, introduced into a cell by a technique such as microinjection, and further, coelenteramide or an analog thereof can be replaced with calcium ions or calcium ions. It can also be used to measure the distribution of the target substance by, for example, generating gFP by contacting in the presence of a chelating agent for removing trivalent ions. Such measurement of the distribution of the target substance can also be performed using a detection method such as fluorescence imaging. In addition, the apoprotein can be expressed in the cell and used in addition to being introduced into the cell by a technique such as a microinjection method.

(3) Amusement equipment materials
gFP can be suitably used as a fluorescent material for amusement goods. Examples of amusement goods include fluorescent soap bubbles, fluorescent ice, fluorescent glaze, fluorescent paints, and the like. The amusement article can be manufactured by a usual method.

2.2. Blue fluorescent protein (BFP)
2.2.1. Blue fluorescent protein (BFP) production method Blue fluorescent protein (BFP) is produced by adding calcium ions or divalent or trivalent ions that can be substituted for calcium ions to the gFP produced by the production method of the present invention. can do. Alternatively, blue fluorescent protein (BFP) is coelenteramide produced by the method of the present invention or an analog thereof (general formula (4)) in the presence of calcium ions or divalent or trivalent ions that can be substituted for calcium ions. It can also be produced by reacting a compound represented) with a calcium-binding apoprotein. The blue fluorescent protein (BFP) produced in the present invention is a complex in which coelenteramide or an analog thereof is coordinated to an apoprotein of a calcium-binding photoprotein. BFP can generate fluorescence upon receiving excitation of light, and can also emit light by reacting with calcium ions or divalent or trivalent ions that can be substituted for calcium ions.

“Calcium ions or divalent or trivalent ions that can be substituted for calcium ions” is the same as described above. The amount of calcium ions or divalent or trivalent ions that can be substituted for calcium ions used for producing BFP is not particularly limited, but calcium ions or divalent that can be substituted for calcium ions used for producing BFP is not limited. Alternatively , the concentration of trivalent ions is not particularly limited as long as it does not affect the BFP regeneration. Since 1 mol of ion apoaequorin is shown to bind 3 mol of calcium ions, 3 mol or more is preferable.

In the production of BFP, the reaction between coelenteramide or its analog and an apoprotein is preferably carried out in the presence of a reducing agent. Examples of the reducing agent used here include dithiothreitol (DTT) and mercaptoethanol.
The amount of reducing agent used for the production of BFP is not particularly limited as long as it does not affect BFP regeneration, but apoaequorin has a concentration that prevents SS bond formation due to the presence of three cysteine residues. Preferably there is. For example, the final concentration is 1 mM dithiothreitol or 0.1% mercapethanol.

Although the reaction temperature and reaction time in the production of BFP are not particularly limited, for example, 0 ° C. to 42 ° C.
And 0.1 to 2 hours, 4 to 37 ° C., 0.1 to 2 hours, or 4 to 15 ° C. and 0.1 to 24 hours.

The BFP thus obtained may be further subjected to purification. Purification of BFP can be performed according to a normal separation / purification method. Separation / purification methods include, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, dialysis, ultrafiltration, etc., alone or in appropriate combination. be able to.

2.2.2. Utilization of blue fluorescent protein (BFP) (1) Utilization as luminescent catalyst The BFP of the present invention acts on a luminescent substrate to cause it to emit light, and thus can be utilized as a luminescent catalyst. Therefore, the present invention includes contacting coelenterazine or an analog thereof with BFP,
A light emitting method is provided. Here, “contact” means that BFP and coelenterazine or an analogue thereof are present in the same reaction system, for example, adding BFP to a container containing coelenterazine or an analogue thereof, For example, adding coelenterazine or an analog thereof to the container accommodated, or mixing BFP and coelenterazine or an analog thereof.

The luminescent substrate used in the luminescent method of the present invention is, for example, coelenterazine or an analog thereof.
The term “coelenterazine analog” refers to a compound that can constitute a calcium-binding photoprotein such as aequorin as an apoprotein, like coelenterazine. Coelenterazine or its analog used as a luminescent substrate is, for example, coelenterazine, h-coelenterazine, f-
Coelenterazine, cl-coelenterazine, n-coelenterazine, cp-coelenterazine,
ch-coelenterazine, hch-coelenterazine, fch-coelenterazine, e-coelenterazine, ef-coelenterazine, ech-coelenterazine, or hcp-coelenterazine, and preferably coelenterazine, h-coelenterazine, or e-coelenterazine. These coelenterazines or analogs thereof are described in, for example, Shimomura et al. (1988
) Biochem. J. 251 , 405-410, Shimomura et al. (1989) Biochem. J. 261 , 913-920, or
It can be produced by the method described in Shimomura et al. (1990) Biochem. J. 270 , 309-312 or a method analogous thereto. Alternatively, since various types are commercially available from Chisso Corporation, Wako Pure Chemical Industries, or Promega Corporation, these commercially available products may be used in the light emitting method of the present invention.

When these coelenterazines and their analogs are brought into contact with BFP, and when the coelenterazine or its analogs are oxidized to the corresponding coelenteramide or its analogs by the catalytic action of the contacted BFP (at this time, carbon dioxide is released) ), Light emission occurs. Normal light emission time is 0.5
Although it is ˜3 hours, the light emission time can be made longer or the light emission time can be made shorter depending on the selection of conditions.

(2) Use as a reporter protein
BFP can also be used as a reporter protein for measuring transcriptional activity of promoters and the like. A vector is constructed in which a polynucleotide encoding an apoprotein is fused to a target promoter or other expression control sequence (for example, an enhancer). The vector is introduced into a host cell, and is further contacted with coelenteramide produced by the method of the present invention or an analog thereof and calcium ion or divalent or trivalent ion that can be substituted with calcium ion, and By detecting fluorescence derived from the fluorescent protein, the activity of the target promoter or other expression control sequence can be measured. Here, “contact” means that a host cell, coelenteramide or an analog thereof, and calcium ions or divalent or trivalent ions capable of substituting calcium ions are present in the same culture system / reaction system. For example, adding coelenteramide or its analog and calcium ion or divalent ion that can replace calcium ion to a host cell culture vessel, host cell and coelenteramide or analog thereof and calcium ion or calcium ion And divalent or trivalent ions that can be substituted, and culturing host cells in the presence of coelenteramide or its analogs and calcium ions or divalent or trivalent ions that can be substituted for calcium ions, etc. Is included.

(3) Use as a detection marker
BFP can be used as a fluorescent detection marker. The detection marker of the present invention is
For example, it can be used for detection of a target substance in an immunoassay or a hybridization assay. The target substance (
It can be used in combination with protein or nucleic acid. A detection method using such a detection marker can be performed by a normal method.

The detection marker of the present invention is expressed, for example, as a fusion protein of an apoprotein and a target substance, introduced into a cell by a technique such as a microinjection method, and further coelenteramide produced by the method of the present invention or It can also be used to measure the distribution of the target substance by contacting the analog and calcium ions or divalent or trivalent ions that can be substituted for calcium ions. Here, “contact” means that cells, coelenteramide or an analog thereof, and calcium ions or divalent or trivalent ions that can be substituted for calcium ions are present in the same culture system / reaction system, For example, coelenteramide or its analogs and calcium ions or divalent ions that can replace calcium ions can be added to cell culture vessels, and cells and coelenteramide or analogs thereof can be replaced with calcium ions or calcium ions Mixing with divalent or trivalent ions, and culturing host cells in the presence of coelenteramide or its analogs and calcium ions or divalent or trivalent ions that can be substituted for calcium ions. .

Such measurement of the distribution of the target substance can also be performed using a detection method such as fluorescence imaging. In addition, the apoprotein can be expressed in the cell and used in addition to being introduced into the cell by a technique such as a microinjection method.

(4) Amusement equipment materials
BFP generates fluorescence under the excitation of light. Therefore, BFP can be suitably used as a fluorescent base material for amusement material. Examples of amusement goods include fluorescent soap bubbles, fluorescent ice, fluorescent glaze, fluorescent paints, and the like. The amusement product of the present invention can be manufactured by a usual method.

3. Calcium-binding photoprotein As shown in FIG. 6, calcium-binding photoproteins such as aequorin are present in the presence of a chelating agent for removing calcium ions such as EDTA or divalent or trivalent ions that can be substituted for calcium ions. Below, it can manufacture by making coelenterazine or its analog react with gFP.

3.1. Production of calcium-binding photoprotein The calcium-binding photoprotein of the present invention can be produced from the gFP of the present invention. That is, the calcium-binding photoprotein of the present invention can be obtained by reacting gFP with coelenterazine, which is a luminescent substrate, or an analog thereof.

The reaction between gFP and coelenterazine or an analog thereof is performed by bringing gFP into contact with coelenterazine or an analog thereof. “Contact” means that the gFP of the present invention and coelenterazine or an analog thereof are present in the same reaction system. For example, the gFP of the present invention is added to a container containing coelenterazine or an analog thereof. , Adding coelenterazine of the present invention or an analog thereof to a container containing the gFP of the present invention, or mixing the gFP of the present invention with coelenterazine or an analog thereof, and the like.

Coelenterazine or its analogs used for producing the calcium-binding photoprotein of the present invention include, for example, coelenterazine, h-coelenterazine, f-coelenterazine, cl-coelenterazine, n-coelenterazine, cp-coelenterazine, ch-coelenterazine, hch-
Coelenterazine, fch-coelenterazine, e-coelenterazine, ef-coelenterazine, ech-coelenterazine, or hcp-coelenterazine, and preferably coelenterazine, h-coelenterazine, or e-coelenterazine. The method for obtaining these coelenterazines or their analogs is as described above.

The amount of coelenterazine or its analog used for producing the calcium-binding photoprotein is not particularly limited, but is, for example, 1.2 mol or more with respect to 1 mol of gFP.

The reaction temperature and reaction time in the production of the calcium-binding photoprotein are not particularly limited. For example, the reaction temperature is 0 to 42 ° C, 0.1 to 2 hours, 4 to 37 ° C, 0.1 to 2 hours, or 4 ℃
At -15 ° C, 0.1 hour to 24 hours.

In a preferred embodiment of the present invention, the reaction between the fluorescent protein and coelenterazine or an analog thereof is performed.
It is carried out in the presence of a reducing agent. The reducing agent used at this time is, for example, dithiothreitol (
DTT), or mercaptoethanol. The amount of the reducing agent used for the production of the calcium-binding photoprotein is not particularly limited as long as it does not affect the regeneration. However, since apoaequorin has three cysteine residues, it can form SS bonds. The concentration to prevent is preferred. For example, the final concentration is 1 mM dithiothreitol or 0.1% mercapethanol.

3.2. Use of calcium-binding photoprotein (1) Detection or quantification of calcium ions The calcium-binding photoprotein of the present invention is a photoprotein (holoprotein) that emits light by the action of calcium ions. Therefore, the photoprotein of the present invention can be used for detection or quantification of calcium ions.

When detecting or quantifying calcium ions, a photoprotein consisting of an apoprotein and a peroxide of coelenterazine analog is used. The photoprotein can be produced according to the method described above. Detection or quantification of calcium ions can be performed by directly adding the sample solution to the photoprotein solution and measuring the generated luminescence. Alternatively, calcium ions can be detected or quantified by adding a photoprotein solution to the sample solution and measuring the generated luminescence. The detection of calcium ions can be performed, for example, by adding the sample solution directly to the photoprotein solution and measuring the generated luminescence. Alternatively, calcium ions can be detected by adding a photoprotein solution to the sample solution and measuring the generated luminescence.

The detection or quantification of calcium ions can be performed by measuring the luminescence of the photoprotein of the present invention by calcium ions using a luminescence measuring device. As the luminescence measuring device, a commercially available device, for example, Centro LB 960 (manufactured by Bertrand) can be used. Quantification of the calcium ion concentration can be measured by creating a luminescence standard curve for a known calcium ion concentration using a photoprotein.

(2) Bioluminescence Resonance Energy Transfer (BRET) Method The calcium-binding photoprotein of the present invention is analyzed for physiological function and measurement of enzyme activity using the principle of intermolecular interaction by bioluminescence resonance energy transfer (BRET) method. It can be used for analysis methods such as.

For example, when the photoprotein of the present invention is used as a donor protein and an organic compound or a fluorescent protein is used as an acceptor protein, bioluminescence resonance energy transfer (BRE) between the two is used.
By causing T), the interaction between proteins can be detected. In one embodiment of the present invention, the organic compound used as the acceptor protein is Hoechist3342, Indo-1, or DAP1.
Etc. In another aspect of the present invention, the fluorescent protein used as the acceptor protein is:
Examples include green fluorescent protein (GFP), blue fluorescent protein (BFP), mutant GFP fluorescent protein, or phycobilin. In a preferred embodiment of the present invention, the physiological function to be analyzed is an orphan receptor (
In particular, G protein-coupled receptors), apoptosis, or transcriptional regulation by gene expression.
In a preferred embodiment of the present invention, the enzyme to be analyzed is a protease, esterase or phosphorylase.

Analysis of physiological functions by the BRET method can be performed by a known method, for example, Biochem.
J. 2005, 385, 625-637, or Expert Opin. Ther Tarets, 2007 11: 541-556. The enzyme activity can also be measured by a known method, for example, according to the method described in Nat Methods 2006, 3: 165-174 or Biotechnol J. 2008, 3: 311-324. Can do.

It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes.

The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily implement the present invention from the description of the present specification. it can. The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for illustration or description, and the present invention is not limited thereto. Not what you want. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

[Example 1]
Synthesis of coelenteramide

4-Methoxyphenylacetyl chloride (Aldrich) used for synthesis, BBr ₃ 1.0M CH ₂ C
l The ₂ solution (Aldrich) and all other chemical reagents were commercially available and were used as they were.
Silica gel was pre-applied to analytical thin layer chromatography (TLC) (0.2%).
5 mm) silica gel plates (MERCK, silica gel 60 F ₂₅₄ , catalog number 1.05715.0009) were used.
For preparative column chromatography, silica gel (Kanto Chemical Co., Inc., silica gel 60N, spherical, neutral, catalog number 37563-84) was used.
Melting point (Mp.) Was measured using YANACO MP-J3 (uncorrected). ¹ H (300 MHz) nuclear magnetic resonance spectrum (NMR spectrum) and ¹³ C (75.5 MHz) NMR spectrum are from Varian Gem
Measurement was performed in DMSO-d6 (CIL) using ini-300. ^As a reference for the chemical shift of ¹ H NMR, the peak of the remaining non-deuterated dimethyl sulfoxide in DMSO-d _{6 as the} measurement solvent was designated as Δ2.49. ¹³
As the standard of chemical shift of C NMR, the peak of DMSO-d ₆ which is a measurement solvent was designated as δ 39.7, and each was expressed in ppm. Further, the coupling constant (J) is shown in unit Hz. The abbreviations s, m and br represent singlet, multiplet and broad, respectively. Infrared spectroscopy (IR) spectrum was measured by a diffuse reflection method using a spectrometer SHIMADZU IRPrestige-21 equipped with DRS-8000A, and the absorption band was shown in the unit cm ⁻¹ . High resolution mass spectrometry (HRMS) is based on the mass spectrometer JE under the conditions of electron impact ionization (EI).
We used OL JMS-700.

Coelenteramide synthesis scheme

Coelenteramide dimethyl ether (IV)
2-amino-3-benzyl-5- (4-methoxyphenyl) pyrazine (II) (also called coelenteramine methyl ether) in pyridine under an argon atmosphere (Kishi et al., Tetrahedron Le
tt., 13, 2747-2748 (1972); Adamczyk et al., Org. Prep. Proced. Int., 33, 477-48
5 (2001)) (502 mg, 1.72 mmol), 4- (dimethylamino) pyridine (DMAP) (21.1 mg, 172 μmol) and 4-methoxyphenylacetyl chloride (III) (527
μL, 3.45 mmol) was subsequently added at room temperature and the mixture was stirred at the same temperature for 22.5 hours. To this, a saturated aqueous solution of sodium hydrogen carbonate (100 mL) was added, and the mixture was extracted with dichloromethane (50 mL × 3). The organic extract was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Residual pyridine was removed azeotropically using toluene (20 mL × 3). The residue was purified by silica gel column chromatography (85 g, dichloromethane / ethyl acetate = 9/1) and coelenteramide dimethyl ether (IV) (617 mg, 81.5% as a pale yellow solid)
) Recrystallization from ethyl acetate gave a colorless solid as an analytically pure sample (total of 2 recrystallizations yielded 458 mg, 60.5%).

Mp. 189.5-191 ° C; ¹ H NMR (300 MHz, DMSO-d ₆ ) δ3.61 (s, 2H), 3.73 (s, 3H), 3.80
(s, 3H), 4.03 (s, 2H), 6.88-6.93 (AA'BB ', 2H), 7.02-7.07 (2 (AA'BB', 4H), 7.12
-7.30 (m, 5H), 8.00-8.05 (AA'BB ', 2H), 8.87 (s, 1H), 10.43 (s, 1H); ¹³ C NMR (75.
5 MHz, DMSO-d ₆ ) δ 41.6, 55.1, 55.3, 113.9 (2C), 114.5 (2C), 126.2, 127.5, 128.0
(2C), 128.1, 128.2 (2C), 129.0 (2C), 130.2 (2C), 137.1, 138.3, 143.7, 148.2, 15
0.5, 158.2, 160.7, 170.3; IR (KBr, cm ^-1 ) 698, 833, 1034, 1177, 1256, 1495, 1514,
1543, 1672, 2833, 2957, 3265; HRMS (EI) m / z 439.1898 (M ⁺ , C ₂₇ H ₂₅ N ₃ O ₃ requires 4
39.1896).

Coelenteramide (I)
Coelenteramide dimethyl ether (IV) (6) in anhydrous dichloromethane under argon atmosphere
60 mg, 1.50 mmol) in solution (20 mL) at 0 ° C. over 10 minutes with boron tribromide (6.01 mL, 6.01
mmol) in 1.0 M dichloromethane and the mixture was stirred at the same temperature for 15 min. The mixture was warmed to room temperature and kept stirring for 21 hours. To this was added saturated aqueous sodium bicarbonate (100 mL) and the mixture was concentrated under reduced pressure to remove dichloromethane. The residual aqueous suspension was filtered and the collected solid was dried in vacuo to give a coelenteramide (I) (
570 mg, 92.3))%) was obtained. Samples of this purity can be fully utilized in the production of gFP and BFP.
Recrystallization from ethanol gave a colorless solid as an analytically pure sample (10
3 mg, 16.7%).

Mp. 242-243 ° C (dec.); ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 3.54 (s, 2H), 4.01 (s, 2H)
, 6.69-6.75 (AA'BB ', 2H), 6.84-6.90 (AA'BB', 2H), 7.00-7.06 (AA'BB ', 2H), 7.11-7
.24 (m, 5H), 7.89-7.95 (AA'BB ', 2H), 8.80 (s, 1H), 9.28 (br s, 1H), 9.85 (br s,
1H), 10.35 (s, 1H); ¹³ C NMR (75.5 MHz, DMSO-d ₆ ) δ41.7, 115.2 (2C), 115.8 (2C),
125.7, 126.2, 126.6, 128.0 (2C), 128.2 (2C), 129.0 (2C), 130.2 (2C), 136.8, 138.
4, 143.4, 148.6, 150.5, 156.2, 159.1, 170.5; IR (KBr, cm ^-1 ) 704, 1157, 1229, 126
7, 1364, 1450, 1493, 1516, 1545, 1593, 1611, 1673, 3022, 3285, 3385; HRMS (EI) m
/ z 411.1582 (M ⁺ , C ₂₅ H ₂₁ N ₃ O ₃ requires 411.1583).

[Example 2]
Expression and purification of recombinant histidine-labeled apoaequorin from bacterial cells Recombinant histidine-labeled apoaequorin was expressed in the periplasm of E. coli. The host strain used is E. coli strain WA802 (CGSC 5610). A recombinant histidine-tagged apoaequorin expression vector piP-His-HE introduced into E. coli strain WA802 was constructed from piP-HEΔ2E (Inouye
& Sahara, Protein Express. Purifi. (2007) 53: 384-389).

That is, piP-HEΔ2E was cleaved with EcoRI and HindIII. And EcoRI / Hi of piP-HEΔ2E
A histidine tag linker set containing 6 histidine residues at the ndIII site (SEQ ID NO: 13
: 5'AAT TCC CAC CAT CAC CAT CAC CAT GGT A 3 'and SEQ ID NO: 14: 5'AG CTT ACC ATG
GTG ATG GTG ATG GTG GG 3 ′) was inserted. Thereby, plasmid piP-His-HE was prepared. This plasmid is under the control of the lipoprotein (lpp) promoter and the lac operator.
It has a signal peptide sequence of outer membrane protein A (OmpA) and 6 histidine residues.

For the expression of apoaequorin, Escherichia coli introduced with piP-His-HE was treated with ampicillin (50
in 16 ml LB medium containing 10 μg / ml) at 30 ° C. for 16 hours, and this was grown in 400 ml LB medium containing 50 μl of antifoam (Disform CE475, Nippon Oil & Fat Tokyo) in a 3 L Sakaguchi flask. Added to.
The cells were cultured at 37 ° C. for 18 hours while performing reciprocal shaking (170 rpm / min), and then centrifuged at 5,000 g for 5 minutes to recover cells from 1 L of the culture solution. The collected cells were washed with 90 ml of 50 mM Tris-HCl (pH 7.
After suspending in 6), sonication was performed using a Model 250 Sonifier from Branson (Danbury, Conn.). The supernatant obtained by centrifugation at 12,000 g for 20 minutes at 4 ° C was added to 50 mM Trim
Nickel chelate column equilibrated with s-HCl (pH 7.6) (Pharmacia, 1.5 x 4.0 cm)
It was used for. The column was washed with 140 ml of 50 mM Tris-HCl (pH 7.6) and 50 m containing 0.1 M imidazole.
The adsorbed apoaequorin protein was eluted using 100 ml of M Tris-HCl (pH 7.6).

  A summary of the purification yield is shown in Table 1. 150 mg of apoaequorin was obtained from 1 L of cultured cells.

Further purification of apoaequorin was achieved by using apoaequorin (26 mg) eluted from a nickel chelate column with 0.1 M imidazole, 30 mM containing 10 mM EDTA and 5 mM dithiothreitol.
It was dissolved in 5 ml of Tris-HCl (pH 7.6) (TED buffer) and left at 4 ° C. for 18 hours. 100 μl in this mixture
Of 0.1 M acetic acid produces a precipitate containing white apoaequorin. The white precipitate was collected by centrifuging at 10,000 xg for 10 minutes. The obtained precipitate was added to 5 ml of TED buffer, and 8 μl of 25% ammonia solution was dissolved therein. Then, the insoluble precipitate was removed by centrifugation, and the supernatant was used as purified apoaequorin.

[Example 3]
Using a commercially available kit for protein analysis (Bio-Rad, Richmond, Calif.) And bovine serum albumin (Pierce, Rockford, Ill.) As a standard, Bradford's dye binding method (
Anal. Biochem. (1976) 72, 248-254), the protein concentration was determined.
Further, as described in Laemmli (Nature (1970) 227, 680-685), SDS-PAGE analysis was performed under reducing conditions using a 12% separation gel (Tefco Corporation, Tokyo). As a result, as shown in FIG. 1, it was revealed that the purity of the purified apoaequorin obtained by this purification method was 95% or more.

[Example 4]
Preparation of synthetic BFP from coelenteramide and apoaequorin
Apoaequorin (0.5 mg) in 1 ml of 50 mM Tris-HCl (pH 7.6) containing 10 mM CaCl ₂ and 1 mM DTT
, 22 nmol) and 10 μl coelenteramide (1.2 μg / μl in anhydrous methanol, 29 nmol), and this mixture was allowed to stand at 4 ° C. for 16 hours to prepare a synthetic BFP (syn-BFP). Subsequently, using a centrifugal concentrator Vivaspin 2 (10,000 MWCO, Sartorius AG, Germany) at 4 ° C, 5,000 x
Treated with g for 20 minutes and concentrated the mixture to 0.1 ml. The concentrated solution is a long wave UV lamp (366
nm).

Preparation of synthetic gFP from coelenteramide and apoaequorin For the preparation of synthetic gFP (syn-gFP) from apoaequorin and coelenteramide, 10 mM
Apoaequorin (0.5 mg, 22 nmo) in 1 ml of 50 mM Tris-HCl (pH 7.6) containing EDTA and 1 mM DTT
l) and 10 μl coelenteramide (1.2 μg / μl in anhydrous methanol, 29 nmol) were mixed, and this mixture was allowed to stand at 4 ° C. for 16 hours to prepare a synthetic gFP (syn-gFP). The fluorescence emission spectrum shown in FIG. 2 was obtained by exciting at 335 nm. Apoaequorin and coelenteramide confirmed the production of syn-gFP with fluorescence.
On the other hand, by adding DTT as a reducing agent to syn-gFP and syn-BFP preparation solutions, Table 2
As summarized in the above, it was revealed that syn-gFP and syn-BFP can be efficiently prepared in a short time.

[Example 5]
Measurement of absorption and fluorescence spectra
Jasco (JASCO Corporation, Tokyo) V-560 spectrophotometer (bandwidth: 0.5nm, response:
The absorption spectrum was measured at 25 ° C. using a quartz cell (optical path length 10 mm) at a medium scan rate of 100 nm / min. Jasco's FP-6500 fluorescence spectrophotometer (emission / excitation bandwidth: 3
nm, response: 0.5 sec, scan speed: 1000 nm / min).
As shown in FIG. 3, the fluorescence spectrum of the obtained syn-gFP is gFP prepared from aequorin.
Consistent with the spectrum. Moreover, as shown in Table 3, the ratio of 280 nm and 330 nm in the absorption spectrum of the prepared syn-BFP is almost the same as or higher than that of BFP prepared from aequorin. Became possible.

[Example 6]
Luminescent activity assay reaction mixture (100 μl) was mixed with coelenterazine (0.5 μl) in 50 mM Tris-HCl (pH 7.6) -10 mM CaCl _2.
g, dissolved in 1 μl of ethanol). The reaction was initiated by adding the protein solution (1 μg of BFP and syn-BFP), and the luminescence intensity was measured using an AB2200 illuminometer manufactured by Atto Corporation (Tokyo) equipped with an R4220P photomultiplier tube manufactured by Hamamatsu Photonics. Was recorded for 1 minute. The maximum intensity (I _max ) of 1 ng of purified recombinant aequorin was 8.8 × 10 ⁵ rlu (relative light units).
As shown in FIG. 4, syn-BFP prepared using synthetic coelenteramide had a luminescent activity equivalent to that of BFP prepared from aequorin.

[Sequence Listing Free Text]
[SEQ ID NO: 1] This is the base sequence of natural apoaequorin.
[SEQ ID NO: 2] is the amino acid sequence of natural apoaequorin.
[SEQ ID NO: 3] This is the base sequence of natural apocrytin-I.
[SEQ ID NO: 4] is an amino acid sequence of natural apocrytin-I.
[SEQ ID NO: 5] This is the base sequence of natural apocrytin-II.
[SEQ ID NO: 6] is the amino acid sequence of natural apocrytin-II.
[SEQ ID NO: 7] is the base sequence of natural apomytrocomin.
[SEQ ID NO: 8] is an amino acid sequence of natural apomytrocomin.
[SEQ ID NO: 9] This is the base sequence of natural apooverin.
[SEQ ID NO: 10] is the amino acid sequence of natural apooverin.
[SEQ ID NO: 11] This is the base sequence of natural apovelvoin.
[SEQ ID NO: 12] is an amino acid sequence of natural apovelvoin.
[SEQ ID NO: 13] This is the base sequence of the histidine tag linker set used in the examples.
[SEQ ID NO: 14] This is the base sequence of the histidine tag linker set used in the examples.

Claims (3)

In the presence of a chelating agent and a reducing agent for removing calcium ions or divalent or trivalent ions that can be substituted for calcium ions,
The following general formula (4)
A compound represented by the formula:
R ³ is alkyl having 1 to 7 carbons, an aliphatic cyclic group, or arylalkyl having 7 to 10 carbons which may be substituted with an aliphatic cyclic group. )
A method for producing a green fluorescent protein (gFP) or an analog thereof , which comprises reacting a fluorescent protein with an apoprotein of a calcium-binding photoprotein. In the presence of a chelating agent and a reducing agent for removing calcium ions or divalent or trivalent ions that can be substituted for calcium ions, the following formula:
A method for producing a green fluorescent protein (gFP), which comprises reacting a compound represented by the formula with apoaequorin. The method according to claim 1, wherein the apoprotein is apoaequorin, apocritin-I, apocrytin-II, apooberin, apomytrocomin, apomineopsin or apovelvoin.