JPH05317043A - Bacterial hololuciferase - Google Patents

Abstract

PURPOSE:To obtain a new bacterial hololuciferase, derived from a luminous bacterium, composed of luciferase, a flavin mononucleotide and a substance exhibiting a specific absorption spectrum and capable of measuring reduced form nicotinamide adenine dinucleotide (NADH) or reduced form nicotinamide adenine dinucleotide phosphate (NADPH) in a simpler manner and at a lower cost than those of a conventional method. CONSTITUTION:The enzyme is obtained by culturing a luminous bacterium such as Vibrio.harveyi or Photobacterium.phosphoreum or transforming a host cell with an expression vector containing a gene capable of coding luciferase derived from the luminous bacterium and then purifying the resultant transformant in the presence of preferably NADH or NADPH by using chromatography, etc. This enzyme is composed of a substance capable of exhibiting an absorption spectrum at 350, 405, 540 and 570nm.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel holoenzyme derived from a luminescent bacterium, more specifically, luciferase and
Flavin mononucleotide, 350nm, 405n
and a substance exhibiting a characteristic absorption spectrum at m, 540 nm and 570 nm with a bacterial hololuciferase.

[0002]

2. Description of the Related Art Coenzyme NA utilizing the luminescent system of luminescent bacteria
D and NADPH are measured based on the following reaction catalyzed by two enzymes possessed by luminescent bacteria.

Oxidoreductase (a) NAD (P) H + H ⁺ + FMN → NAD (P) ⁺ + FMNH ₂

Luciferase (b) FMNH ₂ + RCHO + O ₂ → FMN + RCOOH + H ₂ O + hν

The reaction (a) is performed by NADH: FMN oxidoreductase (EC 1.1.69.3) or NAD.
PH: FMN oxidoreductase (EC 1.6.9
9.1 Reduced flavin mononucleotide (FMN
H ₂ ).

On the other hand, the reaction (b) is a luciferase (E
C. 1.14.14.3) from FMNH ₂ , long-chain aliphatic aldehyde (RCHO) and oxygen (O ₂ ) into FMN
And a fatty acid (RCOOH) corresponding to the aldehyde, water (H ₂ O) and light are generated. Where RCH
As O, a linear saturated aliphatic aldehyde having 8 or more carbon atoms is desirable, and for example, n-decanal, n-tetradecanal and the like are preferably used. Therefore, NAD (P) H can be quantified by measuring the light generated by the above reaction.

Since this luminescence reaction is rapid and has high measurement sensitivity, not only can NADH and NADPH be measured with high sensitivity, but if this reaction system is combined with an enzyme system that produces NADH or NADPH, these enzyme systems Substrate concentration and enzyme activity can be measured with high sensitivity. For example, J. Ford and M. DeLuca; Anal. Biochem., 110.43-4
8 (1981). G. Weinhausen and M. DeLuca; Anal. Bioche
m., 127. 380-388 (1982). MTKarp et al.; Anal. Bi
Ochem., 128. 175-180 (1983), etc., describes measurement examples.

[0008]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In the method for measuring NADH or NADPH by the reaction system of (a) and (b) above,
In particular, NADH: FMN oxidoreductase and NADPH: FMN oxidoreductase used in the reaction (a) are presently cultured and extracted and purified from luminescent bacteria. This purification method is described, for example, in E. Jablonski and M.
DeLuca; Biochemistry. 16.13.2932-2936 (1977). H. Wat
anabe and JWHastings; Molecular and Cellular Bioc
hem., 44.181-187 (1982).

However, NADH: FMN oxidoreductase and N obtained from a luminescent bacterium by the above-mentioned preparation method
ADPH: FMN oxidoreductase is expensive due to its low yield, and therefore has a drawback that the measurement cost is high.

NADH was obtained using only luciferase without using either NADH: FMN oxidoreductase or NADPH: FMN oxidoreductase.
If NADPH and NADPH can be quantified, the measurement operation can be simplified and the cost of NADH: FMN is high.
Since oxidoreductase and NADPH: FMN oxidoreductase are not used, measurement cost can be saved.

The present invention has been devised to meet the above demands.

[0012]

[Means for Solving the Problems] The present inventors have found that luciferase produced by a luminescent bacterium or luciferase produced by a transformant obtained by transforming a host cell with an expression vector containing a gene for a luminescent bacterium. Was found to form a complex with flavin mononucleotide and a specific substance in the cells or cells to form a holoenzyme, and the holoenzyme was separated from the body in vivo without the cofactor being removed from it. -The present invention has been completed through purification and acquisition.

That is, according to the present invention, luciferase, flavin mononucleotide, 350 nm, 405 nm, 540 nm and 57 derived from a luminescent bacterium are used.
A bacterial hololuciferase consisting of a substance exhibiting a characteristic absorption spectrum at 0 nm (hereinafter referred to as the specific absorption substance) is provided (here, the present holoenzyme is LUC to FMN).
-Represented as X).

The present inventors have also found that this holoenzyme is NA
It was found that it controls the reaction of reducing FMN, which is its cofactor portion, to FMNH ₂ by oxidizing DH or NADPH (reaction (c)).

Luciferase and F obtained in the reaction (c)
Holoenzyme consisting of MNH ₂ and iron ion (LUC to FMN
H ₂ · X) controls the reaction represented by the following formula (d).

(C) NAD (P) H + LUC to FMN · X + H ⁺ → NAD (P) ⁺ LUC to FMNH ₂ · X

(D) LUC to FMNH ₂ · X + RCHO + O ₂ → LUC to FMN · X + RCOOH + H ₂ O + hν

In the reaction (d), the holoenzyme LUC to FMNH is used.
LUC~FMN next ₂ · X is reacted with an aldehyde (RCHO) and oxygen (O _2), at the same fatty acids corresponding to the same time the aldehyde (RCOOH) and water (H ₂ O) and light (hv) is produced. Therefore, NADH and NADPH can be quantified by measuring this light.

Therefore, the present inventors have previously required NADH: FMN oxidoreductase and NADP.
We succeeded in quantifying NADH and NADPH using only the hololuciferase according to the invention as the enzyme without using any of the H: FMN oxidoreductases. By this quantification method, NADH and NAD
PH can be measured easily and inexpensively.

The hololuciferase according to the present invention is a holoenzyme derived from a luminescent bacterium. Examples of the luminescent bacterium producing the hololuciferase of the present invention include Vibrio
Harvey (Vibrio harveyi), Vibrio Fuissheri (Vibrio fisheri), Vibrio Supurendidasu (Vibrio splendidus), Vibrio cholerae (Vibrio
cholerae) , Photobacterium phosphoreum , Photobacterium leiognathi, and the like. In addition to the above bacteria, bacteria that produce an enzyme that catalyzes a luminescence reaction can be used for producing the holoenzyme of the present invention.

The bacterial hololuciferase of the present invention is not limited to the hololuciferase obtained by culturing the above-mentioned luminescent bacterium, but a host cell is transformed with an expression vector containing a gene encoding the luciferase derived from the above luminescent bacterium. It also includes hololuciferase produced by the transformant obtained by the transformation. When a hololuciferase is produced using the transformant, the technique may be greatly improved in the expression efficiency of the holoenzyme by a genetic engineering technique, and thus this method is preferably used.

Examples of the transformant include, for example, JP-A-2-
JM109 (pLUX1802) shown in No. 2363
Expresses a large amount of the hololuciferase of the present invention and can be preferably used. The JM109 (pLU
X1802) was obtained by transforming Escherichia coli JM109 with an expression vector containing a gene encoding luciferase of the luminescent bacterium Vibrio harveyi.

Next, the transformant JM109 (pLUX1
A method for preparing 802) will be described.

Vibrio harvey is described, for example, in the method of JW Hastings (Methods in Enzymology,
vol. LVII, 153 (1978)), and from there, for example, the method of C. Miyamoto et al. (Methods in Enzymol
Chromosomal genes are extracted by Ogy, vol.133, 70 (1986)). The extracted chromosomal DNA is then incorporated into a known plasmid vector to create a gene library. As the above plasmid vector,
For example, pUC18 (manufactured by Takara Shuzo), pUC118 (manufactured by Takara Shuzo), pBluescript S having a lac promoter.
K II (+) (Stratagene), pUR278 (Ruth
er etel.; EMBO J., 2,1791 (1983)) etc .; or tac
PK with promoter (trp-lac promoter)
K177-3 (Amann E. and J. Brosius; Gene, 40,
183 (1985)); or pKC30 (Shimatake H and M. Rosenb), which has a P _L promoter of λ phage.
erg; Nature.292.128 (1981)) and the like.

These plasmid vectors are cleaved with appropriate restriction enzymes. Chromosomes D extracted above
NA is also cut with an appropriate restriction enzyme and ligated to the plasmid vector cut with the above restriction enzyme.

For example, as shown in FIG. 1, chromosomal DNA isolated from Vibrio harveyi was treated with restriction enzyme Sa.
pUC1 while partially digested with u 3AI (Takara Shuzo)
8 is digested with a restriction enzyme Bam HI (manufactured by Takara Shuzo), and these are ligated using, for example, a DNA ligase derived from T4 phage (manufactured by Takara Shuzo).

The recombinant plasmid thus obtained can be obtained, for example, by the method of Hanahan (J. Mol. Bio).
l., 166.557 (1983)), or Molecular Cloning
(1.74 to 1.84, Cold Spring Habor Laborato
ry (1989)) and introduced into an appropriate host cell. Examples of the host cell include Escherichia, Bacillus, Pseudomonas strains, yeast, fungi, human and other animal cultured cells, plant cultured cells, and the like. For example, Escherichia coli E. Coli JM109 strain ( (Manufactured by Toyobo Co., Ltd.) is preferably used.

From the transformed microorganism thus obtained, a luciferase-producing strain is screened. This screening uses, for example, expression of drug resistance derived from the used plasmid vector, luminescence reaction by luciferase produced by the strain, or a DNA probe composed of a part of the nucleotide sequence of the gene encoding luciferase derived from Vibrio harveyi. Hybridization is used.

For example, as a plasmid vector, the above p
In the example of using UC18, utilizing the fact that the pUC18 has an ampicillin resistance gene, the obtained coliform group was cultured in an ampicillin and isopropyl-β-D-thiogalactopyranoside (IPTG) -containing medium,
Bacteria that grow in the medium are selected. Furthermore, long-chain aldehydes (eg, n-decanal) against these bacterial cells
To select the bacterial cells that emit light. The transformed microorganism thus obtained has a recombinant plasmid having a luciferase gene derived from Vibrio harveyi incorporated therein. The inventors of the present invention selected the Escherichia coli group having the highest luciferase activity from the transformed Escherichia coli group and named it JM109 (pLUX1801). The plasmid capable of expressing the luciferase activity possessed by the E. coli was named pLUX1801. did.

Transforming microorganism JM109 (pLUX18
01) from the recombinant plasmid pLUX1801 again, and subcloning the DNA fragment containing the luciferase gene inserted in the plasmid to obtain a transformed microorganism having a higher luciferase activity,
And a recombinant plasmid capable of expressing the luciferase activity is obtained.

For example, the above JM109 (pLUX180
1) is cultivated in a large amount, the cells are treated with alkali, the plasmid is isolated, and the above-mentioned plasmid is cleaved using various restriction enzymes alone or in combination of two or more to obtain the following DNA fragment. It was

(1) Hind III D of 4.0 kbp
NA fragment (2) Sal I- Hind III D of 3.7 kbp
NA fragment (3) 3.0 kbp Hind III- Pst ID
NA fragment, and (4) 1.5 kbp HindIII- Eco RI D
NA fragment.

These DNA fragments were again incorporated into the pUC18 plasmid vector according to the above method, and the recombinant plasmid (1) pLU corresponding to each DNA fragment was constructed.
X1802, (2) pLUX1803, (3) pLUX18
04 and (4) pLUX1805 were obtained. Escherichia coli JM109 strain was transformed with each of these recombinant plasmids, and its luciferase activity was examined.
Transformed E. coli harboring pLUX1802 (JM109
(Named pLUX1802), and pLUX180
Transformed E. coli (JM109 (pLUX18
The luciferase activity was confirmed in (03).

FIG. 2 shows the recombinant plasmid pLUX180.
1, pLUX1802, pLUX1803, pLUX1
A restriction enzyme digestion map of 804 and pLUX1805 is shown, and a schematic diagram of pLUX1802 is shown in FIG. From FIG. 2, the luciferase gene was 3.7 kbp SalI- Hi.
nd III DNA fragment and 4.0 kbp Hind
It was confirmed that it was present in the III DNA fragment.

Next, the method for separating and purifying hololuciferase by the present inventors will be described. Since luciferase is in the state of a holoenzyme by binding flavin mononucleotide and iron ion in the bacterial cell, it is either isolated or purified from the luminescent bacterium or isolated or purified from the transformant. It is necessary to separate and purify the hololuciferase using a method in which the flavin mononucleotide and iron ion in the holoenzyme do not deviate from the luciferase.

Culture of microbial cells The luminescent bacteria can be cultured by, for example, the method of Hasting et al.
s in Enzymology vol. LVII, 153 (1978) for culturing to accumulate hololuciferase in the cells. Alternatively, the above format converter JM109 (pLUX1802)
Are cultured, for example, by the method described in JP-A-2-2363. Examples of the medium include LB medium (1% tryptone, 0.5%, which is used for culturing E. Coli JM109 strain).
Yeast extract, 0.5% NaCl) and the like. The culturing temperature is, for example, 30 to 40 ° C., preferably about 37 ° C., and the culturing time is, for example, 2 to 16 hours, preferably about 8 hours. Isopropyl-β-D-thiogalactopyranoside (IPTG) (manufactured by Nacalai Tesque) was added to the medium.
Is added to induce the lac promoter of the vector pUC18, and hololuciferase is produced.

Extraction and purification of holoenzyme The cultured cells are collected by centrifugation or the like, and the collected cells are crushed. As the crushing method, for example, aluminum oxide for cell grinding is added to the cells, a method of grinding the cells in a mortar, or a method in a buffer solution by osmotic shock,
Alternatively, a cell disruption method using the enzyme lysozyme, a surfactant and the like can be mentioned.

A method for purifying the hololuciferase from the obtained extract using a method in which the specific absorbing substance and the flavin mononucleotide in the hololuciferase do not deviate from the luciferase is, for example,
The methods (i), (ii), and (iii) are performed alone or in combination of two or more.

(I) As a first method, chromatography is carried out using an ion exchange column treated with salts containing iron ions. Here, the “salts containing iron ions” are not particularly limited as long as they generate iron ions in the washing or elution buffer of the ion exchange column, and suitable examples include ammonium iron citrate and the like.

As used herein, "treating with a salt containing iron ions" means, for example, stirring the column gel in a buffer solution containing "the salts containing iron ions" before packing the column gel, or, for example, , A treatment including an operation of contacting the surface of gel particles in a column with iron ions by, for example, eluting a certain amount of a buffer solution containing the “salts containing iron ions” after column packing.

As the ion exchange chromatography,
For example, weak anion exchange chromatography such as DEAE (diethylaminoethyl) -cellulofine is preferably used.

(Ii) Another preferable example of the purification method of the hololuciferase is a method of performing chromatography in the presence of NAD (P) H.

Here, "in the presence of NAD (P) H" means, for example, that a chromatography column is NADH or NDH.
Equilibrate with a buffer containing ADPH, mix NADH or NADPH with the crude enzyme solution before applying to the column, and then apply the mixture to the column, in the developing solution (eluent) of the column NADH or N
ADPH is added and the developing solution is used to elute the hololuciferase. A treatment including any one or more of the above operations.

In the presence of NADH or NADPH, the hololuciferase reacts with NADH or NAD.
By converting PH into an oxidized form, FMN, which is its cofactor, is reduced to FMNH ₂ . When FMN is reduced to FMNH ₂ , the binding with luciferase becomes stronger, so that the flavin mononucleotide and the specific absorbing substance are not separated from the luciferase, and the hololuciferase can be purified. As the chromatography, for example, weak anion exchange chromatography such as DEAE / Cellulofine described above is preferably used.

(Iii) Another preferred example of another purification method for the hololuciferase is gel filtration. Examples of gel filtration methods include Sephadex-G.
A method using 75 columns is preferably performed.

Here, since the holoenzyme in which the luciferase, the flavin mononucleotide, and the specific absorption substance are bound to each other has a strong binding force, the flavin mononucleotide and the specific absorption substance are separated by gel filtration using Sephadex G75. Does not deviate from luciferase.

On the other hand, the one in which only flavin mononucleotide is bound to luciferase cannot control the above reaction (c), but this is because the binding force is not strong, so that luciferase and flavin mononucleotide are separated by Sephadex G75. Resulting in. Therefore, with Sephadex G75, it is possible to purify only the holoenzyme to which the three members of the luciferase, the flavin mononucleotide and the specific absorbing substance are bound.

The above method (i) chromatography by an ion exchange column treated with salts containing iron ions and method (iii) gel purification of the hololuciferase purified by a combination of the two methods described below (1) (2) From the confirmation in (3), it was revealed that the luciferase was indeed bound to flavin mononucleotide and the specific absorption substance to be in the state of holoenzyme.

(1) In SDS-PAGE, only two bands corresponding to the α and β subunits of luciferase were confirmed. (2) In the absorption spectrum of the visible region, flavin mononucleotide and the specific absorbing substance were detected. (3) Inductively coupled plasma (ICP) emission spectrometry, in which the corresponding peak was confirmed, confirmed the iron ions that are considered to constitute the hololuciferase.

When NADH and aldehyde were added to the hololuciferase, a luminescence reaction was confirmed. A luminescence reaction was also confirmed when NADPH was added instead of NADH, but in this case, the amount of light was weaker than in the case of NADH. From this, it was revealed that the hololuciferase catalyzes the reduction reaction of FMN (the above reaction (c)) by oxidizing NADH or NADPH. Therefore, using only the hololuciferase, NADH and NAD according to the above reaction (c) (d)
It was found that PH can be quantified.

NADH by hololuciferase and
Quantification of NADPH The hololuciferase of the present invention is obtained as a pure product, for example, by a combination of two methods (i) chromatography on an ion exchange column treated with salts containing iron ions and (iii) gel filtration. By using, it is possible to easily and sensitively measure NADH and NADPH by the following method.

The quantification of NADH and NADPH is carried out by mixing (1) the hololuciferase (2) aldehyde (3) test sample of the present invention and measuring the light generated.

Here, the aldehyde is preferably a linear saturated aliphatic aldehyde having 8 or more carbon atoms, and preferable examples thereof include n-decanal and n-tetradecanal.

The term "measuring light" means, for example, measuring the peak value of the luminescence intensity immediately after the start of the enzyme reaction,
Alternatively, it is possible to use a method in which a value obtained by integrating the amount of emitted light over a unit time is measured, but in addition to this, a method used for ordinary light emission measurement can be used.

Further, the quantification of NADH and NADPH can be carried out with high sensitivity by using an intermediate purified product in the course of purification, without using the above-mentioned completely purified product of the holoenzyme.

The following intermediate products are used, for example.

(1) Cell extract obtained by crushing the above-mentioned cells producing holorluciferase, (2) The cell extract was subjected to at least one treatment of dialysis, ammonium sulfate precipitation and ultrafiltration. (3) The above (i) in the cell extract
(ii) A product which has been subjected to at least one of the purification methods of (iii).

The hololuciferase is NADH or NA.
Not only used for DPH analysis, but also NADH and NA
By combining the enzyme system that produces DPH and the hololuciferase system by a coupling reaction, the substrate concentration and enzyme activity of these enzymes can be measured with high sensitivity. Here, the enzyme system that produces NADH or NADPH
Whether it consists of one enzyme reaction or a plurality of enzyme reactions, it can be coupled to the hololuciferase system.

At this time, NADH: F, which was necessary in the past, was also used.
Since the analysis can be performed without using either MN oxidoreductase or NADPH: FMN oxidoreductase, simple and inexpensive measurement can be realized.

For example, analysis of a dehydrogenase and its substrate of a biological sample for clinical examination is a preferable example. Examples of the dehydrogenase include the following, but the dehydrogenase is not limited to these.

Isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glycerophosphate dehydrogenase, 3-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase Enzyme, alcohol dehydrogenase, aldehyde dehydrogenase, l-glutamate dehydrogenase, l-alanine dehydrogenase, 3α-hydroxysteroid dehydrogenase.

By applying the above-mentioned conjugation reaction, for example, by imaging the light generated by the above-mentioned conjugation reaction system in a tissue or cell on a high-sensitivity film,
The distribution of the above enzyme or substrate can be detected.

It is also possible to quantify the trace components by flow injection with the hololuciferase or the enzyme for the above coupling reaction immobilized on a carrier.

The hololuciferase can also be used as a labeling substance.

For example, by labeling the antigen or antibody with the hololuciferase and detecting the luminescence, a highly sensitive EI can be obtained.
A (enzyme immunoassay) and ELISA (enzyme-Iinke
dimmunosorbent assay) can be realized.

It is also possible to detect the specific base sequence by labeling the hololuciferase with a DNA probe via a suitable linker as necessary and measuring the amount of luminescence.

[0067]

[Examples] Reference example Preparation of recombinant plasmid and transformed microorganism Vibrio harvey ATCC14126 strain was treated with 2% Na
LB medium containing Cl (1 g of bactotryptone, 0.5 g of yeast extract, 2 g of NaCl / 100 ml) 100
After culturing in ml at 28 ° C. for 8 hours, centrifugation was performed at 8,000 rpm for 10 minutes. The obtained bacterial cell precipitate is ED
Saline containing TA (NaCl 0.88 g, ED
TA ・ Na ₂ 1.86 g / 100 ml, pH 8.0)
It was suspended in 10 ml, washed, and centrifuged at 8,000 rpm for 10 minutes. Then, the precipitate was suspended in 5 ml of the above-mentioned physiological saline containing EDTA, lysozyme was added thereto so that the final concentration was 2 mg / ml, and the mixture was incubated at 37 ° C. for 20 minutes.
Incubation was carried out with shaking for a minute. Furthermore, 0.6 ml of 10% SDS was added to this solution,
Incubated at 0 ° C for 10 minutes. After that, an equal amount of a phenol solution (phenol: chloroform: isoamyl alcohol = 25: 24: 1) was added, gently stirred, and then centrifuged to obtain the supernatant (DNA-containing aqueous layer). .. The extraction operation with the above-mentioned phenol solution was repeated twice more on this supernatant.

To the obtained supernatant, twice the amount of ethanol was added, and the floating chromosomal DNA was wound up with a glass rod and collected. The glass rod around which this chromosomal DNA is wrapped is 70% ethanol, 80% ethanol, 90%
Sequentially soak in ethanol and 100% ethanol,
Was dehydrated. After further drying at room temperature, 1
ml TE10-1 (10 mM Tris-HCl (p
It was dissolved in H8.0), 1 mM EDTA.Na ₂ ).

A solution containing 2 μg of the chromosomal DNA of Vibrio harveyi thus obtained (the amount of DNA is 0
D obtained from the absorbance at 260 nm) is 1/1
0 volume of 10 × Sau 3AI buffer (100 mM Tri
s-HCl (pH 7.5), 70 mM MgCl ₂ , l
M NaCl) and the restriction enzyme Sau 3AI (2 units) were added, and the mixture was partially digested at 37 ° C. for 15 minutes. An amount corresponding to 200 ng of DNA in this solution (1 part of the above-mentioned DNA digest
/ 10 amount) and E. coli plasmid DNA completely digested with the restriction enzyme.

This “E. coli plasmid completely digested with restriction enzymes
"Sumid DNA" means the multicopy plasmid pU of E. coli.
100 ng of C18 DNA, 1/10 amount of 10xBa
mHl buffer (100 mM Tris-HCl (pH
8.0), 70 mM MgCl ₂1M NaCl, 2
0 mM 2-mercaptoethanol, and 0.1% sodium
2 units of restriction enzymeBamWith Hl
And incubate at 37 ° C for 2 hours to completely
It has been digested. Add 1/10 volume of 10 to this mixture.
× T_FourLigase buffer (660 mM Tris-HCl
(PH 7.6), 66 mM MgCl₂, 100 mM di
Thiothreitol, and 1 mM ATP), and 1
0 unit of T_FourAdd DNA ligase and incubate at 10 ℃ overnight.
They were connected to each other (see FIG. 1).

The above ligation mixture was contacted with a competent cell of Escherichia coli JM109 strain (manufactured by Takara Shuzo) to transform the Escherichia coli. LB agar medium containing 50 μg / ml of ampicillin and 1 mM of IPTG (100 g of bactotryptone, 0.5 g of yeast extract, 0.5 g of NaCl, and 1.5 g of agar per 100 ml).
Was applied to 10 sheets and plated at 37 ° C. overnight for screening. 10,000 obtained in this culture
To each of the ampicillin-resistant transformed colonies, n-decanal (Hanai Kagaku Co., Ltd.) was added in a mist form using a sprayer, and one colony showing luminescence (having luciferase activity) was obtained. The recombinant plasmid having luciferase activity contained in the strain forming this colony was designated as pLU.
X1801 was named, and after a large amount of the E. coli colony was cultured, it was treated with alkali to obtain the plasmid pL.
UX1801 was isolated and purified. This purified plasmid pLUX1801 was added to the restriction enzymes HindIII and Sal.
The following DNA fragments were obtained by digestion with I, Pst I, and Eco RI in various combinations and electrophoresis on 0.8% agarose gel: (1) 4.0 k
Hind III DNA fragment of bp, (2) S of 3.7 kbp
al I- Hind III DNA fragment, (3) 3.0 kbp
Hind III- Pst I DNA fragment of (4)
HindIII- EcoRI DNA fragment of 1.5 kbp.

(1) to (4) 20 ng of each DNA fragment
And 10 ng of pUC18 DNA digested with the same combination of restriction enzymes as (1) to (4) with 10 units of T
Ligated using 4 DNA ligase and plasmid pLU
Subcloned from X1801 and plasmid pLU
X1802, pLUX1803, pLUX1804, and pLUX1805 were prepared (FIG. 2). That is, each of the above four recombinant plasmids has the following DNA fragment derived from Vibrio harveyi: pL
UX1802: 4.0 kbp HindIII DNA fragment, pLUX1803: 3.7 Kbp Sal I- Hi.
nd III DNA fragment, pLUX1804: 3.0 kbp
Hind III- Pst I DNA fragment and pLU
X1805: Hind III- Eco RI of 1.5 kbp
DNA fragment. Escherichia coli JM109 strain was transformed with each of these recombinant plasmids by the same method as described above. This transformant was cultured on LB agar medium and
-Luciferase activity was investigated by the method using decanal. As a result, the recombinant plasmid pLUX180
Luciferase activity was detected in the cells containing 2 and pLUX1803.

Details of the preparation of the above plasmid pLUX1802 capable of expressing luciferase activity from the above pLUX1801 are as follows. First, pLUX18
To 10 μg of 01 DNA, 1/10 amount of 10 × Hind
III buffer (100 mM Tris-HCl (pH 7.
5), 70 mM MgCl ₂ , 600 mM NaCl)
And add 20 units of the restriction enzyme Hind III to 37
Completely digested at 0 ° C for 2 hours and electrophoresed on 0.8% agarose gel to give 4.0 kbp of Hind III.
Obtain a DNA fragment.

This 4.0 kbp Hind III DNA
20 ng of the fragment and 10 ng of pUC18 DNA digested with 2 units of the restriction enzyme Hind III were used as T ₄ DNA.
Ligation with 10 units of ligase yields recombinant plasmid pLUX1802.

Thus the plasmid pLUX180
Escherichia coli JM109 strain was transformed with the recombinant plasmid pLUX1802 obtained from Example 1 by the same method as above. This transformant was cultured in 1.5 ml of LB medium containing 50 μg / ml ampicillin, and the number of cells was 5 ×.
IPTG was added to 1 mM when 10 ⁸ cells / ml was reached. Since pLUX1802 has a pUC18-derived lactose operon promoter (Plac), it is possible to induce hololuciferase by adding IPTG, and hololuciferase can be efficiently produced. This transformant was allowed to produce hololuciferase by continuing the culture for another 2 hours. This culture was centrifuged at 8000 rpm for 5 minutes, and the resulting precipitate was added to 500 μl of the above TE10-1 buffer solution (pH
8.0). 20 μl was taken from this suspension and centrifuged at 8000 rpm for 5 minutes. The obtained precipitate was suspended in 5 μl of sterilized water, and 5 μl of 2 × sample buffer (0.13M Tris-HCl (pH 6.8), 4
% SDS, 20% glycerol, 10% mercaptoethanol, and 0.002% bromphenol blue)
Was added and heat-treated at 90 ° C. for 3 minutes to heat-denature E. coli, and intracellular protein SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. When the gel after electrophoresis was stained and analyzed by a densitometer, bands were observed at molecular weights of 42,000 and 37,000. These two bands correspond to subunits α and β of luciferase derived from Vibrio harveyi, respectively. When the absorbances of these two bands were summed and compared with the sum of the absorbances of the other bands, luciferase (α + β
The expression of (subunit) was 10% or more of the intracellular protein. This electropherogram is shown in FIG. In FIG. 4, 1 is a molecular weight marker; 2 is a plasmid vector pU.
Intracellular protein obtained by heat treatment of Escherichia coli JM109 strain having C18 in the same manner as above: and 3 is Escherichia coli JM10 having the recombinant plasmid pLUX1802
The heat-treated intracellular protein of 9 strains is shown.

Example Production of hololuciferase using transformed microorganisms and
And purification (a) Culture of transformed Escherichia coli Media A, B, and C were prepared by the following methods.

Medium A (medium for plates) Polypeptone 16 g Yeast extract 10 g Sodium chloride 5 g Agar powder 15 g The above was dissolved in pure water to make 1 L. After sterilization at 120 ° C. for 20 minutes, ampicillin 2.5 g IPTG 0.238 g was added at about 70 ° C. to prepare a plate.

Medium B (Liquid medium for inoculum) Polypeptone 16 g Yeast extract 10 g Sodium chloride 5 g Lactose 2.5 g Riboflavin 30 mg Ammonium iron citrate 100 mg The above was dissolved in pure water to make 1 L in total. 120 ° C, 2
After sterilizing for 0 minutes, when the liquid temperature returned to room temperature, 0.5 g of ampicillin and 0.238 mg of IPTG were added.

Medium C (mass culture medium) Polypeptone 16 g Yeast extract 10 g Sodium chloride 5 g Lactose 4 g Riboflavin 30 mg Ammonium iron citrate 100 mg The above was dissolved in pure water to make a total volume of 1 L. 120 ° C, 2
After sterilizing for 0 minutes, when the liquid temperature returned to room temperature, 0.2 g of ampicillin was added.

Culture of inoculum Transformed Escherichia coli JM109 (pLUX) stored on a plate
1802) was inoculated into each of 2 medium B of 1 L of a platinum loop and cultured at 37 ° C. for 14 hours with shaking.

Large-scale culture 10 cells of 1.5 C of medium were prepared, and 1 inoculum of the previously cultured inoculum was prepared.
200 mL of each book was inoculated. 8 at 37 ° C
The culture was aerated for a period of time, and the culture was centrifuged at 16,000 G for 5 minutes to collect the cells. The wet weight of the obtained bacterial cells is 80 to 100 g.
Met. The cells were immediately frozen and stored.

(B) Extraction of hololuciferase 50 g of frozen preserved cells and aluminum oxide 50 for cell grinding
60 g of 50 mM phosphate buffer (pH 7.0) was added to g, and the cells were well ground using a mortar. The obtained suspension was centrifuged at 45,200 G for 10 minutes to obtain a supernatant. 60 mL of 50 mM phosphate buffer was added again to the precipitate, this was ground in a mortar, and the supernatant was obtained by centrifugation. In this way, the extraction operation was performed 5 times in total to obtain a total of 300 mL of the extract.

(C) Purification of hololuciferase Ion-exchange chromatography DEAE-Cellulofine A-500 1 L was washed,
Then, after equilibrating with a 50 mM phosphate buffer (pH 7.0), 5 g of ammonium iron citrate was added, and after stirring well, it was washed with 3 L of a 350 mM phosphate buffer (pH 7.0). Furthermore, this was added to 50 mM phosphate buffer (pH 7.
0) Wash and equilibrate with 2 L, and then use a column (diameter 5 cm x length 5
0 cm) was created.

Bacterial cell extract 300 previously prepared in this column
ml was applied. 50 mM phosphate buffer (pH 7.
0) 1 L and 350 mM phosphate buffer (pH 7.0)
It was developed and eluted with 1 L of non-linear gradient eluate. After completion of the gradient elution, elution was further carried out with a 350 mM phosphate buffer solution. Column eluate fractions (1
7.5 μL) to obtain 10 μl of protein from each fraction
Take L and add 2 mL of CBB dye solution to develop color.
The absorbance at nm was measured. The elution curve is shown in FIG. Fraction Nos. 114 to 125 were used as the hololuciferase fraction, and the protein purity was assayed by SDS-PAGE. As a result, it was confirmed that there were almost no contaminating proteins. Collect the hololuciferase fractions, add crystalline ammonium sulfate,
The protein was precipitated at 5% saturation. The precipitate is collected by centrifugation, 5
After dialysis against 0 mM phosphate buffer (pH 7.0),
It was subjected to the following purification process.

Gel filtration Sephadex G-75 column (diameter 5 cm x length 90 cm) equilibrated with 50 mM phosphate buffer (pH 7.0).
The purified hololuciferase solution (45 mL) was applied to the above. Elution was performed with 50 mM phosphate buffer, 10 μL was taken from each fraction to determine the amount of protein in each fraction (17.5 mL), 2 mL of CBB dye solution was added, and after color development,
The absorbance at 595 nm was measured. The elution curve is shown in FIG. Fractions Nos. 31 to 39 were collected as a purified hololuciferase fraction, and crystalline ammonium sulfate was added to add 75
It was made to be% saturated and the precipitate was collected by centrifugation. 50 mM precipitation
After dialysis against a phosphate buffer (pH 7.0), insoluble matter was removed by centrifugation and used for the following measurement (18 mL). FIG. 7 shows the results of SDS-PAGE of each fraction, and FIG. 8 shows the visible absorption spectrum of purified hololuciferase. As is clear from FIG. 8, the purified hololuciferase contained 350
nm, 405 nm, 540 nm and 570 nm are observed to include substances exhibiting characteristic absorption spectra.

(D) Confirmation of iron ion in hololuciferase The iron ion in the hololuciferase solution obtained as a result of gel filtration in step (c) was confirmed by inductively coupled plasma (ICP) emission spectrometry. The measurement conditions and the measurement results are shown in FIG. This confirmed that the hololuciferase contained iron ions as a constituent.

(E) Measurement of enzyme activity of hololuciferase 488 μl of 50 mM phosphate buffer (pH = 7.0),
10 μl of the solution of hololuciferase obtained in (c),
2 μl of 10 mg / ml n-tetradecanal in ethanol and 500 μl of 50 mM phosphate buffer (pH = 7.0) containing 3.0 × 10 ⁻⁷ M NADH were mixed, and luminescence immediately after initiation of enzyme reaction Peak height (cp
m) was measured with a luminometer (Aloka Co., Ltd .: bioluminescence reader BLR-102B type).

Further, instead of the above NADH solution, 2.
7 × 10 ^-6 M NADPH, 3.0 × 10 ^-6 M NADP
⁺ , And the addition of 50 mM phosphate buffer (pH = 7.0) containing 2.7 × 10 ⁻⁶ M NADP ⁺ respectively, and the case where only 50 mM phosphate buffer was added as a blank The emission peak value generated immediately after was measured similarly to the case of NADH.

Table 1 shows the results of these measurements.

[0090]

[Table 1] From Table 1, it can be seen that the hololuciferase exhibits high enzymatic activity with NADH as a coenzyme. On the other hand, NA
In the case of a reaction using DPH as a coenzyme, NADPH is replaced with NAD.
Since approximately 10 times the amount of H was added and almost the same amount of luminescence was obtained, it can be seen that although it is not as specific as NADH, it exhibits high enzyme activity also for NADPH.

When NAD ⁺ or NADP ⁺ was added, the amount of luminescence immediately after addition was almost the same as that of the blank (addition of phosphate buffer solution only).

However, in the case of adding NAD ⁺ or NADP ⁺ , luminescence started 2 to 3 minutes after the addition and a luminescence peak was observed 8 to 10 minutes later (peak value was 4 to 6 × 10 6).
⁴ cpm).

This is because the action of the hololuciferase causes the dehydrogenation reaction of the aldehyde to produce NADH or NADPH, and then the reactions (c) and (d) occur, at which time the first dehydrogenation reaction of the aldehyde occurs. Since it is a slow reaction that takes a very long time, light emission does not occur immediately, and it seems to start in a few minutes.

Therefore, in the case of measuring NADH and NADPH, even if NAD ⁺ and NADP ⁺ coexist, they can be measured without being affected by them.

Application Example Quantification of NADH using the hololuciferase 488 μl of 50 mM phosphate buffer (pH = 7.0)
In addition, 10 μl of the hololuciferase prepared in Example 2 was used.
l and 10 mg / ml of n-tetradecanal ethanol solution (2 μl) were added and mixed.

This solution was attached to a luminometer (Bioluminescence Reader BLR-102 type; manufactured by Aloka Co., Ltd.), and 500 μl of 50 mM phosphate buffer solution (pH 7.0) containing NADH of known concentration was added thereto with a microsyringe. The height of the emission peak generated immediately after addition (cp
m) was measured.

The NADH sample concentration was plotted on the abscissa and the emission peak value was plotted on the ordinate, and the calibration curve shown in FIG. 10 was drawn in a logarithmic log graph. From this calibration curve, at least NAD
A straight line was obtained in the H sample concentration range of 3.0 × 10 ^{−9 to} 3.0 × 10 ⁻⁶ M, and the correlation was good (correlation coefficient γ =
0.998).

In addition, the conventional coupling reaction between NADH: FMN oxidoreductase and luciferase (the above reaction)
Quantification of NADH by (a) (b)) (eg Haruo Watanabe; Protein Nucleic Acid Enzyme, 33, 2, 192-199 (1988))
The measurement sensitivity and the measurement region (concentration range) were almost the same even when compared with.

[0099]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, there is provided a bacterial hololuciferase derived from a luminescent bacterium and comprising luciferinase, flavin mononucleotide and the specific absorbing substance.

By using the hololuciferase, both the NADH: FMN oxidoreductase and the NADPH: FMN oxidoreductase, which have been conventionally used, are no longer necessary. Therefore, NADH and NADP are eliminated.
It is possible to measure H more easily and cheaply than ever before.

Further, by coupling various enzymatic reactions for producing NADH or NADPH with the luminescent reaction catalyzed by the forloruciferenase, various trace substances can be analyzed, particularly in vivo trace substances in the field of clinical examination. It is also possible to perform measurement and enzyme activity measurement with high sensitivity.

Also, by using the hololucifernase as a labeling substance, a trace substance can be analyzed with high sensitivity by EIA, ELISA, DNA probe method and the like.

[Brief description of drawings]

FIG. 1 is an explanatory view showing the steps for preparing a recombinant plasmid pLUX1801.

FIG. 2: Recombinant plasmids pLUX1802, pLUX1803, p, in which a DNA fragment encoding a bacterial luciferase contained in the recombinant plasmid pLUX1801 has been cleaved with various restriction enzymes, and a deleted DNA has been inserted.
3 is a restriction enzyme digestion map of LUX1804 and pLUX1805.

FIG. 3 is a schematic diagram showing a recombinant plasmid pLUX1802 in which a DNA fragment encoding bacterial luciferase has been inserted.

FIG. 4 is an electrophoretogram showing SDS-PAGE bands of luciferase produced by E. coli transformed with recombinant plasmid pLUX1802.

FIG. 5 is a graph showing an elution curve diagram in the purification of the hololuciferase of the present invention with DEAE-Cellulofine A-500.

FIG. 6 is a graph showing an elution curve in the purification of hololuciferase of the present invention by Sephadex G-75.

FIG. 7: SD of each fraction in Sephadex G-75
It is S-PAGE.

FIG. 8 is a visible absorption spectrum of the purified hololuciferase.

FIG. 9 is a graph showing an inductively coupled plasma emission assay of the purified hololuciferase.

FIG. 10: N was obtained using the purified hololuciferase.
It is a calibration curve which measured ADH concentration.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:63)

Claims (1)

[Claims] 1. A luciferase derived from a luminescent bacterium, a flavin mononucleotide, 350 nm, 405.
A bacterial hololuciferase consisting of a substance exhibiting a characteristic absorption spectrum at nm, 540 nm and 570 nm.