JPH07112434B2 - Luciferase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to Luciola laterali.
s, Heike firefly) derived luciferase gene.

[Conventional technology]

Luciola luciferase of the genus Luciola is obtained only by separating and purifying it from the collected firefly genus [Proc.
(Proc.Natl.Acad.Sci.), Vol. 74, No. 7, 2799-2
802 (1977)].

The luciferase is an extremely useful enzyme as, for example, an enzyme for quantifying ATP.

[Problems to be Solved by the Invention]

However, since the above-mentioned luciferase is derived from an insect, in order to produce it, it is necessary to collect the firefly of the genus Luciola from the natural world, or to cultivate the firefly and separate and purify the luciferase from the obtained firefly. However, its production requires a lot of time and labor.

[Means for Solving the Problems]

Therefore, the present inventors have conducted various studies to solve the above problems, and obtained a recombinant DNA in which a DNA containing a gene encoding luciferase derived from Luciola lateralis was inserted into a vector DNA, and the recombinant DNA was obtained. A patent application was filed knowing that luciferase can be efficiently produced in a short period of time by culturing in a medium a microorganism having a luciferase-producing ability in which DNA is contained in a microorganism belonging to the genus Escherichia. Patent application specification dated July 1, 63).

Then, as a result of further studying the luciferase gene derived from Luciola lateris, the present inventors succeeded in isolating and determining the structure of the luciferase gene derived from Luciola lateris for the first time, and completed the present invention.
That is, the present invention provides (1) a luciferase gene characterized by encoding the amino acid sequence shown below.

(2) The above (1) represented by the base sequence shown below.
The described luciferase gene.

Hereinafter, the present invention will be described in detail.

First, Luciola lateralis
When searching for a DNA containing a gene encoding a luciferase of (Hayake firefly), Luciola crucata of the same genus, which is one species of firefly, is used as a probe.
iata) (genji firefly) luciferase-encoding gene is used, and it also contains the Luciola cruciata luciferase-encoding gene
When searching for DNA, a DNA containing a gene encoding a luciferase of Photinus pyralis (American firefly), which is one type of firefly, is used as a probe.

Therefore, first, a method for preparing a gene encoding Photinus pyralis luciferase will be described below, followed by a method for preparing a gene encoding a Luciola cruciata luciferase, and finally, a method for Lucifera lateris luciferase. The method for preparing the encoded gene will be described.

To prepare m-RNA from the tail of Photinus pyralis, one of the firefly species, see, for example, "Molecular Cloning", p. 196, "Cold Spring Harbor Laboratory" (Cold S.
pring Harbor Laboratory) (1982) and “Experimental Method of Molecular Genetics” by Haruo Koseki and Rero Shimura, pp. 66-67 (1983).

M-R encoding luciferase from the obtained m-RNA
NA can be concentrated by, for example, the method described in “Biomedical Research”, Volume 3, pp. 534 to 540 (1982).

At this time, anti-luciferase serum against luciferase is used, and the serum can be obtained, for example, by the method described in "Immunochemistry", Yuichi Yamamura, pp. 43-50 (1973).

For synthesizing c-DNA from m-RNA encoding luciferase, for example, "Mole Cell Biol" (Mol. Ce
II Biol.), Vol. 2, p. 161 (1982) and Gene (Gen).
e), Vol. 25, p. 263 (1983).

Then, the c-DNA thus obtained is used as a vector DN.
A, eg plasmid pMCE10DNA, plasmid pKN305
[Agr.Biol.Chem.], No.
50, p. 271 (1986) and a plasmid having a promoter of the E. coli tryptophan operon] and plasmid pMC1843 [“Methods in Enzymology” (M
ethods in Enzymology), 100, 293-308 (198
3) A plasmid having an Escherichia coli β-galactosidase structural gene described above], etc.] to obtain various recombinant plasmid DNAs, and using the DNAs, for example, E. coli DH1 (ATCC33849 ), E. coli HB101 (ATCC33694), etc.
Han's method ["DNA Cloning" (DNA
Cloning), Vol. 1, pp. 109-135 (1985)] to obtain various transformants.

The recombinant plasmid DNA possessed by the transformant thus obtained is a plasmid having c-DNA incorporated in the middle of the Escherichia coli β-galactosidase structural gene, and is a peptide encoded by c-DNA. Is expressed as a protein fused with β-galactosidase.

To detect luciferase-encoding c-DNA from the various transformants described above, it is necessary to culture the transformants to express bacterial cell proteins, and to determine whether or not there is a protein that crosses the anti-luciferase serum. Can be detected by, for example, "Agric Violet Chem" (Ag
ric.Biol.Chem.), 50, 271 (1986) and "Anal. Biochem.", 112, 19
It can be carried out by the method described on page 5 (1981).

Then, the incomplete luciferase c-DNA was subjected to nick translation using ³² P ["Molecular Cloning", pp. 109-112, Cold Spring Harbor Laboratory (Cold Spr.
ing Habor Laboratory) (1982) and "J. Mol. Biol.", 113, 23-251 (1977)], and the c-DNA as a probe. Colony hybridization method ["protein,
Nucleic acid and enzyme ", Vol. 26, pp. 575-579 (1981)] as a vector using plasmid pUC19DNA (manufactured by Takara Shuzo) as a vector. C-DNA encoding luciferase of 1.8 Kb from gene bank library of c-DNA. It is possible to obtain a plasmid DNA containing

And the recombinant plasmid thus obtained
To obtain a DNA containing a gene encoding luciferase derived from Photinus pyralis from DNA, restriction enzymes such as EcoRI and ClaI are added to the plasmid DNA at a temperature of 30.
The reaction-terminated solution obtained by reacting at -40 ° C, preferably 37 ° C for 1-24 hours, preferably 2 hours is subjected to agarose gel electrophoresis ["Molecular cloning" (Molecular cloning)
Cloning), p. 150, Cold Spring Harbor Laboratory (1
982) Description].

Next, a method for preparing a gene encoding Luciola crucita luciferase will be described.

Preparation of m-RNA from the tail of Luciola cruciata and synthesis of c-DNA from m-RNA was carried out in the same manner as, for example, the method for preparing m-RNA of Photinus pyralis and the synthesis of c-DNA described above. Can be done.

Then, the c-DNA thus obtained is used as a vector DN.
A, for example, by incorporating into the plasmid pUC19 DNA and the like to obtain various recombinant plasmid DNA, using the DHA, for example,
E. coli DH1 (ATCC33849), E. coli
HB101 (ATCC33694) and other methods of Hanahan ("DNA Cloning" (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)] to obtain various transformants.

Then, the DNA containing the gene encoding Photinus pyralis-derived luciferase was nick-translated using ³² P [“Molecular Cloning”
(Molecular Cloning), pages 109-112, Cold Spring Harbor Laboratory (Cold Spring Habo)
r Laboratory) (1982) and "J. Mol. Biol.", 113, 237-251 (197).
7)], followed by a colony hybridization method ["protein / nucleic acid.
Enzymes ", Vol. 26, pp. 575-579 (1981)], a c-DNA (2.0 Kb) encoding luciferer from a gene bank library of c-DNA derived from Luciola cruciata prepared using plasmid pUC19DNA as a vector. It is possible to obtain E. coli containing the plasmid DNA containing

To obtain a purified recombinant DNA from the microorganism thus obtained, for example, "Proc. Natl. Acad. Sci.", Volume 62, 1159-116.
It can be obtained by the method described on page 6 (1969).

And the recombinant plasmid thus obtained
To obtain a DNA containing a gene encoding Lucifera cruciata-derived luciferase from DNA, a restriction enzyme such as PstI is added to the plasmid DNA at a temperature of 30 to 40 ° C., preferably 37 ° C. for 1 to 24 hours, preferably Is allowed to act for 2 hours and the reaction-terminated solution is subjected to agarose gel electrophoresis [“Molecular Cloning”,
Page 150, Cold Spring Habor Laboratory (1982))
It can be obtained by treating with.

Next, a method for preparing a gene encoding a luciferase derived from Luciola lateralis will be described.

Since the m-RNA encoding luciferase derived from Luciola lateris is present in the tail of Luciola lateris, the tail is preferable as a source for collecting m-RNA.

The preparation of m-RNA from the tail of Luciola lateralis and the synthesis of c-DNA from m-RAN were carried out in the same manner as, for example, the method for preparing m-RNA of Photinus pyralis and the synthesis of c-DNA described above. Can be done.

Then, the c-DNA thus obtained is used as a vector DN.
A, for example, by incorporating into the plasmid pUC119 DNA and the like, various recombinant plasmid DNA is obtained, and using this DNA, for example,
E. coli DH1 (ATCC33849), E. coli
HB101 (ATCC33694) and other methods of Hanahan (“DNA Cloning” (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)] to obtain various transformants.

Then, the DN containing the gene encoding the luciferase derived from Luciola cruciata obtained as described above.
Nick translation method using A for ³² P ["Molecular Cloning", No. 109-11
Page 2, Cold Spring Harbor Laboratory (1982) and "J. Mol. Biol.", Vol. 113, No. 23.
7-251 (1977)], and the colony hybridization method using the c-DNA as a probe ["Protein / Nucleic Acid / Enzyme" Vol. 26, pp. 575-579 (198).
1)], a luciferase-encoding c library from a gene bank library of c-DNA derived from Luciola lateralis prepared using the plasmid pUC119DNA as a vector.
E. coli containing plasmid DNA containing DNA (2.0 Kb) can be obtained.

Recombinant purified from the microorganism thus obtained (for obtaining DNA, for example, "Proc. Natl. Acad. Sci.", Volume 62, 1159
˜1166 (1969) and the like.

Then, for example, 2 units of the restriction enzyme EcoRI (manufactured by Takara Shuzo) is added to the purified new recombinant DNA at a temperature of 3
A 2,000 bp DNA containing all luciferase genes derived from Luciola latereris after partial digestion by allowing it to act at 0 ° C. or higher, preferably 37 ° C. for 1 to 4 hours, preferably 2 hours, and then performing agarose gel electrophoresis. The fragment can be isolated.

On the other hand, the nucleotide sequence of the luciferase gene was determined by the method as shown in item 18 of the Example to obtain the nucleotide sequence of the luciferase gene shown in FIG. 6, and then the amino acid sequence of the polypeptide translated by the nucleotide sequence. Was confirmed and the results shown in FIG. 7 were obtained.

A gene encoding the amino acid sequence thus determined is one of the present invention.

Then, the above microorganism is cultured in a medium, and luciferase is collected from the culture.

Any medium may be used as long as it is used for culturing microorganisms belonging to the genus Escherichia, for example, tryptone 1% (W / V), yeast extract 0.5.
% (W / V), NaCl 0.5% (W / V) and 1 mM isopropyl-
β-D-thiogalactoside and the like can be mentioned.

The culture temperature is, for example, 30 to 40 ° C, preferably 37 ° C.
The culture time is, for example, 4 to 8 hours, preferably about 4 hours.

Then, for example, the bacterial cells from the culture are collected by centrifugation at 8,000 rpm for about 10 minutes, and the obtained bacterial cells are
For example, "Methods in Enzymology" (Method
s in Enzymology) 133, pp. 3-14 (1986) to obtain a crude enzyme solution.

And the crude enzyme solution can be used as it is,
If necessary, the product is purified by ammonium sulfate fractionation, a hydrophobic chromatography method (for example, a method using butyl-Toyopearl 650C etc.), a gel filtration method (for example, a method using Ultrogel AcA34 etc.) to obtain a purified luciferase.

The physicochemical properties of the luciferase thus obtained are as shown below.

Action: It is an enzyme that catalyzes the oxidation of luciferin by oxygen molecules as shown in the following enzymatic reaction formula.

Substrate specificity: Does not act on ADP, CTP, UTP and GTP.

Optimum pH and stable pH range: The optimum pH is 25 mM glycylglycine with luciferin as the substrate, the pH is changed from pH 6.5 to 11.5, the reaction is performed at a temperature of 30 ° C, and the luminescence (photon number) for 20 seconds is set. If measured, first
As shown in the figure, the pH is 7.5 to 9.5, and the stable pH range is a buffer containing luciferin (pH 4.6 to 8.0: 25 mM phosphate buffer, pH 8.0 to 11.5: 25 mM glycine / sodium chloride-
Uses sodium hydroxide buffer respectively. In addition, ammonium sulfate was added to each of the buffer solutions so as to achieve 10% saturation. ) And add the enzyme to
When it is operated for a time, it is 6.0 to 10.5 as shown in FIG. In addition, in FIG. The activity is shown when 25 mM phosphate buffer and 25 mM glycine / sodium chloride-sodium hydroxide buffer are used, respectively.

Titration method: 25 mM glycylglycine (pH 7.8) 8 ml, magnesium sulfate solution [0.5 ml glycylglycine (pH 7.8) with magnesium sulfate added to 0.1 M] 0.5 ml and luciferin solution [25 mM 0.8 ml of a solution in which luciferin is added to glycylglycine (pH 7.8) to a concentration of 1 mM] is mixed to prepare a luciferin mixed solution.

A mixture of 400 μl of the luciferin mixed solution thus obtained and 10 μl of luciferase to be measured was added to an ATP solution [25 mM glycylglycine (pH 7.8) to which ATP was adjusted to 10 mM. At the same time as injecting 80 μl, a luminometer (Aloka Co., Luminescence Reader BLR-2
Calculate the total number of photons generated in 01) for 20 seconds.

Optimum temperature range of action: When the amount of luminescence (number of photons) is measured for 20 seconds by reacting at each temperature under pH 7.8, it is in the range of 0 to 50 ° C.

 Conditions for inactivation by pH: Complete inactivation after 4 hours at pH 5.0 or lower and pH 12.0 or higher.

〔The invention's effect〕

As is apparent from the above, according to the present invention, by culturing in a medium a microorganism belonging to the genus Escherichia containing recombinant DNA in which the luciferase gene derived from Luciola lateris of the present invention is incorporated, a very short period of time is obtained. The luciferase derived from Luciola lateris can be efficiently produced, and the present invention is industrially very useful.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Examples In addition, in items 1 to 10 described below, a DNA containing a gene encoding a luciferase of Photinus pyralis, which is one species of firefly (the DNA contains a gene encoding a luciferase of Luciola crucita). DNA
Is used as a probe when searching for. ), And in the following items 11 to 13, DNA containing a gene encoding luciferase derived from Luciola cruciata (the DNA contains a gene encoding luciferase from Luciola lateris).
It is used as a probe when searching DNA. ) Is described.

1. Preparation of m-RNA Photinus pyralis (Photinus)
pyralis) 1 g of dried tail (manufactured by Sigma) was sufficiently crushed using a mortar and pestle, and 5 ml of lysis buffer [20 mM Tris-HCl buffer (pH 7.4) / 10 mM NaCl / 3 mM magnesium acetate / 5% (W / V) Sucrose / 1.2% (V / V) Triton X-10
0/10 mM vanadyl nucleoside complex (manufactured by New England Biolabs)] was added and further crushed in the same manner as described above to obtain a solution containing a crushed tail of Photinus pyralis.

5 ml of the solution thus obtained was placed in a cup blender (manufactured by Nippon Seiki Seisakusho Co., Ltd.) and treated at 5,000 rpm for 5 minutes, and then 12 ml of a guanidine isothiocyanate solution (6M guanidine isothiocyanate / 37.5 mM sodium citrate was added. (PH 7.0) /0.75% (W / V) N-lauroylsarcosine sodium / 0.15M β-mercaptoethanol) was added, and further 3,000 rpm using the above blender.
The solution obtained after 10 minutes of treatment was filtered with a triple layer of gauze to obtain a filtrate, and 1.2 ml of 5.7M chloride was preliminarily added to four ultracentrifuge tubes (manufactured by Hitachi Koki Co., Ltd.). Layer the cesium solution on top of each other, and pour each of the above filtrates on top of each other, and centrifuge at 30,000 rpm for 16 hours at 15 ° C using an ultracentrifuge (Hitachi Koki Co., Ltd., SCP55H). A precipitate was obtained.

The obtained precipitate was washed with cold 70% (V / V) ethanol and then used as a 10 mM Tris buffer solution (10 mM Tris-hydrochloric acid buffer solution (pH 7.4) / 5 mM EDTA / 1% sodium dodecyl sulfate). To the suspension in 4 ml, the same amount of n-butanol and chloroform mixed at a ratio of 4 to 1 (volume ratio) was added and extracted, followed by centrifugation at 3,000 rpm for 10 minutes by a conventional method, and then water. Layer and an organic solvent layer are separated, 4 ml of the above 10 mM Tris buffer is added to this organic solvent layer, and the extraction and separation operations are repeated twice, and the aqueous layer obtained is 1/10 volume of 3M. It was obtained by adding sodium acetate (pH5.2) and twice the amount of cold ethanol and leaving it at a temperature of -20 ° C for 2 hours, and then centrifuging at 8,000 rpm for 20 minutes by a conventional method to precipitate RNA. Dissolve RNA in 4 ml water,
After performing the above ethanol precipitation procedure, the RNA obtained was
It was dissolved in ml of water to obtain 3.75 mg of RNA.

And by repeating the above operation again, a total of 7 mg
RNA was prepared and 7 mg of RNA was subjected to an oligo (dT) -cellulose (New England Biolabs) column chromatogram in order to select m-RNA from this RNA.

A 2.5 ml Terumo syringe (manufactured by Terumo Corp.) was used as a column, and 0.5 g of resin was an elution buffer [10 mM Tris-hydrochloric acid buffer (pH 7.6) / 1 mM DETA / 0.1% (W / V) sodium dodecyl sulfate]. After swelling, it was packed in a column, and the binding buffer [10 mM Tris-hydrochloric acid (pH 7.6) / 1 mM EDTA / 0.4 M NaCl / 0.1 was added.
% Sodium dodecyl sulfate].

To 7 mg of RNA, the same amount of buffer solution (10 mM Tris-HCl (pH 7.
6) / 1mM EDTA / 0.8M NaCl / 0.1% sodium dodecyl sulfate] was added, and the mixture was heat-treated at a temperature of 65 ° C. for 10 minutes, quenched in ice, and applied to an oligo (dT) -cellulose column,
Wash the resin with binding buffer to remove unbound r-RNA and tR
NA was thoroughly washed, and m-RNA was further eluted with an elution buffer to obtain 40 μg of luciferase m-RNA.

2. Concentration of luciferase m-RNA Next, luciferase m-RNA was concentrated by a sucrose density gradient centrifugation method.

A sucrose density gradient of 10 to 25% (W / V) was obtained by using a 40% (W / V) sucrose solution [50 mM Tris-hydrochloric acid buffer solution (pH 7.5) / 20 mM NaCl /
1 mM EDTA / 40% (W / V) sucrose] 0.5 ml, and put it on it
2.4 ml each 25% (W / V), 20% (W / V), 15% (W / V) and
It was prepared by overlaying 10% (W / V) sucrose solution and leaving it at a temperature of 4 ° C. for 24 hours. 30 μg of m-RNA obtained in item 1 was overlaid on this sucrose density gradient, and S-Beckman S
Using W41 rotor, 30,000 rpm, temperature 18
Centrifugation was carried out at 18 ° C for 18 hours. After the centrifugation operation,
Fractions of 0.5 ml each were collected, m-RNA was recovered by the ethanol precipitation method, and dissolved in 10 μl of water.

Next, the protein encoded by m-RNA was examined to identify the fraction enriched in luciferase m-RNA. Fractionated RNA (1 µl), rabbit reticulocyte lysate (Amersham) 9 µl and [ ³⁵ S] methionine 1 µl (Amersham) were mixed, and the temperature was 30 ° C.
Reaction for 30 minutes, add 150 μl of NET buffer [150 m
M NaCl / 5mM EDTA / 0.02% (W / V) NaN 3 / 20mM Tris - HCl buffer (pH7.4) /0.05% (W / V ) Nonidet P-40 ((Bethesda Research Laboratories, Inc., surfactants )], And 1 μl of anti-luciferase serum (prepared as described below) was added, and the mixture was allowed to stand at a temperature of 4 ° C. for 18 hours, and then 10 mg of protein A sepharose (Pharmacia) was added. What was added and left to stand at a temperature of 20 ° C. for 30 minutes was centrifuged at 12,000 rpm for 1 minute by a conventional method to recover the resin.

The recovered resin was washed 3 times with 200 μl of NET buffer, and 40 μl of the sample buffer for SDS-PAGE [62.5 m
M Tris-HCl buffer (pH6.8) / 10% (V / V) glycerol / 2% (W / V) sodium dodecyl sulfate / 5% (V / V) mercaptoethanol / 0.02% (W / V) bromine Phenol blue], and boil for 3 minutes at a temperature of 100 ° C.
Centrifuge at 2,000 rpm for 1 minute, collect the supernatant,
The entire amount was loaded on a 7.5% (W / V) sodium dodecyl sulfate-polyacrylamide gel.

Gel electrophoresis was performed by the Laemmli method [Nature, p. 227, p. 680 (1970)], and the gel after electrophoresis was diluted with 10% (V / V) acetic acid. 3
After soaking for 0 minutes to fix the protein, soaking in water for 30 minutes, further soaking in 1M sodium salicylate solution for 30 minutes and drying to obtain a dried gel, X-ray film (Fuji Photo Film Co., RX) Was used for fluorography.

By the above operation, the band of luciferase protein was observed on the X-ray film only when the RNA of the fraction containing luciferase m-RNA was used, and the fraction enriched in luciferase m-RNA could be identified. It was

3. Preparation of antiserum Rabbit antiluciferase serum against purified luciferase was prepared by the following method.

A luciferase solution with a concentration of 3.2 mg / ml [a luciferase manufactured by Sigma Co., which was dissolved in a 0.5 M glycylglycine solution (pH 7.8)] 0.7 ml was suspended in an equal amount of Freund complete adjuvant 2.24 mg 2k as an antigen
g of the Japanese white rabbit was administered to the palm of the hand, and after 2 weeks of breeding, the same amount of antigen as the initial dose was administered intradermally on the dorsal skin, and after 1 week of breeding, the same operation was performed. Furthermore, after 1 week of breeding, whole blood was collected.

The blood thus obtained was allowed to stand at a temperature of 4 ° C. for 18 hours and then centrifuged at 3,000 rpm for 15 minutes by a conventional method to obtain anti-luciferase serum as a supernatant.

4. Synthesis of c-DNA The synthesis of c-DNA was performed using a kit manufactured by Amersham.

Using 2 μg of m-RNA obtained as described above, “Mol. Cell Bol” (Mol. Cell B) instructed by Amersham Co.
iol.), Volume 2, 161 (1982) and "Gene" (Gen).
e), Vol. 25, p. 263 (1983). As a result, 300 ng of double-stranded c-DNA was obtained.

150 ng of this c-DNA was added to 7 μl of TE buffer [10 mM Tris-
11 dissolved in hydrochloric acid buffer (pH 7.5) / 1 mM EDTA]
μl mixed solution [280 mM sodium cacodylate (pH6.8) / 6
0 mM Tris-HCl buffer (pH 6.8) / 2 mM cobalt chloride] and 3.8 μl tilling mixture [10 mM dithiothreitol
7.5 μl / 10 ng / ml poly A 1 μl / 5 mM dCTP 2 μl / water 110
μl], and then 29 units of terminal transferase (Boehringer Mannheim) were added and reacted at a temperature of 30 ° C. for 10 minutes, and then 2.4 μl
The reaction was stopped by the addition of 0.25 M EDTA and 2.4 μl of 10% (W / V) sodium dodecyl sulfate, respectively.

After deproteinizing with 25 μl of water-saturated phenol as a stop solution, 25 μl of 4M ammonium acetate and 100 μl of cold ethanol were added to the recovered aqueous layer, and the mixture was left at −70 ° C. for 15 minutes. , C-DNA was recovered by centrifugation at 12,000 rpm for 10 minutes and dissolved in 10 μl of TE buffer to obtain a c-DNA solution.

As described above, c- with a deoxycytidine tail
100 ng of DNA was obtained.

5. Recombinant plasmid pMCE10DNA used for vector
Preparation of E. coli W3110 strain (ATCC27325), plasmid pBR325DNA (B
RL) and plasmid pBR322DNA (Takara Shuzo), T. Masuda et.al. "Agricultural Biological Chemistry" (Agrica)
ltural Biological Chemistry), Volume 50, 271-279
Plasmid pKN305 prepared by the method described on page (1986).
DNA and plasmid pMC1403-3DNA (JP-A-61-274683)
No. The publication) respectively 1 [mu] g, 10 [mu] l of mixture - was added to the [50mM Tris-HCl buffer _{(pH7.5) / 10mM MgCl 2 /} 100mM NaCl / 1mM dithiothreitol], further, to this, Hind III
And Sal I (both manufactured by Takara Shuzo Co., Ltd.) were added at a rate of 2 units each, and the mixture was allowed to react at a temperature of 37 ° C. for 1 hour for cleavage, and phenol extraction and ethanol precipitation were carried out by a conventional method to obtain a precipitate. The precipitate, ligation buffer 10μl [20 mM MgCl _2/66 mM Tris - HCl buffer (pH 7.
6) / 1 mM ATP / 15 mM dithiothreitol] to obtain a solution, 1 unit of T4 DNA ligase (Takara Shuzo) was added, and ligation reaction was carried out at a temperature of 20 ° C. for 4 hours.
Then, using this reaction solution, Escherichia coli JM101 (A) was transformed by the transformation method described in "J. Bacteriology", Vol. 119, pp. 1072 to 1974 (1974)].
TCC33876) strain was transformed, drug resistance (ampicillin resistance and tetracycline sensitivity) and β-galactosidase activity were examined, a transformant was obtained, and the recombinant plasmid DNA contained in the strain was named pMCE10. Escherichia coli JM101 strain containing this recombinant plasmid pMCE10DNA, tryptone 1% (W / V), yeast extract 0.5% (W / V)
V) and NaCl 0.5% (W / V) in Escherichia coli JM obtained by preculturing the medium 1 at 37 ° C. for 16 to 24 hours.
20 ml of 101 (pMCE10) culture solution was inoculated, shake-cultured at a temperature of 37 ° C. for 3 hours, 0.2 g of chloramphenicol was added, and the same culture was performed at the same temperature for 20 hours to obtain a culture solution. .

Then, this culture solution was centrifuged at 6,000 rpm for 10 minutes by a conventional method to obtain 2 g of wet cells, which was added to 20 ml of 25% (W /
V) 350 mM Tris-HCl buffer containing sucrose (pH 8.
0), and then lysozyme 10mg,
8 ml of a 0.25 M EDTA solution (pH 8.0) and 8 ml of a 20% (W / V) sodium dodecyl sulfate solution were added, and the mixture was kept warm at 60 ° C for 30 minutes to lyse the cells to obtain a lysate.

To this lysate, add 13 ml of 5M NaCL solution and
What was treated for 6 hours was centrifuged at 15,000 rpm for 30 minutes by a conventional method to obtain an extract. Phenol extraction and ethanol precipitation were performed by a conventional method to obtain a precipitate.

Then, this precipitate was subjected to ordinary vacuum drying treatment, and 6 ml of 10 mM Tris-HCl buffer containing 1 mM EDTA (p
H7.5) and further added with 6 g of cesium chloride and 0.2 ml of ethidium bromide solution (10 mg / ml), equilibrium density using an ultracentrifuge at 39,000 rpm for 42 hours by a conventional method. After carrying out a gradient centrifugation treatment to isolate the recombinant plasmid pMCE10DNA and further removing ethidium bromide using n-butanol,
Recombinant plasmid pM purified by dialysis against 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA
500 μg of CE10 DNA was obtained.

6. Preparation of vector DNA Recombinant plasmid pMCE10DN obtained as described above
Dissolve 15 μg of A in 90 μl of TE buffer described in item 4, and
0 μl of Med buffer [100 mM Tris-HCl buffer (pH 7.5)
/ 100mM MgCl ₂ / 10mM dithiothreitol / 500mM NaCl], then 30 units of restriction enzyme AccI (Takara Shuzo)
Was further added, and a cutting treatment was performed at a temperature of 37 ° C. for 1 hour to obtain a cut treatment product. After deproteinizing by adding 100 μl of water-saturated phenol to this cleaved product, the aqueous layer was recovered, and 1/10 volume of 3M sodium acetate (pH 7.5) and twice volume of cold ethanol were collected. Was added, the mixture was allowed to stand at a temperature of -70 ° C for 15 minutes, and then centrifuged at 12,000 rpm for 10 minutes to recover DNA.

This DNA was dissolved in 10 μl of TE buffer, and mixed with 15 μl of a mixture [280 mM sodium cacodylate (pH 6.8) / 60 mM Tris-
Hydrochloric acid buffer solution (pH 6.8) / 2 mM cobalt chloride] was added, and then 5 μl of the tilling mixture solution (described in item 4) (5 m
MdGTP was used), 5 units of terminal transferase (Takara Shuzo) was further added, and the mixture was reacted at 37 ° C. for 15 minutes. By performing the same post-treatment as the c-DNA tilling reaction described in item 4, a DNA having a tail of deoxyguanosine at the AccI site of the recombinant plasmid pMCE10DNA was prepared.

On the other hand, a DNA having a deoxyguanosine tail attached to the PstI site of the plasmid pUC19DNA was prepared at the same time as described below.

30 μg of plasmid pUC19DNA (manufactured by Takara Shuzo), 350 μl
40 μl of Med buffer and 120 units of restriction enzyme PstI (manufactured by Takara Shuzo Co., Ltd.) were added to the solution dissolved in TE buffer solution of, and the digestion treatment was performed at 37 ° C. for 1 hour, followed by deproteinization with phenol by a conventional method. DNA was collected by and ethanol precipitation treatment.

The obtained DNA was dissolved in 35 μl of TE buffer,
0 μl mixed solution [280 mM sodium cacodylate (pH 6.8) / 60
mM Tris-HCl buffer (pH 6.8) / 1 mM cobalt chloride], 1
Add 9 μl of the tilling mixture described in item 4 (containing dGTP) and 60 units of terminal transferase (Takara Shuzo Co., Ltd.), react at 37 ° C. for 10 minutes, and then perform phenol treatment and ethanol precipitation by a conventional method. As a result, the target DNA was recovered.

7. Annealing and transformation 15ng of the above-synthesized c-DNA and 200ng of vector DNA were
μl of annealing buffer (10 mM Tris-HCl buffer (pH 7.
5) / 100mM NaCl / 1mM EDTA], temperature 65 ℃ for 2 minutes, temperature 46 ℃ for 2 hours, temperature 37 ℃ for 1 hour and temperature 20 ℃
The c-DNA and the vector DNA were annealed by leaving them to stand for 18 hours.

Using the annealed DNA, the Hanahan method [“DNA cloning” (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)], Escherichia coli DH1
The strain (ATCC33849) was transformed with plasmid pUC19DNA and recombinant plasmid pMCE10DNA as vectors.
-Created each DNA bank.

8. Search for luciferase c-DNA Since the AccI site of the recombinant plasmid pMCE10DNA is located at the site encoding the Escherichia coli β-galactosidase gene, the c-DNA incorporated in this site forms a fusion protein with β-galactosidase. In addition, the promoter of the β-galactosidase gene of the recombinant plasmid pMCE10 has been converted to the promoter of Escherichia coli driptophan gene as described above.

Using recombinant plasmid pMCE10DNA as a vector c
-96 96 DNA bank colonies in 10 ml of M9 casamino acid medium [Molecular Cloning]
g), pages 440-441, Cold Spring Harbor Laboratory (1
982)] and thiamine (10 μg / ml) were added to the medium and shake-cultured at a temperature of 37 ° C. for 10 hours.
It was suspended in 200 μl of the sample buffer for SDS-PAGE described in item 2 and boiled at a temperature of 100 ° C. for 5 minutes.

40 μl of this suspension was subjected to electrophoresis by a conventional method using 7.5% (W / V) polyacrylamide gel. After the electrophoresis was completed, the protein developed on the gel was analyzed by Western blotting ["Anal. Biochem."
Vol., P. 195 (1981)] to a nitrocellulose filter, and the nitrocellulose filter is immunoblot assay kit (manufactured by Bio-Rad).
Was used to stain with anti-luciferase serum. The method followed the operating method of Hyorad.

That is, use a nitrocellulose filter with 100 ml of blocking solution [TBS buffer [20 mM Tris-HCl buffer / 500 m
M NaCl (pH 7.5)] in which 3% (W / V) gelatin was dissolved] at a temperature of 25 ° C. for 30 minutes. Next, this nitrocellulose filter was transferred to 25 ml of a primary antibody solution [a luciferase antiserum diluted 25 times (V / V) with a solution of 1% (W / V) gelatin in TBS buffer]. ,
After shaking for 90 minutes at a temperature of 25 ° C, 100 ml of Tween (T
ween) -20 washing solution [solution containing 0.05% (W / V) Tween-20 in TBS buffer] and transferred at 25 ° C for 1
The operation of shaking for 0 minutes was performed twice. Then, the nitrocellulose filter thus obtained was treated with 60 ml of a secondary antibody solution [horseradish peroxidase-labeled anti-rabbit antibody (Bio-Rad) 1% (W / V) gelatin in TBS buffer. The solution was diluted 3000 times (V / V) with the dissolved solution] and shaken at a temperature of 25 ° C for 60 minutes, then 10
The above operation of washing the nitrocellulose filter with 0 ml of Tween-20 was repeated twice, and the thus obtained nitrocellulose filter was replaced with 120 ml of the coloring solution [60 mg of 4-chloro-1-naphthol in 20 ml. Solution mixed with cold methanol and 60 μl of 30% (V / V) hydrogen peroxide solution added to 100 ml of TBS buffer solution], and color was developed at a temperature of 25 ° C. for 10 minutes.

In this way, when 96 colonies were treated as one group and the same method was carried out for 4 groups, a protein band stained with luciferase antiserum was observed in 2 groups. Next, 96 colonies belonging to these two groups were divided into 8 groups of 12 colonies, and the same operation was performed. As a result, a protein reacting with anti-luciferase serum was observed in each group. Finally, the 12 colonies contained in this group were subjected to the same operation one colony at a time to identify colonies producing a protein that reacts with the luciferase antiserum. Two colonies having luciferase c-DNA were obtained by the above operation.
From these two colonies, a plasmid is prepared by the method described in item 5.
DNA was prepared. The obtained recombinant plasmid DNA is
They were named pALf2B8DNA and pALf3A6DNA, respectively.

9. Search for large luciferase c-DNA-preparation of DNA probe 100 μg of recombinant plasmid pALf3A6DNA was added to 330 μl of
Was dissolved in TE buffer, to which 40μl of Low buffer [100mM Tris - HCl buffer _{(pH7.5) / 100mM MgCl 2 /} 10mM dithiothreitol], 130 units of PstI (Takara Shuzo Co., Ltd.) and 1
20 units of SacI (Boehringer Mannheim) was added, and the mixture was cut at a temperature of 37 ° C. for 1.5 hours.

The total amount of this DNA was separated by electrophoresis using 0.7% (W / V) agarose gel. Agarose gel electrophoresis
Methods such as T.Maniatis ["Molecular
"Cloning" (Molecular Cloning), 156-161
Page, Cold Spring Harbor Laboratory (1984)]. A DNA band containing luciferase c-DNA was cut out, placed in a dialysis tube, 2 ml of TE buffer was added, the dialysis tube was sealed, and the DNA was eluted from the gel into the buffer solution by electrophoresis. After adding an equal volume of water-saturated phenol to this solution and stirring, the aqueous layer was recovered, and DNA was recovered by ethanol precipitation according to the above.

10 µg of the obtained DNA fragment was dissolved in 126 µl of TE buffer, and 16 µMed buffer and 64 units of Sau3AI were added.
(Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at a temperature of 37 ° C. for 2 hours, and then the total amount was separated by electrophoresis using a 5% (W / V) polyacrylamide gel. Polyacrylamide gel electrophoresis is performed by A. Ma
xam) method [“Methods in Enzymology” (M
ethds in Enzymology), Vol. 65, p. 506 (1980)]. A 190 bp DNA fragment was isolated in the same manner as described above, and 1 μg of Sau3AI luciferase c-DN was isolated.
An A fragment was obtained.

1 μg of this luciferase c-DNA was added to [α- ³² P] dC
It was labeled by the nick translation method using TP (manufactured by Amersham). Nick Translation uses a kit made by Takara Shuzo Co., Ltd.
J. Mol. Biol., Volume 113, 237-2
Page 51 (1977) and "Molecular Cloning" (Mo
lecular Cloning), pages 109-112, Cold Spring Habor La
boratory) (1982).

10. Search for large luciferase c-DNA-colony hybridization Luciferase c labeled with ³² P prepared by the above method
-A DNA fragment was used as a probe and a Photinus pyralis tail c-DNA bank using the recombinant plasmid pUC19DNA as a vector was prepared by the colony hybridization method {"Protein / nucleic acid / enzyme" vol. 26, pp. 575-579 (1981). )}, And a colony having luciferase c-DNA was obtained. The recombinant plasmid DNA contained in one of the colonies was named pALf3, and the plasmid DNA was prepared by the method described in item 5. E. coli containing the recombinant plasmid DNA was named E. coli DH1 (pALf3). The transformant has been deposited as ATCC67462.

Then, the above recombinant plasmid pALf3DNA was treated with XbaI,
DNA fragments obtained by single digestion and double digestion using Hind III, BamHI, EcoRI and PstI (all manufactured by Takara Shuzo) were analyzed for mobility patterns by agarose gel electrophoresis and the resulting migration And the standard mobility pattern of the DNA fragment obtained by digesting λDNA (Takara Shuzo Co., Ltd.) with Hind III, the molecular weight obtained was 1,77 bp. ,
It was as shown in FIG.

11. Luciola Cruciata MR
Preparation of NA 10 g of living Luciola cruciata (purchased from Genjibotaru Co., Ltd., Seibu Department Store) was placed in an ultra-low temperature freezer, frozen, and the tail was cut off using scissors.
To this, 18 ml of a guanidine isothiocyanate solution was added, and 1.1 mg of RNA was prepared according to the method described in item 1.
1.1 mg of this RNA was oligo (dT) according to the method described in item 1.
-Perform column chromatography of cellulose at 30μ
g of Luciola cruciata tail m-RNA was prepared.

12. Preparation of Luciola cruciata tail c-DNA bank Use the kit purchased from Amersham for the synthesis of c-DNA and use the "Mole Cell Violet" specified by Amersham.
(Mol. Cell. Biol.), Volume 2, p. 161 (1982) and Gene, 25, p. 263 (1983).

0.9 μg of double-stranded c-DNA was synthesized from 2 μg of RNA from tail of Luciola cruciata. A tail of polydeoxycytidine was added to 0.3 μg of this c-DNA using the method described in item 4.

PUC19 plasmid DNA in which 20 ng of this c-DNA and the tail of polyguanosine prepared in item 6 were added to its PstI site
Anneal 500 ng by the method described in item 7, and
anahan) method ["DNA Cloning" (DN
A Cloning), Vol. 1, pp. 109-135 (1985)], and the E. coli DH1 strain (ATCC33849) was transformed with the annealed DNA to prepare a Luciola crucita tail c-DNA bank.

13. Luciferase c-DNA from Luciola cruciata
Recombinant plasmid pALf3DNA obtained in Item 10 of pALf3DNA 10 μg
Was dissolved in 90 μl of TE buffer and 10 μl of Med buffer, 2
5 units of restriction enzyme EcoRI and 25 units of restriction enzyme
ClaI (both manufactured by Takara Shuzo Co., Ltd.) was added, and the reaction was carried out at a temperature of 37 ° C. for 2 hours to cut the DNA. The cleaved recombinant plasmid pALf3 DNA contains a luciferase c-DNA portion derived from Photinus pyralis (American firefly). 8
The 00 bp EcoRI / ClaI DNA fragment was isolated according to the method using agarose gel electrophoresis described in item 9,
1 μg of EcoRI / ClaI DNA fragment was obtained. This 1μ
[α- ³² P] dCTP triphosphate (manufactured by Amersham)
Was labeled with ³² P by the nick translation method described in item 9. ^{Using the} 32P-labeled EcoRI / ClaI DNA fragment as a probe, Luciola cruciata tail c
-Escherichia coli having luciferase c-DNA derived from Luciola cruciata was selected by searching the DNA bank by the colony hybridization method described in item 10. Several E. coli colonies hybridizing with the probe were obtained. The plasmid DNA of one colony in this is p
It was named GLf1 and the recombinant plasmid DNA was isolated according to the method described in item 5.

This recombinant plasmid pGLf1DNA was cloned into HpaI, HindIII, Ec
oRV, DraI, Afl II, Hinc II, PstI (all manufactured by Takara Shuzo)
And SspI (manufactured by New England Biolabs) were used to analyze the mobility patterns of the DNA fragments obtained by single digestion and double digestion by agarose gel electrophoresis, and the obtained mobility patterns and λ phages were analyzed. The molecular weight obtained by comparing the standard mobility pattern of the DNA fragment obtained by digesting DNA (Takara Shuzo Co., Ltd.) with Hind III was 2,000 bp. As shown in the figure.

14. Preparation of Luciola lateralis m-RNA 5 g of live Luciola lateris (purchased from Heikebotaru and Kawahara Bird and Animal Trading) was placed in an ultra-low temperature freezer, frozen, and the tail was cut off with scissors. 18 ml of a guanidine isothiocyanate solution was added, and 340 μg of RNA was prepared according to the method described in item 1. This RNA340
According to the method described in item 1, μg was subjected to column chromatography of oligo (dT) -cellulose to prepare 6 μg of Luciola lateralis tail m-RNA.

15. Preparation of Lucio lateraris tail c-DNA bank c-DNA was synthesized using a kit manufactured by Amersham.

2.0 μg of m-RNA obtained as described above was used to instruct "Amersham""Mole Cell Violet" (Mol.Cel).
l.Biol., Vol. 2, p. 161 (1982) and Gene (Gen.
e), Vol. 25, p. 263 (1983). As a result, 250 ng of double-stranded c-DNA was obtained.

Thus obtained 250 ng of c-DNA was added to c of Amersham Co.
-A DNA cloning kit was used to methylate the restriction enzyme EcoRI cleavage site by the method specified by Amersham, and EcoRI linkers were attached to both ends of c-DNA.

Added - [HCl buffer (pH7.5) 100mM MgCl ₂ / 10mM dithiothreitol / 500 mM NaCl 100 mM Tris] Plasmid PUC119DNA (Takara Shuzo Co., Ltd.) 100 ng, is dissolved in a water 8 [mu] l, 1 [mu] l of Med buffer After that, 1 more to this
0 unit (1 μl) of restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.) was added, and a cleavage treatment was performed at a temperature of 37 ° C. for 1 hour.

Next, 1 μl of 1 M Tris-hydrochloric acid buffer (pH 8.0) and 0.3 unit (1 μl) of alkaline phosphatase (Takara Shuzo) were added to the cleaved product, and the temperature was adjusted to 1 at 65 ° C.
After enzymatic treatment for a period of time, dephosphorylation of both ends of the digested product was performed, and 12 μl of water-saturated phenol was added to deproteinize, and then 1 μl of 3M sodium acetate (pH 5) was added to the recovered aqueous layer. .8) and 26 μl of cold ethanol were added, respectively, and the mixture was allowed to stand at a temperature of −70 ° C. for 15 minutes, and a microcentrifuge [Tomy Seiko Co., Ltd., MRX-150] was used at 12,000 rpm.
DNA was collected by centrifugation for 5 minutes.

The thus obtained plasmid pUC119DNA100, which had been digested with the restriction enzyme EcoRI and dephosphorylated on both ends,
ng and 250 ng of the c-DNA prepared in item 15 were suspended in 8 μl of water, and 1 μl of this was added.
Ligation buffer - in that the addition of [200 mM MgCo _2/660 mM Tris-HCl buffer (pH7.6) / 10mM ATP / 150mM dithio Sui les Torr], T4 DNA of one unit (1 [mu] l)
Ligase (Takara Shuzo Co., Ltd.) was added and the mixture was allowed to stand at a temperature of 16 ° C. for 16 hours to ligate the plasmid vector DNA and c-DNA to obtain a reaction product.

Using this reaction, the method of Hanahan ["DNA Cloning" (DNA Clonin
g), Vol. 1, pp. 109-135 (1985)], Escherichia coli DH1
Strain (ATCC33849) was transformed, and c-DN derived from the tail of Luciola lateralis using plasmid pUC119DNA as a vector
A bank was made.

16. Luciola lateralis-derived luciferase c-DNA
Recombinant plasmid pGLf1DNA 10 μg obtained in Item 13
Was dissolved in 90 μl of TE buffer and 10 μl of Med buffer, 2
5 units of restriction enzyme PstI (manufactured by Takara Shuzo) was added, and the reaction was carried out at a temperature of 37 ° C. for 2 hours to cut the DNA. 2,000 bp Ps containing luciferase c-DNA part derived from Luciola cruciata from the digested recombinant plasmid pGLf1 DNA
The tI DNA fragment was isolated according to the method using the agarose gel electrophoresis described in item 9, and 1 μg of PstIDN was isolated.
A fragment was obtained. 1 μg of this DNA was added to [α-
³² P] dCTP (manufactured by Amersham) was used to label with ³² P by the nick translation method described in item 9. ^By using the PstI DNA fragment labeled with ³² P as a probe, the Escherichia coli having luciferase c-DNA derived from Luciola lateris was selected by searching the tail c-DNA bank of Luciola lateris by the colony hybridization method described in item 10. Several E. coli colonies hybridizing with the probe were obtained. The plasmid DNA contained in one of the colonies was named pHLf7, and the recombinant plasmid DNA was isolated according to the method described in item 5.

The E. coli DH1 (p.
HLf7) has been deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, as Micro Engineering Research Article No. 1917 (FERM BP-1917).

The above recombinant plasmid pHLf7DNA was replaced with HpaI, EcoRV, ApaI,
Hind III and EcoRI (both manufactured by Takara Shuzo) and SspI
(Manufactured by New England Biolabs), the DNA patterns obtained by single digestion and double digestion are analyzed by agarose gel electrophoresis for mobility patterns, and the obtained mobility patterns and λ phage DNA ( Hind the Takara Shuzo)
The molecular weight of the gene encoding luciferase of Luciola lateralis obtained by comparing with the standard mobility pattern of the DNA fragment obtained by digestion with III was 2,
The restriction map of the above plasmid is
As shown in the figure.

Then, 10 μg of the above recombinant plasmid pHLf7DNA,
To the solution dissolved in 45 μl of TE buffer, 5 μl of Med buffer and 2 units of EcoRI (Takara Shuzo) were added respectively, and the reaction was carried out at 37 ° C. for 2 hours to obtain a partial digestion product of DNA. It was

Then, this partially digested product was subjected to agarose gel electrophoresis described in item 9 to isolate 1 μg of a 2,000-bp EcoRI fragment containing all luciferase genes derived from Luciola tetheralis.

17. E. coli DH1 (pHLf7) (FERM BP-1917) culture and preparation of crude enzyme solution E. coli DH1 (pHLf7) (FERM BP-1917) LB
-amp medium [Bactotryptone 1% (W / V), yeast extract
0.5% (W / V), NaCl 0.5% (W / V), and ampicillin (50 μg / ml)] 3 ml, and shaking culture was performed at a temperature of 37 ° C. for 18 hours. 0.5 ml of this culture was inoculated into 10 ml of the above LB-amp medium, 1 mM isopropyl-β-D-thiogalactoside was further added thereto, and the mixture was cultivated at 37 ° C. for 4 hours with shaking, and then at 8,000 rpm. 20 mg of wet cells were obtained by centrifugation for 10 minutes.

Collected cells were treated with 0.1M KH ₂ PO ₄ (pH7.8), 2mM EDTA, 1m
Suspend in 0.9 ml of a buffer consisting of M dithiothreitol and 0.2 mg / ml protamine sulphate, and add 100 μl of 10
A mg / ml lysozyme solution was added and left in ice for 15 minutes. Next, this suspension was frozen in a methanol / dry ice bath and subsequently left at a temperature of 25 ° C. to be completely thawed. Further, centrifugation was carried out at 12,000 rpm for 5 minutes to obtain 1 ml of a crude enzyme solution as a supernatant.

The luciferase activity in the thus obtained crude enzyme solution was measured by the method described below, and the results are shown in Table 1.

The luciferase activity in the obtained crude enzyme solution was measured by a method such as Kricka [“Achievements of Biochemistry and Biophysics” (Arch
ives of Biochemistry and Biophysics), Volume 217, Vol.
674 (1982)], and the number of photons generated was measured.

That is, 260 μl of 25 mM glycylglycine buffer (pH
7.8), 16 μl of 0.1 M magnesium sulphate, 24 μl of 1 mM luciferin (manufactured by Sigma) and 10 μl of the crude enzyme solution were mixed, then 100 μl of 20 mM ATP was added, and the number of photons generated was integrated for 20 seconds. The results are shown in Table 1.

For comparison, the luciferase activity was similarly measured for the Escherichia coli DH1 strain [E. coli DH1 (pUC119)] having the plasmid pUC119 DNA, and the results are shown in Table 1.

As is clear from the above table, E. coli DH1 (pHLf containing the recombinant plasmid pHL57 containing the luciferase gene of the present invention
7) has an increased number of photons compared to the control,
It was revealed that luciferase was produced in E. coli cells.

18. Luciferase c-DNA derived from Luciola lateralis
Analysis of nucleotide sequence of recombinant plasmid pHLf7DNA (10 μg) with restriction enzyme EcoRI
Cleaved with (Takara Shuzo) to obtain 2.0 μg and 0.5 μg of 1.7 Kb and 0.3 Kb DNA fragments containing luciferase c-DNA, respectively.
Each of these DNA fragments was subcloned into the EcoRI site of plasmid pUC118DNA (manufactured by Takara Shuzo Co., Ltd.) to obtain 4 clones depending on the type of insert fragment (1.7 Kb and 0.3 Kb) and the insertion direction.
Types of plasmids, pHLf11, pHLf12, pHLf13 and pHLf14
(The 1.7 Kb fragment was subcloned into pHLf11 and pHLf12, and 0.3 Kb into pHLf13 and pHLf14.
The fragment of b is subcloned. ).

Recombinant plasmid pHLf7DNA and plasmid pUC118DN
Cleavage of A with EcoRI (method described in item 6), isolation of luciferase c-DNA fragment using agarose gel electrophoresis (method described in item 9), plasmid pUC119DNA
Ligation reaction of luciferase c-DNA fragment (method described in item 5), E. coli JM101 strain (ATCC3
3876) transformation (method described in item 5), and recombinant plasmids pHLf11, pHLf12, pHLf13 and pHLf14 DNA
Was prepared (the method described in item 5) according to the method described in parentheses.

Then recombinant DNA, pHLf11, pHLf12, pHLf13 and p
Using HLf14DNA, a deletion kit for kilosequencing (manufactured by Takara Shuzo Co., Ltd.), and various luciferase c-DNAs according to the method of Henikoff (Gene, 28, 351-359 (1984)]. A plasmid DNA into which the deletion of is introduced is prepared, and the E. coli JM101 strain (AT
CC33876). The thus obtained Escherichia coli was infected with Perperphage M13KO7 (manufactured by Takara Shuzo Co., Ltd.) to perform the method of Messing (Methods in Enzymology, No. 1).
01, pp. 20-78 (1983)] to prepare single-stranded DNA. Sequencing with the obtained single-stranded DNA
13 Sequencing kit (manufactured by Takara Shuzo Co., Ltd.) was used according to the method of Messing. Gel electrophoresis for the analysis of the nucleotide sequence was performed using 8% (W / V) polyacrylamide gel (manufactured by Fuji Photo Film Co., Ltd.). The nucleotide sequence of the luciferase-derived luciferase c-DNA thus obtained is shown in FIG. 6, and the amino acid sequence of the polypeptide translated from the c-DNA is shown in FIG.
-The corresponding sequences of DNA and amino acid sequences are shown in Fig. 8, respectively.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH range of luciferase derived from Luciola lateris, and FIG. 2 is a diagram showing the stable pH of luciferase derived from Luciola lateris.
FIG. 3 is a diagram showing a digestion map of recombinant plasmid pALf3DNA with a restriction enzyme, FIG. 4 is a diagram showing a digestion map of recombinant plasmid pGLf1DNA with a restriction enzyme, and FIG. 5 is a recombinant plasmid. FIG. 6 is a diagram showing a digestion map of pHLf7DNA with a restriction enzyme, FIG. 6 is a diagram showing a nucleotide sequence of a luciferase gene derived from Luciola lateralis of the present invention, and FIG. 7 is a diagram showing Luciola lateris of the present invention. FIG. 8 is a diagram showing the amino acid sequence of a polypeptide translated from the luciferase gene derived from FIG. 8. FIG. 8 is a diagram showing a sequence in which the nucleotide sequence of the luciferase gene derived from Luciola latereris corresponds to the amino acid sequence.

Claims (2)

[Claims] 1. A luciferase gene characterized by encoding the amino acid sequence shown below. 2. The luciferase gene according to claim 1, which is represented by the base sequence shown below.