JPH02171189A - Luciferase gene - Google Patents

Abstract

PURPOSE:To isolate a gene of luciferase useful as an enzyme for ATP determination from luciferase of Luciola lateralis and to analyze structure of the gene. CONSTITUTION:A DNA containing a gene to code luciferase of Luciola cruciata) is searched by using a DNA containing a gene to code luciferase of Photinus pyralis as a probe and this DNA is used as a probe to search a DNA containing a gene to code luciterase of Luciola lateralis. The luciferase gene has restriction scission map shown by the figure (EI is EcoRI, S is Sspl, EV is EcoRV, A is ApaI and E is HpaI).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is directed to Luciola lateralis (Luciola lateralis).
teralis, Heike firefly).

[Conventional technology]

The luciferase of fireflies of the genus Luciola has only been obtained by isolation and purification from collected fireflies of the genus Luciola [Proc, Natl, Acad, Sci.
Vol. 74, No. 7, pp. 2799-2802 (1977
)).

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

[Problem to be solved by the invention]

However, since the above-mentioned luciferase is derived from insects, its production requires collecting Luciola fireflies from nature or cultivating the fireflies, and then separating and purifying the luciferase from the resulting fireflies. However, its production requires a great deal of time and effort.

[Means to solve the problem]

Therefore, as a result of various studies to solve the above problems, the present inventors transferred DNA containing a gene encoding luciferase derived from Luciola lateralis to vector D.
A recombinant DNA inserted into NA was obtained, and this recombinant DNA was transferred to f!5cherichia.
After discovering that luciferase can be efficiently produced in a short period of time by culturing in a medium a microorganism with luciferase-producing ability contained in a microorganism belonging to the genus, he filed a patent application (dated July 1, 1988). patent application specification).

After that, the present inventors further investigated the luciferase gene derived from Luciola lateralis, and as a result, succeeded in isolating and determining the structure of the luciferase gene derived from Luciola lateralis for the first time, thereby completing the present invention.

That is, the present invention is derived from (1) Luciola lateralis,
Luciferase gene defined by the restriction enzyme cleavage map below.

EI 5EVA HHEISEIAsn T
yr Gly Leu Val Val Asp Gl
y Arg l1eAla Lea Cys Ser
Glu Asn Cys Glu Glu PheSe
r Lys Lys Gly Leu Asp Lys
Vat lie Thr (2) The above (1) characterized in that it encodes the amino acid sequence shown below.
Luciferase gene described.

Met Glu Asn MetVat Ty
r Gly Pr.

Glu Glu Gly 5er Lys Tyr Met Asp Ala Ile Ala PheVal As
p Tyr Thr Lys Ser Cys Cys Glu Asn Asp Glu Glu Pro Phe Tyr Ala Gly Ala Gln Arg Tyr Ala LysThr Asn
Ala Leu Tyr Ala Glu Tyr Leu Gly Glu Ala Asn 1ie Pro Ire Leu Arg Leu Gly Thr Gly Leu Glu Leu Lys Gly Phe Arg Ile Vat
Met Leu Thr Lys PheVa
l Ala Gly Val Pro Asp
Pro Ile Ala GlyPro Thr Leu Phe Ala le Leu As口Arg Ser Leu Arg ry 1e Pr.

Phe Cys Glu Cys Ser　Lys Arg　Phe Tyr　Gly 11e l1e Gly Ala Lys Ala Lys Thr Val　Cys Gly Tyr Glu　Ile　Gly Asn Leu Pr.

Leu Thr Glu Thr Pro Glu Ser Gly Lys Lys Val l1e Leu Gly Pr.

Val Lys Gly Val Asp Asn Glu Ala Gly Val Thr Thr Gly Asp Vat Vat Asp Leu Asn Arg Pro Met Pro Glu Vat Ala Arg Gln 5er Ala A sp Lys Pro Leu Asp Thr ^rg Gly Leu Met Ala Thr Ala Lys Arg Leu Arg
Gly Gly Val Arg PheLe
u Lys Lys Pro Val Al
a Lys Met (3) The luciferase gene according to (1) or (2) above, which is represented by the base sequence shown below.

ATG GAA AACATG GAG AA
CGATGTG TAT GGT CCT G
AA CCA TTTGAA GAG GGA
TCT GCT GGA GCAGAA
^heT ATT TACCCT ATT CAA TTG CGC Thr Cys Ser Pr.

Gln Gly is Leu Pr.

1s Asp l1e Phe Phe 11e Lys Ala Glu Pro Asn Gly Tyr 11e Val Tyr Lys Leu Glu IIs Phe Tyr Asp Glu Asp Arg Leu Gly Tyr Gln 5e r Val Leu Asp Ala Gly GCG TTA TGCAGT G^^ ^AC
TGT GAA GAA TTCCCG AC
T TTG TTT GCA ATT CT
T AAT AGA AGTGGCATC TCT AAC ACA ACA TT GTA TT ACT TTA GTT GAA ATT GCA
TCT GGCGGA GCA CCT^TT TT GTA CTG GAG ACC AAA CTG CAT CAT 8TT ATT ATCACA CCG GA
A GGCGAT GAT AAACCA G
GT GCT TCT GGCAA GTT
GTG CCA TTATCG GGT
TCA ACCGGT TTG CCA AA
A GGT GTGCAA CTT ACT C
AT GAA AAT GCA GTCACT A
GAATG TTT 8CT 8CT TT8GGCTAT CT^ ACT TGT GAA ^^A CAT TTCTTT AT
CGTG GAT CGT TTGTACTCT
TTA ATCAAA To TAC A GG
A TAT CAAGACGAA GAG AC
T TTT TTA AAA ACA CTG C
AAGAT TACAAA TGT TCA A
GCGTT ATT CTT GTAGAG CTT
CCG GGA GCT GTT GTT
GTA CTT G8^CTG AAG
AAA CCA GTT GCT AAG
ATG The present invention will be described in detail below.

First, Luciola lateralis (Luciola 1at)
When searching for DNA containing the gene encoding luciferase of Luciola cruciaLa (Heike firefly), which is a type of firefly of the same genus, as a probe In addition, when searching for DNA containing the gene encoding the luciferase of Luciola cruciata, Photinus pyralis, a species of firefly, was used as a probe.
(American firefly) DNA containing a gene encoding luciferase is used.

Therefore, below we first describe the method for preparing the gene encoding luciferase from Photinus viralis, and then describe the method for preparing the gene encoding luciferase from Photinus viralis, and then describe
A method for preparing a gene encoding luciferase of Luciola lateralis will be described, and a method for preparing a gene encoding luciferase of Luciola lateralis will be described.

To prepare m-RNA from the feet of Photinus viralis, a type of firefly, for example, "Molecular
Cloning” (Molecular C1C1on
1n, p. 196, [Cold Spring Harbor Laboratory J
arbor Laboratory) (19B2) and “Molecular Genetics Experimental Methods” Haruo Koseki, Reibe Shimura, No. 66-6
It can be obtained by the method described on page 7 (1983).

m-RNA encoding luciferase was obtained from the obtained m-RNA.
- To reduce RNA, for example, Biomedical Research J (Bioa+edical Re5e
arch), Volume 3, Pages 534-540 (1982)
This can be done by the method described.

In this case, an anti-luciferase serum against luciferase is used, and the serum can be obtained, for example, by the method described in "Immuno Chemistry", Yu Yamamura, pp. 43-50 (1973).

C-DNA from m-RNA encoding luciferase
For example, to synthesize ``Mole Cell Paiol J
(Mo1. Ce1l Biol,), Volume 2, No. 161
(1982) and Gene, Vol. 25, p. 263 (1983).

Next, the c-DNA thus obtained is transformed into a vector DNA, for example, plasmid pMcE10DNA, plasmid pKN305 [r Agul Paiol Chem J
(Agr, Biol, Chem,), Volume 50, No. 2
71 (1986)] and plasmid pMC C1843 (rMethods in Enzymology)
”(Methods in f!nzymology)
, Vol. 100, pp. 293-308 (1983), which was prepared using a plasmid containing the E. coli β-galactosidase structural gene].
Various recombinant plasmid DNAs are obtained, and using the DNAs, for example, Escherichia coli (E, coli) D II l
(AT CC33849), E. coli
) HB 101 (AT CC33694) etc. were transformed by the Hanahan method [[DNA Cloning J (DNA (janiB), Vol. 1, pp. 109-135 (1985))], and various transformations were performed. Get convertible shares.

The recombinant plasmid DNA of the transformant thus obtained is a plasmid in which c-DNA is inserted into the middle of the E. coli β-galactosidase structural gene, and the peptide encoded by the c-DNA is is expressed as a protein fused with β-galactosidase.

In order to detect c-DNA encoding luciferase from the various transformed strains mentioned above, the transformed strains are cultured to express the bacterial protein, and the presence or absence of the protein that intersects with the anti-luciferase serum is determined. For example, [Ageric Paiol Chem J
(Agric, Biol.

Chem, ), Vol. 50, p. 271 (1986) and Anal, Biochem J.
), Vol. 112, p. 195 (1981).

Then, the incomplete luciferase c-DNA was 32p
The nick translation method [[Molecular
Cloning J (Molecular Clonin
g), 109th-112th JK, Cold Spring
Harbor Laboratory (Cold Spring l)
1abor Laboratory), (1982) and J. Mol.

Biol, ), Vol. 113, pp. 23-251 (197
After labeling with 7) ), the 8 pUc-DNA was transformed into plasmid pUc19DNA (manufactured by Takara Shuzo Co., Ltd.) using the probe knee hybridization method [Protein, Nucleic Acids, Enzymes, Vol. 26, pp. 575-579 (1981)]. A plasmid DNA containing c-DNA encoding 1.8 Kb luciferase can be obtained from a Scene Bank library of c-DNA created using the vector.

In order to obtain DNA containing the gene encoding Photinus viralis-derived luciferase from the recombinant plasmid DNA thus obtained, restriction enzymes such as EcoRI and Clal are added to the plasmid DNA at a temperature of 30 to 40°C. The reaction solution obtained by reacting at 1 to 24 hours, preferably 2 hours at 37°C, preferably 37°C, was subjected to agarose gel electrophoresis [Molecular Cloning].
g), page 150, Cold Spring Harbor
Laboratory (Cold Spring Laboratories)
Laboratory) (1982)).

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

The preparation of m-RNA from the leg of Luciola cruciata and the synthesis of c-DNA from m-RNA are, for example, exactly the same as the methods for preparing m-RNA and synthesizing c-DNA of Photinus viralis described above. It can be done by

Next, the c-DNA thus obtained is integrated into vector DNA, such as plasmid ρUC19 DNA, to obtain various recombinant plasmid DNAs.
For example, E. coli (E.

coli) DHI (ATCC33849), Escherichia coli ([i.coli) HB 101 (A T C
C 33694) etc. using the method of Ianahan [``DNA Cloning J (DNA
Cloning), Volume 1, pp. 109-135 (1
985)) to obtain various transformed strains.

Next, DNA containing the gene encoding luciferase derived from Photinus viralis was subjected to the nick translation method using izp [Molecular Cloning J, No. 109-1121, Cold Spring Harbor.
Laboratory (Cold Spring Harbor)
Laboratory) (19B2) and [J. Mol.

Biol. ), Volume 113, Volumes 237-251i
(1977)), and then the colony hybridization method using the c-DNA as a probe ["
"Proteins, Nucleic Acids, Enzymes" Volume 26, pp. 575-579 (
Luciola cruciata-derived c that was created using plasmid pUc19 DNA as a vector by (1981) ).
Escherichia coli containing plasmid DNA containing c-DNA (2.OKb) encoding lucifera can be obtained from the -DNA Scene Bank library.

In order to obtain purified recombinant DNA from the microorganism thus obtained, for example, "Proc. Natl. Acad. Sci.
), Vol. 62, pp. 1159-1166 (1969).

In order to obtain DNA containing the gene encoding the luciferase derived from Luciola cruciata from the recombinant plasmid DNA thus obtained, restriction enzymes such as E
After reacting at 40°C, preferably 37°C, for 1 to 24 hours, preferably 2 hours, the reaction solution was subjected to agarose gel electrophoresis ["Molecular Cloning J (M
olecular Cloning), page 150, Cold Spring Harbor Laboratory (Col.
d Spring flavor Laborator
y) described in (19B2)].

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

Since m-RNA encoding luciferase derived from Luciola lateralis is present in the leg of Luciola lateralis, this leg is preferable as a source of m-RNA collection.

The preparation of m-RNA from the leg of Luciola lateralis and the synthesis of c-DNA from m-RNA are, for example, completely similar to the methods for preparing m-RNA and synthesizing c-DNA of Photinus viralis described above. It can be done by

Next, the c-DNA thus obtained is integrated into vector DNA, such as plasmid pUc119 DNA, to obtain various recombinant plasmid DNAs, and the D
For example, Escherichia coli (E. coli) D H using NA
1 (AT CC33849), E. coli
) HB 101 (AT CC33694) etc. by the method of Hanahan (DNA Cloning, Vol. 1, pp. 109-135 (1985)), Obtain various transformed strains.

Next, D containing the gene encoding luciferase derived from Luciola cruciata obtained as described above was used.
Nick translation method using NA and 32P [“Molecular Turning” (Molecular Cl
oning), pp. 109-112, Cold Spring Harbor Laboratory (ColdSprin
g Labor Laboratory) (1982)
and “JoMol, Bi
ol, ), Vol. 113, pp. 237-251 (197
7)), and then the colony hybridization method using the CDNA as a probe [Protein, Nucleic Acids, Enzymes, Vol. 26, pp. 575-579 (1981)
), c-DNA derived from Luciola lateralis was created using plasmid pUC119DNA as a vector.
Escherichia coli containing plasmid DNA containing C-DNA (2.0 Kb) encoding luciferase can be obtained from the Scene Bank library.

In order to obtain recombinant DNA purified from the microorganism thus obtained, for example, "Proc, Natl, Acad, Sci J.
), Vol. 62, pp. 1159-1166 (1969)
It can be obtained by the method described above.

Then, to the above purified new recombinant DNA,
For example, after partially digesting the enzyme by applying 2 units of the restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.) at a temperature of 30"C or higher, preferably 37°C, for 1 to 4 hours, preferably 2 hours,
2, which contains all the luciferase genes derived from Luciola lateralis, was determined by agarose gel electrophoresis.
000 bp DNA fragments can be isolated.

On the other hand, the nucleotide sequence of this luciferase gene was determined by the method shown in Item 18 of the Examples to obtain the nucleotide sequence of the luciferase gene shown in FIG.
The amino acid sequence of the polypeptide translated from the above base sequence was determined, and the results shown in FIG. 7 were obtained.

A gene encoding the amino acid sequence determined in this way is one aspect of the present invention.

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

As the medium, any medium may be used as long as it is used for culturing microorganisms belonging to the genus Edzierisia, such as tryptone 1% cap/v), yeast extract 0
．． 5% (W/V), NaCl 10.5% (W/V), and 111M isopropyl-β-D-thiogalactoside.

Further, the culture temperature is, for example, 30 to 40''C1, preferably about 37°C, and the culture time is, for example, 4 to 8 hours.
Preferably it is about 4 hours.

Then, the bacterial cells are collected from the culture at, for example, 8.00 Or, po.
m.

Collect bacteria by centrifugation for about 10 minutes, and collect the bacteria using, for example, "Methods in Enzymology"
(Methods in [!nzywology) Vol. 133, pp. 3-14 (1986) to crush to obtain a crude enzyme solution.

The crude enzyme solution can be used as is, but
If necessary, purified luciferase is obtained by purification by ammonium sulfate fractionation, hydrophobic chromatography (for example, using Butyl-Toyopearl 6500, etc.), gel filtration (for example, using Ultrogel AcA34, etc.).

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

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

This enzyme: /+ A T P + Oz b Oxidyl luciferin + AMP + Pyrroline M + CO Substrate specificity: Does not act on ADP, CTP, UTP and GTP.

■Optimal pH and stable pH range: The optimal pH is determined by using luciferin as a substrate and changing the pH of 25mM glycylglycine from pH 6.5 to 11.5.
React at a temperature of 30℃ and emit light amount (number of photons) for 20 seconds.
When measuring <pH 7.5 to 9.5 as shown in Figure 1.
The stable pH range is luciferin-containing buffer (pH 4.6-8.0: 25mM phosphate buffer, pH 8.0-11.5: 25+wM glycine.
Sodium chloride-sodium hydroxide buffers were used, respectively. Note that ammonium sulfate was added to each buffer solution to achieve 10% saturation. ) is added to the enzyme and allowed to act at a temperature of 0° C. for 4 hours, the value is 6.0 to 10.5, as shown in FIG. In FIG. 2, -1 and H indicate the activity when using 25 mM phosphate buffer and 25 mM glycine/sodium chloride/sodium hydroxide buffer, respectively.

■Method for measuring titer: 25a+M glycylglycine (pH 7,8) 8mf, magnesium sulfate solution (25+nM glycylglycine (p
H7,8) with magnesium sulfate added to a concentration of 0.1 M] 0.5- and luciferin solution (25n
A luciferin mixture is prepared by mixing +M glycylglycine (pH 7, 8) with 0.8- solution of luciferin added to 1 mM.

400μ of the luciferin mixture obtained in this way! A mixture of 10 μl of luciferase and 10 μl of luciferase to be measured,
TP solution [25a+M glycylglycine (pH 7, 8)
to which ATP was added to give a concentration of 10mM.・〕80
At the same time as μl is injected, the number of photons generated by a luminometer (Luminescence Reader BLR-201, manufactured by Aloka) is integrated and determined for 20 seconds.

(2) Suitable temperature range for action: When the reaction is carried out at each temperature at pH 7 and 8 and the amount of light emitted (photon number) is measured for 20 seconds, it is within the range of 0 to 50°C.

(2) Conditions for inactivation by po: Completely inactivated after 4 hours at pH 5.0 or lower and pH 12.0 or higher.

〔Effect of the invention〕

As is clear from the above, according to the present invention, by culturing in a medium a microorganism belonging to the genus Ethzierisia containing the recombinant DNA into which the luciferase gene derived from Luciola lateralis of the present invention has been integrated, a very short period of time can be achieved. The present invention is industrially extremely useful because luciferase derived from Luciola lateralis can be efficiently produced.

〔Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Examples Items 1 to 10 described below include DNA containing a gene encoding luciferase from Photinus viralis, a type of firefly (the DNA containing a gene encoding luciferase from Luciola cruciata). The preparation of DNA containing the gene encoding luciferase derived from Luciola cruciata is described in items 11 to 13 below.
A is used as a probe when searching for DNA containing the gene encoding Luciola lateralis luciferase. ) preparation is described.

1. Preparation of m-RNA Photinus viralis, a species of firefly.
inus pyralis) dried reams (manufactured by Sigma)
Thoroughly crush lg with a mortar and pestle, add 5 ml of lysis buffer (20n+M Tris-HCl buffer (
pH7,4)/10mM NaCl/3o+M Magnesium Acetate 15% (W/V) Shea $Ft/1.2
%(V/V) )IJ door X-100/10a
hM Ha+Dil nucleoside complex (manufactured by New England Biolabs)] was added and further crushed in the same manner as above to obtain a solution containing crushed Photinus pyralis tail.

Put 5 ml of the solution obtained in this way into a cup-shaped blender (manufactured by Nippon Seiki Seisakusho Co., Ltd.), and put 5.00 Or, ρ, -
9 for 5 minutes, 12af guanidine isothiocyanate solution (6M guanidine isothiocyanate)/37.5s+M sodium citrate (pH 7,0)
10.75% (W/V) N-lauroylsarcosine sodium (10.15M β-mercaptoethanol) was added, and further, using the above blender, 3.00 Or, p
The solution obtained by treating with .
1.2 d of 5.7M cesium chloride solution was layered in advance on each of the four tubes (manufactured by Hitachi Ichiki Co., Ltd.), and the above filtrate was dispensed onto each tube so as to be layered. Temperature: 15°C, 30.00 Or
, p, m for 16 hours to obtain a precipitate.

The obtained precipitate was washed with cold 70% (V/V) ethanol, and the precipitate was washed with 10111M Tris buffer (10%
mM Tris-HCl buffer (pH 7,4) 15mMEDT
A/1% sodium dodecyl sulfate) 4- was suspended in the same amount of n-butanol and chloroform in a ratio of 4:1.
(volume ratio) is added and extracted.
3.00 Or by conventional method, 9. Centrifuge for 10 minutes at m, to separate into an aqueous layer and an organic solvent layer, add the above 10a+M Toljs buffer 4- to this organic solvent layer, and repeat the above extraction and separation operation twice. To the resulting aqueous layer, 1,710 amounts of 3M sodium acetate (pH 5,2) was added.
and added with twice the amount of cold ethanol at a temperature of -20
After leaving it at °C for 2 hours, 8.00 Or
Centrifuge at p, ah for 20 minutes to precipitate RNA;
The obtained RNA was dissolved in 4 liters of water and subjected to the above-mentioned ethanol precipitation operation, and then the obtained RNA was dissolved in 1 vi of water to obtain 3.75 μ of RNA.

Then, by repeating the above operation again, a total of 7■
To select m-RNA from this RNA, 7 mg of RNA was subjected to oligo(dT)-cellulose column chromatography (manufactured by New England Biolabs).

A 2.5-thermo syringe (manufactured by Chiru Shichi Co., Ltd.) was used as a column, and 0.5 g of resin was mixed with an elution buffer [10 mM Tris-HCl buffer (pH 7,6)/1s MDETA 10.1
% (W/V) sodium dodecyl sulfate], packed into a column, and added with binding buffer (10n+M).
Lith-hydrochloric acid (pH 7,6) / 1 n+M E D T
A/0.4M NaCl/o, t% sodium dodecyl sulfate].

7 RNA and the same amount of buffer (10 sM) Lis-HCl (pH 7,6)/1+ME DTAlo, 8M
NaC110, 1% sodium dodecyl sulfate] was added, heat treated at a temperature of 65°C for 10 minutes, and rapidly cooled in water.
After applying to an oligo(dT'')-cellulose column, the resin was washed with a binding buffer to completely remove unbound r-RNA and t-RNA, and then m-R was washed with an elution buffer.
NA was eluted to obtain 40 μg of luciferase m-RNA.

2. In the concentrated coating of luciferase m-RNA, see! Luciferase m-RNA was concentrated by sugar density gradient centrifugation.

A 10-25% (W/V) sucrose density gradient was applied to a Beckman rotor SW41 polyaromatic tube.
0% (W/V) Sea J$J! Solution [50IIIM Tris-HCl buffer (pH 7,5)/20mM NaCl/1
mME D TA/40% (W/V) sucrose) 0.5
ml and add 2.4-25% (W/V) on top of it.
20% (revised ν), 15% (W/V) and 10% (W/V
) (7) It was produced by layering the sucrose solution and leaving it at a temperature of 4°C for 24 hours. This shi! ! tJ! 30 μg of m-RNA obtained in item 1 was layered on a density gradient, and centrifuged at 30.00 Or, p, m, and 18°C for 18 hours using a Beckman SW410-tar in a conventional manner. Ta.

After centrifugation, m-RNA was fractionated into 0.5-unit fractions, and m-RNA was collected by ethanol precipitation and dissolved in 10 μl of water.

Next, by examining the proteins encoded by the m-RNA, we identified both regions in which luciferase m-RNA was enriched. Fractionated RNA II! , rabbit reticulocyte lysate (manufactured by Amajam) 9μ! and [3SS] methionine 1μ! (manufactured by Amajam) and reacted at a temperature of 30'C for 30 minutes.
μ! NET buffer (150mM NaCl/5m
ME D T A10.02% (W/V) NaNi/
2On+M) Lis-HCl buffer (pH 7,4) 10.0
5% (W/V) Nonidet P-40 (manufactured by Bethesda Research Laboratory, surfactant)], and further 1
μ! of anti-luciferase serum (prepared as described below) was added and left at a temperature of 4°C for 18 hours, 1O of protein A Sepharose (manufactured by Pharmacia) was added and the mixture was incubated at a temperature of 20°C. The resin was left to stand for 30 minutes and then centrifuged for 1 minute at 12.0 OOr, p, m in a conventional manner to recover the resin.

The recovered resin was washed three times with 200μ2 NET buffer, and 40p! 5DS-PAGE sample buffer (62,5mM Tris-HCl buffer (pH
6,8) / 10% (V/V) glycerol / 2% (
W/V) sodium dodecyl sulfate 15% (V/V) mercaptoethanol 10.02% (-ha) bromophenol blue] and boiled at a temperature of 100°C for 3 minutes.
Centrifuge for 1 minute at 12.0 OOr, p, m using a conventional method, collect the supernatant, and adjust the total volume to 7.5% (W/V).
It was loaded onto a sodium dodecyl sulfate-polyacrylamide gel.

Gel electrophoresis was performed using the method of Laemlli (Nature J (Nature), p. 227, p. 680 (1970)), and the gel after electrophoresis was 10% (V/ν) Soak in acetic acid for 30 minutes,
After fixing the protein, it was immersed in water for 30 minutes, and
The gel was immersed in M sodium salicylate solution for 30 minutes and dried to obtain a dry gel, which was then subjected to fluorography using an X-ray film (RX, manufactured by Fuji Photo Film Co., Ltd.).

As a result of the above procedure, the luciferase protein band was observed on the X-ray film only when both RNAs containing luciferase m-RNA were used, and the two regions in which luciferase m-RNA was enriched could be identified. Ta.

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

A 0.5 M glycylglycine solution (pH
0.7@l dissolved in 7, 8) was suspended in an equal volume of Freund's complete adjuvant.
．． 24■ was administered as an antigen to the digits of a Japanese white rabbit weighing 2 kg, and after 2 weeks of rearing, the same amount of antigen as the first time was administered to the dorsal skin. After 1 week of rearing, the same amount was administered. After one week of rearing, whole blood was collected.

Then, the obtained blood was left at a temperature of 4°C for 18 hours, and then centrifuged for 15 minutes at 3+00 Or, p, m by a conventional method to obtain anti-luciferase serum as a supernatant.

4. Synthesis of c-DNA c-DNA was synthesized using a kit manufactured by Amajam.

Using 2 μg of m-RNA obtained as described above, “Mol Cell Paiol J” (Mo
1. Ce1l Biol, ), Volume 2, Page 161 (1
9B2) and the method described in Gene, Vol. 25, p. 263 (1983).
Double-stranded c-DNA of 300 nH was obtained.

150 ng of this c-DNA was added to 1 ul of TE buffer [1 ml of Tris-HCl buffer (pH 7,5)/1
11μ2 of the mixture (280mM sodium cacodylate (pH 6,8)/
60+nM Tris-HCl buffer (pH 6,8)/2m
M cobalt chloride) and 3.8μ2 tilling mixture (1
011M dithiothreitol 7,5 u 1/10
ng/d poly (poly) A 1 tt l
/ 5mMdcTP2μm/water 110μ! ] 10μ hydrogen! ], 29 units of terminal transferase (manufactured by Boehringer Mannheim) were added and reacted for 10 minutes at a temperature of 30°C.
10% (W/V) of 25 MEDTA and 2.4 μN
The reaction was stopped by adding sodium dotecyl sulfate respectively.

After deproteinizing the reaction stop solution using 25 μl of water-saturated phenol, 25 μl of 4
M ammonium acetate and 1001! of cold ethanol was added to each, and left at a temperature of -70°C for 15 minutes.
c-DNA was collected by centrifugation at 00r,p,m for 10 minutes and dissolved in lOμi of TE buffer to obtain a C-DNA solution.

As described above, c with a deoxycytidine tail is
-DN Aloong was obtained.

5. Recombinant plasmid pMcE used in vector
Preparation of IODA Using Escherichia coli strain W3110 (AT CC27325), plasmid pBR325DNA (manufactured by BRL) and plasmid pBR322DNA (manufactured by Zenshuzo Co., Ltd.), Masuda et al.
r Agricultural Biological Chemistry J
Chea+1stry), Volume 50, Nos. 271-2
Plasmid pK8305 DNA and plasmid pMC140 prepared by the method described on page 79 (1986)
3-3D NA (described in Japanese Patent Application Laid-Open No. 61-274683) lag was added to a mixture of lOμi (50mM l-Lis-HCl buffer (pH 7.5)/10mM MgCh/
10M NaC1/1mM dithiothreitol] and further added Hindll [and Sal
(2) (both manufactured by Zenshuzo Co., Ltd.) were added, reacted for 1 hour at a temperature of 37°C, cut, and subjected to phenol extraction and ethanol precipitation in a conventional manner to obtain a precipitate. The precipitate was dissolved in 10μ2 of Ligae buffer (20mM MgC1g/66mM Tris-
Hydrochloric acid buffer (pH 7,6) / 1 a+MAT
P/15mM dithiothreitol) to obtain a solution, and 1 unit of T4 DNA ligase (manufactured by Zenshuzo Co., Ltd.) was added to carry out a ligation reaction at a temperature of 20°C for 4 hours.

Next, using this reaction solution, [J, Bacteriology, Vol. 119,
1072-1074 (1974)], E. coli J MIOI (AT CC3
3B76) strain was transformed and examined for drug resistance (ampicillin resistance and tetracycline sensitivity) and β-galactosidase activity, a transformed strain was obtained, and the recombinant plasmid DNA contained in the strain was named pMcEIO.

E. coli JMIOI strain containing this recombinant plasmid pMcE10 DNA was treated with tryptone 1% (-/ν),
Yeast extract 0.5% (W/V) and NaC10.5
% (W/V) using the medium 11 at a temperature of 3.
E. coli JM101 obtained by pre-culturing at 7°C for 16-24 hours
After inoculating 20ra1 of culture solution of (pMCElo) and culturing with shaking at a temperature of 37°C for 3 hours, 0.2 g of chloramphenicol was added, and the culture solution was further incubated at the same temperature for 20 hours to obtain a culture solution. Ta.

Next, this culture solution was heated to 6.00 Or, p, by a conventional method.
a+.

Centrifuge for 10 minutes to obtain 2 g of wet bacterial cells, which are then
25% (W/V) of m1 350s containing ci=I sugar
+M) After suspending in lys-hydrochloric acid buffer (pH 8,0), lysozyme 10μ, 0.25M
8 ml of EDTA solution (pH 8,0) and 20% (W
/V) Sodium dodecyl sulfate solution 8- was added to each and kept at a temperature of 60°C for 30 minutes to lyse the bacteria to obtain a lysate.

To this lysate, 13ml of 5M NaCl solution was added and treated at a temperature of 4°C for 16 hours.
The extract was centrifuged at R, P, M for 30 minutes to obtain an extract, which was then subjected to phenol extraction and ethanol precipitation in a conventional manner to obtain a precipitate.

Next, this precipitate was dried under normal vacuum and dissolved in 6 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 1 dEDTA, and further, 6 g of cesium chloride and ethidium bromide solution were added thereto. (1
0 mg/ad) 0.2- was added using an ultracentrifuge for 42 hours at 39.00 Or, p, m in a conventional manner to perform equilibrium density gradient centrifugation to isolate the recombinant plasmid pMcE10 DNA. After removing the ethidium bromide using n-butanol, the recombinant plasmid pMcEIO DNA50 was purified by dialysis against 10mM) Lis-HCl buffer (pH 7.5) containing 1a + MEDTA.
0 μg was obtained.

6. Preparation of vector DNA Recombinant plasmid pMcE obtained as above
TE1 described in item 4 of 90 μi of 15 μg of 10 DNA
1 out of Med buffer (1100
III Tris-HCl buffer (pH 7,5)/100++
+M MgC1g/10d dithiothreitol 150
After adding 0@M NaCl), 30 units of restriction enzyme Acc I (manufactured by Zenshuzo Co., Ltd.) was added, and the temperature was raised to 37.
Cutting was performed at °C for 1 hour to obtain a cut product. After adding 100 μl of water-saturated phenol to this cut product and performing a protein removal operation, the aqueous layer was collected, and
Add 1O volume of 3M sodium acetate (pH 7,5) and 2 volumes of cold ethanol, leave at -70°C for 15 minutes, and centrifuge at 12.00 Or, p, m for 10 minutes to remove DNA. Recovered.

Dissolve this DNA in 10 μl of TE buffer, add 15 μl
l mixture (280mM sodium cacodylate (pH 6,
8) / 60mM) Lis-HCl buffer (pH 6,
8) / 2mM cobalt chloride), and then add 5μl of Tilling mixture (described in item 4) (5mM dG
TP) was added thereto, and furthermore, 5 units of terminal transferase (manufactured by Zenshuzo Co., Ltd.) were added, and the mixture was allowed to react at a temperature of 37°C for 15 minutes. c-DN described in item 4
A post-treatment similar to the A-tiling reaction was performed to prepare recombinant plasmid pMcE10 DNA with a deoxyguanosine tail attached to the Acc■ site.

On the other hand, DNA with a deoxyguanosine tail attached to the Pstl site of plasmid pUc19 DNA was also prepared at the same time as described below.

Plasmid pUc19 DNA (manufactured by Zenshuzo Co., Ltd.) 30μg
Dissolved in 350 μl of TE buffer, add 40 μl of
1 of Med buffer and 5 tl of restriction enzyme (manufactured by Zenshuzo Co., Ltd.)
After adding 120 units each and cutting at a temperature of 37°C for 1 hour, the DNA was recovered by deproteinization with phenol and ethanol precipitation in a conventional manner.

The obtained DNA was dissolved in 35μ2 of TE buffer, and 50μl of a mixture (280mM sodium cacodylate (pH 6,8)/60mM) in Lis-HCl buffer (
pH 6,8)/11 IM cobalt chloride), 19 μl of the tilling mixture described in item 4 (containing dGTP) and 60
Unit's terminal transferase (manufactured by Zenshuzo Co., Ltd.) was added to each, and the mixture was allowed to react at a temperature of 37°C for 10 minutes, followed by phenol treatment and ethanol precipitation in a conventional manner to recover the desired DNA.

7.7 Two-Ring and Transformation 15 ng of C-D l'J A synthesized above and 200 ng of vector DNA were added to 35 ul of annealing buffer (10d) Lis-HCl buffer (pH 7,5)/100a.
+M NaC1/1mME DTA) at a temperature of 65°C for 2 minutes, at a temperature of 46°C for 2 hours, at a temperature of 37°C.
The c-DNA and vector DNA were annealed by leaving the mixture at 20° C. for 1 hour and 18 hours at a temperature of 20° C.

Using the annealed DNA, Hana-h
an) method [“DeNA Cloning” (D
NA C1ons), Volume 1, Nos. 109-13
5 (1985)), Escherichia coli strain DHI (A T
CC33849) was transformed into plasmid pUc19.
A C-DNA bank was created using the DNA and the recombinant plasmid pMcEIODNA as vectors.

8. Search for luciferase c-DNA The Acc■ site of the recombinant plasmid pMcEIO DNA is located at the site encoding the E. coli β-galactosidase gene, so the C-DNA integrated into this site creates a fusion protein with β-galactosidase. . Furthermore, the promoter of the β-galactosidase gene in the recombinant plasmid pMCEIO was converted to the E. coli tryptophan gene promoter as described above.

96 colonies of c-DNA bank using recombinant plasmid pMcE10 DNA as a vector were transferred to 10th M9 casamino acid medium ["Molecular Cloning"]
(Molecular Cloning), No. 440
~441 pages, Cold Spring Harbor Laboratory
(1982)) and thiamin (1
0 ug/rd) at a temperature of 37°C for 10
After culturing with shaking for an hour and collecting bacteria using a standard method, 200 μl
It was suspended in the sample buffer for 5DS-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 using a 7.5% (-8) polyacrylamide gel in a conventional manner.

After the electrophoresis, the proteins developed on the gel were analyzed by Western blotting ["Anal, Biochem"].
chm, ), Volume 112, Page 195 (1981)]
was transferred to a nitrocellulose filter, and the nitrocellulose filter was stained with anti-luciferase serum using an Immune Blot Assay Kit (manufactured by Bioland). The method followed Bioland's operating instructions.

That is, a nitrocellulose filter, 100 ml of blocking solution [TBS buffer [20 μlM Tris-HCl buffer 150 (1 wM NaCl (pH 7,5)])
A solution of 3% (-8) gelatin dissolved in] medium humidity 25
The nitrocellulose filter was shaken for 30 minutes at ℃.Then, the nitrocellulose filter was diluted with 25 ml of primary antibody solution [luciferase antiserum 25 times (V/V) in a solution of 1% (W/V) gelatin dissolved in TBSII buffer. The solution was transferred to a diluted solution) and shaken for 90 minutes at a temperature of 25°C.
The nitrocellulose filter obtained in this way was then transferred to a secondary antibody solution of 60 [labeled with horseradish peroxidase] and shaken twice for 10 minutes at a temperature of 25°C. The anti-rabbit antibody (manufactured by Bio-Rand) was diluted 3000 times (V/V) with a solution of 1% (W/V) gelatin dissolved in TBS buffer] and incubated at a temperature of 25°C. After shaking for 60 minutes at
Wash the nitrocellulose filter with the washing liquid. Repeat the above steps 2.
Repeatedly, the nitrocellulose filter thus obtained was mixed with 120 mg of coloring solution [60 μ of 4-chloro-
A solution of 1-naphthol dissolved in 20 m of cold methanol and 60 μ2 of 30% (V) hydrogen peroxide solution were added to 100-
The solution added to TBS buffer was transferred to a mixed solution] and color was developed at a temperature of 25°C for 10 minutes.

When the same method was carried out on 4 groups each consisting of 96 colonies, a protein band stained with the luciferase antiserum was observed in 2 groups. Next, 96 colonies belonging to these two groups were divided into 8 groups of 12 colonies each and the same procedure was performed, and a protein that reacted with the anti-luciferase serum was observed in each group. Finally, 12 colonies included in this group were subjected to the same procedure one by one to identify colonies that produce a protein that reacts with the luciferase antiserum. colonies were obtained. Plasmid DNA was prepared from these two colonies by the method described in Item 5. The obtained recombinant plasmid DNAs were named pAL f 2 B B DNA and pAL f 3A6 DNA, respectively.

9. Search for large luciferase c-DNA - Preparation of DNA probe Recombinant plasmid pALf3A6 DNA 100 μg
, 330μ! Dissolve in TEJl street solution and add 40μ
1 of Lo-buffer [1100III Tris-HCl buffer (pH 7,5)/100mM MgC1g/10mM
dithiothreitol], 130 units of Pstl
(manufactured by Takara Shuzo Co., Ltd.) and 120 units of 5acl (manufactured by Boehringer Mannheim) were added, and
．． Cut for 5 hours.

The total amount of this DNA was separated by electrophoresis using a 0.7% (W/V) agarose gel. Agarose gel electrophoresis was performed using the method of T. Maniatis et al.
arCloning), pp. 156-161, Cold Spring Harbor Laboratory (Cold S
pring Habor Laboratory) (1
984)). luciferase c-D
Cut out the DNA band containing NA, put it into a dialysis tube, add 2-TE buffer, seal the dialysis tube, and electrophoresis remove the DNA from the gel into the buffer.
was eluted.

After adding an equal volume of water-saturated phenol to this solution and stirring, the aqueous layer was collected, and DNA was collected by ethanol precipitation from above.

10 μg of the obtained DNA fragment was dissolved in 126 μl of TE buffer, 16 μl of Med buffer and 64 units of 5au3AI (manufactured by Takara Shuzo Co., Ltd.) were added, and the mixture was heated at a temperature of 37 μl.
After reacting at °C for 2 hours, the DNA fragments were separated by electrophoresis using a 5% (W/V) polyacrylamide gel. Polyacrylamide gel electrophoresis was performed using the method of A. Maxa [MeLhods in Enzymology].
Enzyaology), Volume 65, Page 506 (
1980)]. 190bp DNA
The fragment was isolated in a manner similar to that described above, and 1 gg of S
An au3AI luciferase c-DNA fragment was obtained.

1 μg of this luciferase c-DNA was added to [α-”P
) Labeling was performed by the nick translation method using dCTP (manufactured by AmaJam).

Nick Translator Seigon uses a kit made by Takara Shuzo Co., Ltd., and uses Takara Shuzo Co.'s instructions for "J Mol Paiol J (
j, Mo1. Biol,), Volume 113, Volume 237~
251 (1977) and “Molecular Cloning”, Vol.
Pages 09-112, Cold Spring Harbor Laboratory
(1982).

10. Search for large luciferase c-DNA - Colony hybridization was prepared using the method described above! Using a luciferase c-DNA fragment labeled with 2P as a probe, a Photinus pyralis foot c-DNA bank using the recombinant plasmid ρUC19 DNA as a vector was subjected to colony hybridization method ([Proteins, Nucleic Acids, Enzymes] Vol. 26, 575-579 (1981)) and a colony containing luciferase c-DNA was obtained. Recombinant plasmid DN of one of the colonies
A was named pALf3, and plasmid DNA was prepared by the method described in Item 5. E. coli containing the recombinant plasmid DNA was named E. coli DHI (pALf3). The transformed strain has been deposited as ATCC67462.

Then, the above recombinant plasmid pALf3 DNA was infected with Xbal, , ind m, BamHI, and EcoRI.
and Pstl (both manufactured by Takara Shuzo Co., Ltd.), the mobility patterns of the DNA fragments obtained by single and double digestion were analyzed by agarose gel electrophoresis. company) and 1n
The molecular weight obtained by comparing with the standard mobility pattern of the DNA fragment obtained by digestion with dlI [
, 700 bp, and the restriction enzyme map of the above plasmid was as shown in FIG.

11. Luciola Cru
ciata) Preparation of m-RNA Place 10 g of live Luciola crucciata (purchased from Genji Firefly Co., Ltd. and Seibu Department Store) into an ultra-low temperature freezer.
After freezing, the tail was cut off using scissors, and 18-guanidine isothiocyanate solution was added to 2 g of the obtained tail to prepare 1.1 μ RNA according to the method described in item 1. This RNA1.1 was subjected to oligo(dT)-cellulose column chromatography according to the method described in Item 1, and 30 ug of Luciola cruciata tail m
- RNA was prepared.

12. Preparation of c-DNA bank of Luciola cruciata series c-DNA was synthesized using a kit purchased from Amajam Co., Ltd., using "Mo1. Ce1l Biol" (Mo1. Ce1l Biol), No. 2 Volume, No. 16
1 (1982) and Gene, Vol. 25,
It was synthesized according to the method described on page 263 (1983).

0.9μ from 2μg of Luciola cruciata tail RNA
Two 144 c-DNAs were synthesized. A polydeoxycytidine tail was added to 0.3 μg of this c-DNA using the method described in Item 4.

20 ng of this c-DNA and p which added the polyguanosine tail prepared in item 6 to its Pst1 site.
500 ng of Llc19 plasmid DNA was added to item 7.
Annealed as described, Hanahan
Method of DNA cloning
Cloning), Vol. 1, pp. 109-135 (19
85) E. coli D
HI strain (ATCC33849) was transformed to produce Luciola.
A C. cruciata continuous c-DNA bank was created.

13. Luciferase c- from Luciola cruciata
Recombinant plasmid pALf 3 obtained in DNA search item 10
Dissolve 10ug of DNA in 90ul of TE buffer,
0 μl Med buffer, 25 units Eco restriction enzyme
RI and 25 units of the restriction enzyme C1al (both manufactured by Takara Shuzo Co., Ltd.) were added, and the reaction was carried out at a temperature of 37°C for 2 hours to cleave the DNA. Photenus viralis (
An 800 bp EcoRr/C1aI DNA fragment containing the luciferase c-DNA portion derived from the American firefly was isolated according to the method using agarose gel electrophoresis described in item 9, and 1 gg of EcoRI/C was isolated.
A 1a I DNA fragment was obtained. 1 gg of this DNA was labeled with ff12p by the nick translation method described in item 9 using [α-”P)dCTP triphosphate (manufactured by Amajam).EcoR labeled with tP
I/C1al DNA fragment as a probe,
Item 10: Luciola cruciata c-DNA bank
Luciferase c derived from Luciola cruciata was obtained by searching with the colony hybridization method described.
-Escherichia coli containing DNA was selected. Several E. coli colonies that hybridized with the probe were obtained.

The plasmid DNA of one of these colonies was converted into pGL.
The recombinant plasmid DNA was named fl and was isolated according to the method described in Item 5.

This recombinant plasmid pGLflDNA and 1 animal,
Hindll [, Ashinami RV, engineering is Dan 1. Afyu ■,
In addition, ■, Uedoko 1 (all manufactured by Zenshuzo Co., Ltd.) and 5''l
The mobility patterns of the DNA fragments obtained by single and double digestion were analyzed by agarose gel electrophoresis using New England Biolabs (manufactured by New England Biolabs), and the mobility patterns and λ phage DNA (manufactured by Zenshuzo Co., Ltd.)
The molecular weight obtained by comparing the standard mobility pattern of the DNA fragment obtained by digestion with indlll was 2.000 bρ, and the restriction enzyme map of the above plasmid is as shown in FIG.

14. Preparation of m-RNA of Luciola lateralis 5 g of live Luciola lateralis (purchased from Heikebotaru Kawahara Choju Trading) was placed in an ultra-low temperature freezer, frozen, and the tail was cut off using scissors. 18-guanidine isothiocyanate solution was added, and 340 gg of RNA was prepared according to the method described in item 1. 340 gg of this RNA was added to the oligo(
dT)-cellulose column chromatography was performed to prepare 6 gg of Luciola lateralis foot m-RNA.

15. Preparation of Luciola lateralis foot c-DNA bank c-DNA was synthesized using a kit manufactured by Amarjam.

Using 2.0 gg of m-RNA obtained as described above, "Mol Cell Paiol J"
(Mo1.Ce11.Biol,), Volume 2, No. 161
(1982) and Gene, Vol. 25, p. 263 (1983).
Two-terminal c-DNA of 250 nH was obtained.

Restriction enzyme Ec was added to 250 ng of the c-DNA obtained in this manner using a c-DNA cloning kit manufactured by Amajam Co., Ltd. according to a method instructed by Amajam Co., Ltd.

The R1 cleavage site was methylated, and EcoRI linkers were attached to both ends of the c-DNA.

Plasmid pUc119 DNA (manufactured by Zenshuzo Co., Ltd.) 11
00n dissolved in 8 μl of water, add 1 μl of Me
d buffer [1001M Tris-HCl buffer (pH 7,5
) 100mM MgC1z /10a + M dithiothreitol 1500 + wM NaC1), and then 10 units (1 μl) of restriction enzyme map R
I (manufactured by Zenshuzo Co., Ltd.) was added, and cutting treatment was performed at a temperature of 37°C for 1 hour.

Next, 1μ2 of IM) Lis-HCl buffer (pH 8,0) and 0.3 units (1μm) of the cut product were added.
alkaline phosphatase (manufactured by Zenshuzo Co., Ltd.) was added,
Enzyme reaction treatment was performed at a temperature of 65°C for 1 hour to dephosphorylate both ends of the cleaved product, and 12μ! After deproteinization was performed by adding 1 ml of water-saturated phenol, 1 pl of 3M sodium acetate (pH 5, 8) and 2
Add 6 μl of cold ethanol to each and incubate at -70°C.
Leave it for 5 minutes, then use a microcentrifuge [Tomy Seiko, MRX-1]
503 at 12.00 Or, p, m for 5 minutes to recover the DNA.

Digested with the restriction enzyme EcoRI obtained in this way,
and plasmid vector pUc1 with both ends dephosphorylated
A mixture of 1100n of 19 DNA and 250ng of the sea DNA prepared in item 15 was suspended in 8μ2 of water, and then 1μl of ×10 ligation buffer (200mM MgCh/660mM Tris-HCl buffer (pH 7, 6)/IOIIIM ATP/150
111M dithiothiretol] and 1 unit (1 μm of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.)
was added and left at a temperature of 16°C for 16 hours to perform ligation of plasmid vector DNA and c-DNA to obtain a reaction product.

Using this reaction product, the method of Hanahan ["DNAC Cloning J (DNAC
loning), Vol. 1, pp. 109-135 (19
85) ) to E. coli DHI strain (A T CC338
49) and transform it into plasmid pUc119 DNA.
c- derived from the tail of Luciola lateralis using the vector
A DNA bank was created.

16. Luciferase c- from Luciola lateralis
Recombinant plasmid pGLf I obtained in DNA search item 13
DNA bank g was diluted with 90 u 12 (7) TE buffer i 8 M and 10 μ! of Med buffer and 25 units of restriction enzyme PsLI (manufactured by Takara Shuzo Co., Ltd.) were added, and the reaction was carried out at a temperature of 37°C for 2 hours to cleave the DNA. Luciola cruciata-derived luciferase c-DNA from the cut recombinant plasmid pat, r I DNA
Contains parts2. The Pstl DNA fragment of ooobp was isolated according to the method using agarose gel electrophoresis described in Item 9, and 1 μg of Pstl DNA fragment was obtained. This 1 μg of DNA was transferred to [α-”P)d
It was labeled with 32p by the nick translation method described in item 9 using CTP (manufactured by Amajam). 3t
E. coli having luciferase c-DNA derived from Luciola lateralis was selected by searching the Luciola lateralis foot c-DNA bank using the colony hybridization method described in item 10 using the P-labeled Pstl DNA fragment as a probe. Several E. coli colonies that hybridized with the probe were obtained. The plasmid DNA of one of these colonies was named pHLf7, and the recombinant plasmid DNA was isolated according to the method described in Item 5.

In addition, Escherichia coli (E. coli) obtained in this way
DHI (pHLf?) was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 1917 (FERM HP-19).
17).

The above recombinant plasmid pHLf? DNA and animal I,
Ishi Liao RV, l mutual 1. The DNA fragments obtained by single and double digestion were subjected to agarose gel electrophoresis using ■1ndlII and Ashinari R1 (both manufactured by Takara Shuzo Co., Ltd.) and button ■ (manufactured by New England Hyolabo Co., Ltd.). Luciola, which was obtained by analyzing the mobility pattern and comparing the obtained mobility pattern with the standard mobility pattern of a DNA fragment obtained by digesting λ phage DNA (manufactured by Takara Shuzo Co., Ltd.) with Hind m. The molecular weight of the gene encoding luciferase in L. lateralis is 2.000b.
p, and the restriction enzyme map of the above plasmid is as shown in FIG.

And the above recombinant plasmid pHLf7DNAI
50 μg dissolved in 45 μl of TE buffer.
μ2 of Med buffer and 2 units of restriction enzyme EcoR
I (manufactured by Takara Shuzo Co., Ltd.) was added, and the reaction was carried out at a temperature of 37°C for 2 hours to obtain a partially digested DNA.

Next, this partial digest was subjected to agarose gel electrophoresis as described in item 9, and a 2,000bp EcoRI fragment 1. ug was isolated.

17. Escherichia coli (E, coli) D H1 (pHL f
7) Culture of (FERM BP-1917) and preparation of crude enzyme solution Escherichia coli (f!, coli) D H1 (pHL f 7)
) (FERM BP-1917), L B-amp
Medium [Batatotributon 1% (W/V), yeast extract 0
．． 5% (W/V), NaClO, 5% (W/V).

and ampicillin (50 pg//) 3-, shaking culture was performed at a temperature of 37°C for 18 hours. This culture solution 0
．． 5d to 10m! inoculated into the above LB-amp medium of
Furthermore, 1mM isopropyl-β-D-thiogalactoside was added to this, and after culturing with shaking at a temperature of 37°C for 4 hours, the wet bacterial cells were centrifuged at 8,000 Or, p, m for 10 minutes. 20 mg was obtained.

The collected bacterial cells were reduced to 0. I M KIIzPO4 (pH
7,8), a buffer consisting of 21mM EDTA, 1mM dithiothreitol and 0.2mm/-protamine sulfate.
Suspend in 9 ml, and add 100 μ2 of 10 ml to this.
g/m/norizozyme solution was added and left in water for 15 minutes. This suspension was then frozen in a methanol/dry ice bath and then left at a temperature of 25°C to completely thaw.

Furthermore, by centrifuging for 5 minutes at 12.00 Or, p, n+, crude enzyme solution 1- was obtained as a supernatant.

The luciferase activity in the crude enzyme solution thus obtained 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 the method of Ricka et al.
(Archives of Biochemist
ry and Biophysics), Volume 217,
This was done by measuring the number of photons generated according to No. 674 (1982)).

In other words, 260μ! 25mM glycylglycine buffer? fi, (pH 7,8), 16 μl of 0.1 M magnesium sulfate, 24 μl! After mixing 1mM luciferin (manufactured by Sigma) and 10μ2 of crude enzyme solution, 100μ
1 of 2On+MATP was added, and the number of photons generated was integrated over 20 seconds and the values are shown in Table 1.

For comparison, an E. coli DHI strain [E. coli DHI (pUC119)] having plasmid pUc119 DNA was also used.
) was similarly measured for luciferase activity, and the results are shown in Table 1.

As is clear from the table above in Table 1, E. coli DH containing the recombinant plasmid pHL57 containing the luciferase gene of the present invention
I (pHLf7) had an increased number of photons compared to the control, which revealed that luciferase was produced in the E. coli cells.

18. Luciferase c- from Luciola lateralis
Analysis of DNA base sequence Recombinant plasmid pHL f? DNA10μg
was digested with the restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.) to obtain 2.0 μg and 0.5 μg of 1.7 Kb and 0.3 Kb DNA fragments containing luciferase c-DNA, respectively, and these DNA fragments were transformed into plasmid pUc118 DNA (
(manufactured by Takara Shuzo Co., Ltd.) into the EcoR1 site, and four types of plasmids, pHL f 11, were generated depending on the type of inserted fragment (1.7 Kb and 0.3 Kb) and the insertion direction.
pHL f 12, pHL f13 and pHL f14
were obtained respectively (pHLfll and pHLf12 had 1.
A 7 Kb fragment has been subcloned and pHLf
A 0.3 Kb fragment has been subcloned into pHLf13 and pHLf14. ).

Cleavage of recombinant plasmid pHL f 7 DNA and plasmid pUc118 DNA with EcoRI (method described in item 6), isolation of luciferase c-DNA fragment using agarose gel electrophoresis (method described in item 9), plasmid ρUC119 DNA and Ligation reaction of luciferase c-DNA fragment (method described in item 5), E. coli JMIOI strain (A T
CC33876) (method described in item 5), and recombinant plasmid pHL FIL pHL
Preparation of f12, pHLf13 and pHLf14 DNA (
The method described in item 5) was carried out according to the method described in parentheses.

Next, recombinant DNA, pHLflll, pHL[1
2. °ζζ using pHLf13 and pHLf14 DNA
Using Kiroshifence Deletion Kit (manufactured by Takara Shuzo Co., Ltd.),
Henikoff's method [Gen
e), Vol. 28, pp. 351-359 (1984))
Plasmid DNAs in which various deletions were introduced into the luciferase c-DNA were prepared according to the procedure, and introduced into Escherichia coli JMIOI strain (AT CC33B76) by the method described in Item 5. The meshing method [Men's in.
Enzymology (Methods in Enzym)
101, pp. 20-78 (1983
) Single-stranded DNA was prepared according to ). Sea fencing using the obtained single-terminal strand DNA was performed using the M13 sequencing kit (manufactured by Takara Shuzo Co., Ltd.) according to the method of Messing described above. Gel electrophoresis for base sequence analysis was performed using 8% (W/V) polyacrylamide gel (manufactured by Fuji Photo Film Co., Ltd.). The base sequence of the obtained Luciola lateralis-derived luciferase C-DNA is shown in FIG. 6, and the c-D
The amino acid sequence of the polypeptide translated from NA is shown in FIG. 7, and the corresponding sequence between the c-DNA and the amino acid sequence is shown in FIG. 8, respectively.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the optimal pH range of luciferase derived from Luciola lateralis, Figure 2 is a diagram showing the stable pH of luciferase derived from Luciola lateralis, and Figure 3 is a diagram showing the stable pH range of luciferase derived from Luciola lateralis. pALf3DN
FIG. 4 is a diagram showing a restriction enzyme cleavage map of recombinant plasmid ρGLflDNA, and FIG. 5 is a diagram showing a restriction enzyme cleavage map of recombinant plasmid pHL f 7 DNA. FIG. 6 is a diagram showing the cleavage map, FIG. 6 is a diagram showing the base sequence of the luciferase gene derived from Luciola lateralis of the present invention, and FIG. 7 is a diagram showing the nucleotide sequence of the luciferase gene derived from Luciola lateralis of the present invention. FIG. 8 is a diagram showing the amino acid sequence of the polypeptide to be translated, and FIG. 8 is a diagram showing the correspondence between the base sequence and the amino acid sequence of the luciferase gene derived from Luciola lateralis.

Claims (3)

[Claims] (1) A luciferase gene derived from Luciola lateralis and defined by the restriction enzyme cleavage map shown below. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, E I represents EcoR I, S represents Ssp I, EV represents EcoRV, A represents Apa I, and H represents Hpa I.) (2) The luciferase gene according to claim (1), which encodes the amino acid sequence shown below. [There is a gene sequence] (3) Claim (1) represented by the base sequence shown below
) or the luciferase gene described in (2). [There is a gene sequence]