Luciferase gene and novel recombinant DNA as well as a method of producing luciferase

A luciferase gene isolated from Luciola cruciata (Japanese firefly) coding for an amino acid sequence shown in FIG. 4 and a novel recombinant DNA characterized by incorporating a gene coding for luciferase into a vector DNA are disclosed. There is also disclosed a method of producing luciferase which comprises culturing in a medium a microorganism containing a recombinant DNA having inserted a gene coding for luciferase in a vector DNA and belonging to the genus Escherichia capable of producing luciferase and collecting luciferase from the culture.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a luciferase gene derived from Luciola 
cruciata and a novel recombinant DNA having inserted therein the 
luciferase gene as well as a method of producing luciferase. 
2. Discussion of Related Art 
Luciferase from fireflies belonging to the genus Luciola is obtained simply 
by isolating and purifying from the collected fireflies belonging to the 
genus Luciola [Proc. Natl. Acad. Sci., 74 (7), 2799-2802 (1977)]. 
Luciferase is an enzyme which is extremely useful as an enzyme, e.g., for 
ATP assay. 
However, the luciferase described above is derived from insects and hence, 
for producing luciferase, fireflies belonging to the genus Luciola must be 
collected from the natural world or such fireflies must be cultivated and 
luciferase should be isolated and refined from the fireflies so that much 
time and labors are required for the production. 
As a result of various investigations to solve the foregoing problems, the 
present inventors have found that by obtaining a recombinant DNA having 
incorporated DNA containing a gene coding for luciferase into a vector DNA 
and culturing in a medium a luciferase-producing microorganism belonging 
to the genus Escherichia containing the recombinant DNA, luciferase can be 
efficiently produced in a short period of time, and the like. Also as a 
result of further investigations on luciferase gene derived from Luciola 
cruciata, the present inventors have succeeded in isolating a luciferase 
gene derived from Luciola cruciata and determining its structure, for the 
first time and, have thus accomplished the present invention. 
SUMMARY OF THE INVENTION 
That is, a first aspect of the present invention lies in a luciferase gene 
coding for an amino acid sequence shown in FIG. 4. 
A second aspect of the present invention resides in a novel recombinant DNA 
characterized by incorporating a gene coding for luciferase into a vector 
DNA. 
A third aspect of the present invention resides in a method of producing 
luciferase which comprises culturing in a medium a microorganism 
containing a recombinant DNA having inserted a gene coding for luciferase 
in a vector DNA and belonging to the genus Escherichia capable of 
producing luciferase and collecting luciferase from the culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It has been hitherto attempted to isolate luciferase from Luciola cruciata; 
however, the enzyme per se is unstable and such causes a problem that the 
enzyme could be provided for practical use only with difficulty. 
Luciferase obtained in accordance with the present invention is 
advantageous in that it is stable and its activity is also high. 
Hereafter the present invention will be described in detail. 
In survey of DNA containing a gene coding for luciferase of Luciola 
cruciata, DNA containing a gene coding for luciferase derived from 
Photinus pyralis which is one of fireflies is used as a probe. Therefore, 
preparation of the DNA is described below. 
Preparation of m-RNA from the tail of Photinus pyralis which is one of 
fireflies can be effected by methods described in, for example, Molecular 
Cloning, page 196, Cold Spring Harbor Laboratory (1982), Haruo Ozeki and 
Reiro Shimura, BUNSHI IDENGAKU JIKKENHO (Experimental Molecular Genetics), 
pages 66-67 (1983), etc. 
Concentration of m-RNA coding for luciferase from the obtained m-RNA can be 
performed by a method described in, for example, Biomedical Research, 3, 
534-540 (1982) or the like. 
In this case, anti-luciferase serum to luciferase is used. This serum can 
be obtained by, for example, Yuichi Yamamura, MEN-EKI KAGAKU 
(Immunochemistry), pages 43-50 (1973), etc. 
Synthesis of c-DNA from m-RNA coding for luciferase can be performed by 
methods described in, for example, Mol. Cell Biol., 2, 161 (1982) and 
Gene, 25, 263 (1983). 
Then, the thus obtained c-DNA is incorporated into, for example, plasmid 
pMCE10 DNA [plasmid prepared using plasmid pKN 305 [plasmid having a 
promoter of Escherichia coli tryptophane operator described in Agr. Biol. 
Chem., 50, 271 (1986)] and plasmid pMC 1843 [plasmid containing 
Escherichia coli .beta.-galactosidase structural gene described in Methods 
in Enzymology, 100, 293-308 (1983)]], etc. to obtain various recombinant 
plasmid DNAs. Using these DNAs, transformation of Escherichia coli (E. 
coli) DH1 (ATCC 33849), Escherichia coli (E. coli) HB 101 (ATCC 33694), 
etc. is effected by the method of Hanahan [DNA Cloning, 1, 109-135 (1985)] 
to obtain various transformants. 
The recombinant plasmid DNAs possessed by the thus obtained transformants 
are plasmids wherein c-DNA has been incorporated in the middle of 
Escherichia coli .beta.-galactosidase structural gene. A peptide encoded 
by c-DNA is expressed as a protein fused with .beta.-galactosidase. 
In order to detect c-DNA coding for luciferase from the various 
transformants described above, the transformants are cultured thereby to 
express cell protein. By determining if any protein crossing over 
anti-luciferase serum is present, the detection can be made. Methods 
descried in, for example, Agric. Biol. Chem., 50, 271 (1986) and Anal. 
Biochem., 112, 195 (1981), etc. can be used for the detection. 
Next, after labeling c-DNA of incomplete luciferase with .sup.32 P by the 
nick translation method [Molecular Cloning, pages 109-112, Cold Spring 
Harbor Laboratory (1982) and J. Mol. Biol., 113, 237-251 (1977)], using 
the colony hybridization method [Protein, Nucleic Acid & Enzyme, 26, 
575-579 (1981)], an Escherichia coli strain having plasmid DNA containing 
Photinus pyralis luciferase c-DNA of 1.8 Kb can be obtained from a 
Photinus pyralis-derived c-DNA library prepared using plasmid pUC19 DNA 
(manufactured by Takara Shuzo Co., Ltd.) as a vector. 
To obtain the purified plasmid DNA, there is used, for example, a method 
described in Proc. Natl. Acad. Sci., 62 1159-1166 (1969), etc. 
To obtain DNA containing the gene coding for luciferase derived from 
Photinus pyralis from the thus obtained recombinant plasmid DNA, 
restriction enzymes, e.g., EcoR I and Cla I, are acted on the plasmid DNA 
at temperatures of 30.degree. to 40.degree. C., preferably at 37.degree. 
C., for 1 to 24 hours, preferably 2 hours; the solution obtained after 
completion of the reaction is subjected to agarose gel electrophoresis 
[which is described in Molecular Cloning, page 150, Cold Spring Harbor 
Laboratory (1982)] to obtain DNA containing the gene coding for luciferase 
derived from Photinus pyralis. 
Next, preparation of the luciferase gene and the like in accordance with 
the present invention are described below. 
Firstly, source from which the gene coding for luciferase is derived may be 
any source and, mention may be made of, for example, Luciola cruciata, 
etc. Particularly preferred is the tail part of this firefly. 
And preparation of m-RNA from the tail of the firefly and synthesis of 
c-DNA from m-RNA can be conducted, for example, in quite the same manner 
as in the preparation of m-RNA of Photinus pyralis and synthesis of c-DNA 
described above. 
Then, the thus obtained c-DNA is incorporated into a vector DNA, for 
example, plasmid pUC 19 DNA (manufactured by Takara Shuzo Co., Ltd.), etc. 
to obtain various recombinant plasmid DNAs. Using these DNAs, 
transformation of Escherichia coli (E. coli) DH1 (ATCC 33849), Escherichia 
coli (E. coli) HB 101 (ATCC 33694), etc. is effected by the method of 
Hanahan [DNA Cloning, 1, 109-135 (1985)] to obtain various transformants. 
Next, after labeling DNA containing the gene coding for luciferase derived 
from Photinus pyralis with .sup.32 P by the nick translation method 
[Molecular Cloning, pages 109-112, Cold Spring Harbor Laboratory (1982) 
and J. Mol. Biol., 113, 237-251 (1977)], using the colony hybridization 
method [Protein, Nucleic Acid & Enzyme, 26, 575-579 (1981)], an 
Escherichia coli strain having plasmid DNA containing Luciola cruciata 
luciferase c-DNA of 2.0 Kb can be obtained from the gene library of 
Luciola cruciata-derived c-DNA. 
To obtain the purified plasmid DNA, there is used, for example, a method 
described in Proc. Natl. Acad. Sci., 62 1159-1166 (1969), etc. 
By acting on the purified plasmid DNA, for example, restriction enzyme Pst 
I (manufactured by Takara Shuzo Co., Ltd.) at a temperature of 30.degree. 
C. or higher, preferably 37.degree. C. in an enzyme concentration of 200 
to 400 units/ml for 1 to 4 hours, preferably 4 hours to effect digestion, 
a DNA fragment mixture is obtained. 
Isolation from the DNA fragment mixture described above of DNA containing 
the gene coding for luciferase derived from Luciola cruciata can be 
performed by quite the same manner as in the isolation of DNA containing 
the gene coding for luciferase derived from Photinus pyralis. 
Next, by acting a restriction enzyme, e.g., Ssp I [manufactured by New 
England Biolab Co., Ltd.] on the plasmid DNA containing c-DNA encoding 
Luciola cruciata of 2.0 Kb in a conventional manner, DNA containing c-DNA 
coding for luciferase of 1.6 Kb is obtained. The DNA is incorporated into 
a vector DNA to give a novel recombinant DNA. 
As the vector DNA described above, any vector DNA may be used. Mention may 
be made of, for example, plasmid vector DNA, bacteriophage vector DNA, 
etc. Specific examples are pUC 18 (manufactured by Takara Shuzo Co., 
Ltd.), pUC 19 (manufactured by Takara Shuzo Co., Ltd.), 
.lambda.cI857h80att.lambda.sRI.lambda..sub.3.sup.0 sRI.lambda..sub.2.sup.0 
sRI.lambda..sub.1.sup.0 (described in Japanese Patent Publication KOKOKU 
No. 61-37917), etc. 
Using the thus obtained novel recombinant DNA, microorganism belonging to 
the genus Escherichia, for example, Escherichia coli JM 101 (ATCC 33876) 
or the like, is transformed by the method of Cohen et al. [J. Bac., 119, 
1072-1074 (1974)] or transfected by the method described in Molecular 
Cloning, pages 256-268, Cold Spring Harbor Laboratory (1982)] to give 
luciferase-producing microorganism belonging to the genus Escherichia 
containing the novel recombinant DNA having inserted the 
luciferase-encoding gene into vector DNA. 
To obtain a purified novel recombinant DNA from the thus obtained 
microorganism, there is used, for example, a method described in Proc. 
Natl. Acad. Sci., 62, 1159-1166 (1969), etc. 
By acting on the purified novel recombinant DNA described above, for 
example, restriction enzyme Pst I (manufactured by Takara Shuzo Co., Ltd.) 
at a temperature of 30.degree. C. or higher, preferably 37.degree. C. in 
an enzyme concentration of 200 to 400 units/ml for 1 to 4 hours, 
preferably 4 hours to effect digestion, a DNA fragment mixture is 
obtained. 
Isolation from the DNA fragment mixture described above of DNA containing 
the gene coding for luciferase derived from Luciola cruciata can be 
performed by quite the same manner as in the isolation of DNA containing 
the gene coding for luciferase derived from Photinus pyralis. 
Next, the microorganism described above is cultured in a medium and 
luciferase is collected from the culture. 
Any medium may be used as far as it is used for culture of microorganism 
belonging to the genus Escherichia. Mention may be made of, for example, 
1% (W/V) of Tripton, 0.5% (W/V) of yeast extract, 0.5% (W/V) of NaCl and 1 
mM of isopropyl-.beta.-D-thiogalactoside, etc. 
Temperature for the cultivation is between 30.degree. and 40.degree. C., 
preferably about 37.degree. C. and a time period for the cultivation is, 
for example, 4 to 8 hours, preferably about 4 hours. 
The cells are collected from the culture by centrifugation at 8,000 r.p.m. 
for about 10 minutes. The obtained cells are homogenized by the method 
described in, for example, Methods in Enzymology, 133, 3-14 (1986) to 
obtain a crude enzyme solution. 
The crude enzyme solution may be usable as it is; if necessary and desired, 
the crude enzyme solution can be purified by fractionation with ammonium 
sulfate, hydrophobic chromatography, for example, using Butyl Toyo Pearl 
650 C, etc., gel filtration using, e.g., Ultrogel AcA 34, etc. thereby to 
give purified luciferase. 
Physicochemical properties of the thus obtained luciferase are quite the 
same as those described in Photochem. Photobiol., 42, 609-611 (1985). 
As is clear from the foregoing description, according to the present 
invention, luciferase can be efficiently produced in an extremely short 
period of time by culturing the microorganism belonging to the genus 
Escherichia which contains the recombinant DNA having incorporated therein 
the luciferase gene of the present invention. Therefore, the present 
invention is extremely useful from an industrial point of view. 
Hereafter the present invention will be described in more detail by 
referring to the examples below. 
EXAMPLE 
In Items 1 to 10 below, preparation of DNA containing a gene coding for 
luciferase of Photinus pyralis as one of fireflies (this DNA is used as a 
probe upon survey of DNA containing a gene coding for luciferase of 
Luciola cruciata) is described. 
1. Preparation of m-RNA 
Using a mortar and a pestle, 1 g of the dry tail (manufactured by Sigma 
Co., Ltd.) of Photinus pyralis as one of fireflies was thoroughly 
homogenized, to which 5 ml of dissolution buffer [20 mM Tris-hydrochloride 
buffer (pH 7.4)/10 mM NaCl/3 mM magnesium acetate/5% (W/V) sucrose/1.2 % 
(V/V) Triton X-100/10 mM vanadyl nucleoside complex (manufactured by New 
England Biolab Co., Ltd.)] was added. The mixture was further homogenized 
as in the manner described above to give a solution containing the 
homogenized tail of Photinus pyralis. 
In a cup blender (manufactured by Nippon Seiki Seisakusho) was charged 5 ml 
of the thus obtained solution. After treating at 5,000 r.p.m. for 5 
minutes, 12 ml of guanidine isothiocyanate solution (6M guanidine 
isothiocyanate/37.5 mM sodium citrate (pH 7.0)/0.75% (W/V) sodium 
N-lauroylsarcocine/0.15 M .beta.-mercaptoethanol) was added to the system. 
The mixture was treated with the blender described above at 3,000 r.p.m. 
for 10 minutes. The resulting solution was filtered using a threefold 
gauze to give the filtrate. The filtrate was gently poured in layers onto 
4 tubes for ultracentrifuging machine (manufactured by Hitachi Koki Co., 
Ltd.) in which 1.2 ml each of 5.7 M cesium chloride solution had 
previously be laid in layers. Using the ultracentrifuging machine 
(manufactured by Hitachi Koki Co., Ltd., SCP55H), centrifugation was 
performed at 30,000 r.p.m. for 16 hours to give precipitates. 
The obtained precipitates were washed with chilled 70% (V/V) ethanol and 
suspended in 4 ml of 10 mM Tris buffer [10 mM Tris-hydrochloride (pH 
7.4)/5 mM EDTA/1% sodium dodecyl sulfate]. A mixture of the same amount of 
n-butanol and chloroform in 1:4 (volume ratio) was added to the mixture to 
perform extraction. The extract was centrifuged at 3,000 r.p.m. for 10 
minutes in a conventional manner to separate into the aqueous phase and 
the organic solvent phase. To the organic solvent phase was added 4 ml of 
10 mM Tris buffer described above. The extraction and separation 
operations described above were repeated twice. To the aqueous phase 
obtained were added a 1/10 amount of sodium 3 M sodium acetate (pH 5.2) 
and a 2-fold amount of cold ethanol were added. After allowing to stand at 
a temperature of -20.degree. C. for 2 hours, the mixture was centrifuged 
at 8,000 r.p.m. for 20 minutes in a conventional manner to precipitate 
RNA. The obtained RNA was dissolved in 4 ml of water. After the operation 
for precipitation with ethanol described above was carried out, the 
obtained RNA was dissolved in 1 ml of water to give 3.75 mg of RNA. 
By repeating the foregoing operations again, 7 mg in total of RNA was 
prepared. To select m-RNA from the RNA, 7 mg of RNA was subjected to oligo 
(dT)-cellulose (manufactured by New England Biolab Co., Ltd.) column 
chromatography. 
As the column, 2.5 ml of Terumo syringe (manufactured by Terumo Co., Ltd.) 
was used. After 0.5 g of resin was swollen with elution buffer [10 mM 
Tris-hydrochloride buffer (pH 7.6)/1 mM DETA/0.1% (W/V) sodium 
dodecylsulfate], the resin was packed in the column and equilibrated with 
binding buffer [10 mM Tris-hydrochloride (pH 7.6)/1 mM DETA/0.4 M 
NaCl/0.1% (W/V) sodium dodecylsulfate]. 
To 7 mg of RNA was added the same amount of buffer [10 mM 
Tris-hydrochloride (pH 7.6)/1 mM DETA/0.8 M NaCl/0.1% (W/V) sodium 
dodecylsulfate]. The mixture was heat-treated at a temperature of 
65.degree. C. for 10 minutes and then quenched in ice water. After 
subjecting to oligo(dT)-celluose column, the resin was washed with binding 
buffer to completely wash unbound r-RNA and t-RNA out. Further m-RNA was 
eluted with eluting buffer to give 40 .mu.g of m-RNA. 
2. Concentration of luciferase m-RNA 
Sucrose density gradient of 10 to 25% (W/V) was prepared by charging 0.5 ml 
of 40% (W/V) sucrose solution [50 mM Tris-hydrochloride buffer (pH 7.5)/20 
mM NaCl/1 mM EDTA/40% (W/V) sucrose] in a polyaroma tube for Rotar SW 41 
manufactured by Beckmann Co., Ltd., laying 2.4 ml each of 25% (W/V), 20% 
(W/V), 15% (W/V) and 10% (W/V) of the sucrose solution in layers and 
allowing to stand the system at a temperature of 4.degree. C. for 24 
hours. To the sucrose density gradient, 30 .mu.g of m-RNA was laid to form 
a layer. Using SW 41 Rotar manufactured by Beckmann Co., Ltd., 
centrifugation was conducted at 30,000 r.p.m. at a temperature of 
18.degree. C. for 18 hours in a conventional manner. After the 
centrifuging operation, 0.5 ml each was fractionated and m-RNA was 
recovered by the ethanol precipitation method and dissolved in 10 .mu.l of 
water. 
Next, protein encoded by m-RNA was examined, whereby the fraction 
concentrated on m-RNA of luciferase was identified. The fractionated RNA, 
1 .mu.l, 9 .mu.l of rabbit reticular erythrocyte lysate (manufactured by 
Amersham Co., Ltd.) and 1 .mu.l of [.sup.35 S] methionine (manufactured by 
Amersham Co., Ltd.) were mixed and reacted at a temperature of 30.degree. 
C. for 30 minutes. To the reaction mixture was added 150 .mu.l of NET 
buffer [150 mM NaCl/5 mM EDTA/0.02% (W/V) NaN.sub.3 /20 mM 
Tris-hydrochloride buffer (pH 7.4)/0.05% (W/V) Nonidet P-40 (manufactured 
by Besesda Research Laboratories Co., Ltd., surface active agent)] and, 1 
.mu.l of anti-luciferase serum (prepared as will be later described) was 
added to the mixture. After allowing to stand at a temperature of 
20.degree. C. for 30 hours, 10 mg of Protein A Sepharose (manufactured by 
Pharmacia Fine Chemicals Inc.) was added to the mixture. The resulting 
mixture was then centrifuged at 12,000 r.p.m. for a minute in a 
conventional manner to recover the resin. 
The recovered resin was washed three times with 200 .mu.l of NET buffer. To 
the resin was added 40 .mu.l of sample buffer for SDS-PAGE [62.5 mM 
Tris-hydrochloride buffer (pH 6.8)/10% (V/V) glycerol/2% (W/V) sodium 
dodecylsulfate/5% (V/V) mercaptoethanol/0.02% (W/V) bromophenol blue]. The 
mixture was boiled at a temperature of 100.degree. C. for 3 minutes and 
centrifuged at 12,000 r.p.m. for a minute in a conventional manner to 
recover the supernatant. The whole amount was applied onto 7.5% (W/V) 
sodium dodecylsulfate-polyocrylamide gel. 
Gel electrophoresis was performed by the method of Laemmli [Nature, 227, 
680 (1970)]. After the electrophoresis, the gel was immersed in 10% (V/V) 
acetic acid for 30 minutes to immobilize protein. Then, the gel was 
immersed in water for 30 minutes and further immersed in 1 M sodium 
salicylate solution for 30 minutes and then dried to give a dry gel. The 
dry gel was subjected to fluorography using an X ray film (manufactured by 
Fuji Photo Film Co., Ltd., RX). 
From the foregoing operations, the band of luciferase protein was 
recognized on the X ray film only in the case of using RNA in the fraction 
in which luciferase m-RNA was present and, the fraction wherein luciferase 
m-RNA was concentrated could be identified. 
3. Preparation of anti-serum 
Rabbit anti-luciferase serum to purified luciferase was prepared by the 
following method. 
Luciferase solution having a 3.2 mg/ml concentration [solution obtained by 
dissolving luciferase manufactured by Sigma Co., Ltd. in 0.5 M 
glycylglycine solution (pH 7.8)], 0.7 ml, was suspended in an equivalent 
amount of Freund's complete adjuvant. 2.24 mg of the suspension was 
administered to the palm of Japanese white rabbit weighing 2 kg as an 
antigen. After feeding for 2 weeks, the same amount of antigen as in the 
initial amount was intracutaneously administered to the back. After 
feeding for further one week, similar operation was performed. Further one 
week after feeding, whole blood was collected. 
The obtained blood was allowed to stand at a temperature of 4.degree. C. 
for 18 hours and centrifuged at 3,000 r.p.m. for 15 minutes in a 
conventional manner to give anti-luciferase serum as the supernatant. 
4. Synthesis of c-DNA 
Synthesis of c-DNA was carried out using a kit manufactured by Amersham 
Co., Ltd. 
Using 2 .mu.g of m-RNA obtained as described above, synthesis of c-DNA was 
carried out in accordance with the methods described in Mol. Cell Biol., 
2, 161 (1982) and Gene, 25, 263 (1983). As a result, 300 ng of double 
stranded c-DNA was obtained. 
This c-DNA, 150 ng, was dissolved in 7 .mu.l of TE buffer [10 mM 
Tris-hydrochloride buffer (pH 7.5)/1 mM EDTA]. To the solution were added, 
respectively, 11 .mu.l of a mixture [280 mM sodium cacodylate (pH 6.8)/60 
mM Tris-hydrochloride buffer (pH 6.8)/2 mM cobalt chloride] and 3.8 .mu.l 
of a tailing mixture [7.5 .mu.l of 10 mM dithiothreitol/1 .mu.l of 10 
ng/ml poly(A)/2 .mu.l of 5 mM dCTP/110 .mu.l of water]. Further 29 units 
of terminal transferase (manufactured by Boehringer Mannheim AG) was added 
to the mixture. After reacting at a temperature of 30.degree. C. for 10 
minutes, 2.4 .mu.l of 0.25 M EDTA and 2.4 .mu.l of 10% (W/V) sodium 
dodecyl sulfate were added to the mixture to discontinue the reaction. 
The solution which reaction had been discontinued was subjected to a 
treatment for removing protein using 25 .mu.l of water-saturated phenol. 
Then, 25 .mu.l of 4 M ammonium acetate and 100 .mu.l of cold ethanol were 
added to the recovered aqueous phase, respectively. The mixture was 
allowed to stand at a temperature of -70.degree. C. for 15 minutes and 
centrifuged at 12,000 r.p.m. for 10 minutes to recover c-DNA. c-DNA was 
dissolved in 10 .mu.l of TE buffer to give a c-DNA solution. 
As described above, 100 ng of c-DNA with the deoxycytidine tail was 
obtained. 
5. Preparation of recombinant plasmid pMCE10 DNA used in vector 
Plasmid pKN305 DNA prepared by the method described in T. Masuda et al., 
Agricultural Biological Chemistry, 50, 271-279 (1986) using Escherichia 
coli W3110 strain (ATCC 27325) and plasmid pBR 325 (manufactured by BRL 
Co.) and plasmid pBR 322 DNA (manufactured by Takara Shuzo Co., Ltd.) as 
well as pMC 1403-3 DNA (described in Japanese Patent Publication KOKAI No. 
61-274683) were added by 1 .mu.g each to 10 .mu.l of a mixture [50 mM 
Tris-hydrochloride buffer (pH 7.5)/10 mM MgCl.sub.2 /100 mM NaCl/1 mM 
dithiothreitol]. Further, 2 units each of Hind III and Sal I (both 
manufactured by Takara Shuzo Co., Ltd.) were added to the mixture. By 
reacting at a temperature of 37.degree. C. for an hour, a cleavage 
treatment was effected. Extraction with phenol and precipitation with 
ethanol were conducted in a conventional manner to give precipitates. The 
precipitates were dissolved in 10 .mu.l of ligation buffer [20 mM 
MgCl.sub.2 /66 mM Tris-hydrochloride buffer (pH 7.6)/1 mM ATP/15 mM 
dithiothreitol] to give a solution. Further 1 unit of T4DNA ligase 
(manufactured by Takara Shuzo Co., Ltd.) was added thereto to perform 
ligation at a temperature of 20.degree. C. for 4 hours. Then, using this 
reaction solution, Escherichia coli JM 101 (ATCC 33876) was transformed 
according to the transformation method described in [J. Bacteriology, 119, 
1072-1074 (1974)]. By examination of chemical resistance (ampicillin 
resistance and tetracycline resistance) and .beta.-galactosidase activity, 
a transformant was obtained. Recombinant plasmid DNA contained in the 
strain was named pMCE 10. Escherichia coli JM 101 strain containing this 
recombinant plasmid DNA pMCE 10 DNA was cultured in medium composed of 1% 
(W/V) of tripton, 0.5% (W/V) of yeast extract and 0.5% (W/V) of NaCl at a 
temperature of 37.degree. C. for 16 to 24 hours. 20 ml of the thus 
obtained culture solution of Escherichia coli JM 101 (pMCE 10) was 
inoculated on 1 liter of the medium followed by shake culture at a 
temperature of 37.degree. C. for 3 hours. After the addition of 0.2 g of 
chloramphenicol, cultivation was conducted at the same temperature for 
further 20 hours to give a culture solution. 
Next, the culture solution was centrifuged at 6,000 r.p.m. for 10 minutes 
in a conventional manner to give 2 g of wet cells. After the cells were 
suspended in 20 ml of 350 mM Tris-hydrochloride buffer (pH 8.0) containing 
25% (W/V) sucrose, 10 mg of lysozyme, 8 ml of 0.25 M EDTA solution (pH 8.) 
and 8 ml of 20% (W/V) sodium dodecyl sulfate were added to the suspension, 
respectively. The mixture was kept at a temperature of 60.degree. C. for 
30 minutes to give a lysate solution. 
To the lysate solution was added 13 ml of 5 M NaCl solution. The mixture 
was treated at a temperature of 4.degree. C. for 16 hours and then 
centrifuged at 15,000 r.p.m. in a conventional manner to give an extract. 
The extract was subjected to an extraction treatment with phenol and a 
precipitation treatment with ethanol in a conventional manner to give 
precipitates. 
Then, the precipitates were dried under reduced pressure in a conventional 
manner and dissolved in 10 mM Tris-hydrochloride buffer (pH 7.5) 
containing 1 mM EDTA. To the solution were further added 6 g of cesium 
chloride and 0.2 ml of ethydium bromide solution (10 mg/ml). The resulting 
mixture was subjected to an equilibrated density gradinent centrifugation 
treatment using a ultracentrifuging machine at 39,000 r.p.m. for 42 hours 
in a conventional manner thereby to isolate recombinant plasmid pMCE 10 
DNA. After ethydium bromide was removed using n-butanol, dialysis was 
performed to 10 mM Tris-hydrochloride buffer (pH 7.5) containing 1 mM EDTA 
to 500 .mu.g of purified recombinant plasmid pMCE 10 DNA. 
6. Preparation of vector DNA 
The thus obtained recombinant plasmid pMCE 10 DNA, 15 .mu.g, was dissolved 
in 90 .mu.l of TE buffer described in Item 4. After 10 .mu.l of Med buffer 
[100 mM Tris-hydrochloride buffer (pH 7.5)/10 mM MgCl.sub.2 /10 mM 
dithiothreitol/500 mM NaCl] was added to the solution, 30 units of 
restriction enzyme Acc I (manufactured by Takara Shuzo Co., Ltd.) was 
further added to the mixture. A cleavage treatment was conducted at a 
temperature of 37.degree. C. for an hour to give the cleavage product. To 
the cleavage product was added 100 .mu.l of water-saturated phenol, 
whereby protein was removed. Then, the aqueous phase was recovered and a 
1/10-fold amount of 3 M sodium acetate (pH 7.5) and a 2-fold amount of 
cold ethanol were added to the aqueous phase. After allowing to stand at a 
temperature of -70.degree. C. for 15 minutes, the mixture was centrifuged 
at 12,000 r.p.m. for 10 minutes to recover DNA. 
This DNA was dissolved in 10 .mu.l of TE buffer and 15 .mu.l of a mixture 
[280 mM sodium cacodylate (pH 6.8)/60 mM Tris-hydrochloride buffer (pH 
6.8)/2 mM cobalt chloride] was added to the solution. Then, 5 .mu.l of a 
tailing solution mixture (described in Item 4) (5 mM dGTP was used instead 
of 5 mM dCTP) was further added to the mixture. Furthermore, 5 units of 
terminal transferase (manufactured by Takara Shuzo Co., Ltd.) was added to 
react at a temperature of 37.degree. C. for 15 minutes. By after-treatment 
in a manner similar to the c-DNA tailing reaction described in Item 4, DNA 
with the tail of deoxyguanosine at the Acc I site of recombinant plasmid 
pMCE 10 DNA was prepared. 
On the other hand, preparation of DNA with a tail of deoxyguanosine at the 
Pst I site of plasmid pUC 19 DNA was performed at the same time. 
To a solution of 30 .mu.g of plasmid pUC 19 DNA (manufactured by Takara 
Shuzo Co., Ltd.) in 350 .mu.l of TE buffer were added 40 .mu.l of Med 
buffer and 120 units of restriction enzyme Pst I (manufactured by Takara 
Shuzo Co., Ltd.). After a cleavage treatment at a temperature of 
37.degree. C. for an hour, DNA was recovered by a treatment of removing 
protein with phenol and a precipitation treatment with ethanol in a 
conventional manner. 
The obtained DNA was dissolved in 35 .mu.l of TE buffer. To the solution 
were added 50 .mu.l of a mixture [280 mM sodium cacodylate (pH 6.8)/60 mM 
Tris-hydrochloride buffer (pH 6.8)/2 mM cobalt chloride], 19 .mu.l of the 
tailing mixture (containing dGTP instead of dCTP) described in Item 4 and 
60 units of terminal transferase (manufactured by Takara Shuzo Co., Ltd.). 
After reacting at a temperature of 37.degree. C. for 10 minutes, DNA was 
recovered by a treatment of removing protein with phenol and a 
precipitation treatment with ethanol in a conventional manner. 
7. Annealing and transformation 
The synthesized c-DNA, 15 ng and 200 ng of vector DNA were dissolved in 35 
.mu.l of annealing buffer [10 mM Tris-hydrochloride buffer (pH 7.5)/100 mM 
NaCl/1 mM EDTA]. The solution was allowed to stand at a temperature of 
65.degree. C. for 2 minutes, at a temperature of 46.degree. C. for 2 
hours, at a temperature of 37.degree. C. for an hour and at a temperature 
of 20.degree. C. for 18 hours thereby to anneal c-DNA and vector DNA. 
Using the annealed DNA, Escherichia coli DH1 strain (ATCC 33849) was 
transformed by the method of Hanahan [DNA Cloning, 1, 109-135 (1985)] to 
prepare c-DNA bank containing plasmid pUC 19 DNA and recombinant plasmid 
pMCE 10 DNA as vectors, respectively. 
8. Survey of luciferase c-DNA 
The Acc I site of recombinant plasmid pMCE 10 DNA is present at a site 
which codes for Escherichia coli .beta.-galactosidase gene. Therefore, 
c-DNA incorporated into this site forms a fused protein with 
.beta.-galactosidase. Further a promoter of .beta.-galactosidase gene of 
the recombinant plasmid pMCE 10 DNA has been converted into a promoter of 
Escherichia coli tryptophane gene, as described above. 
96 colonies of c-DNA having recombinant plasmid pMCE 10 DNA as a vector 
were shake cultured in 10 ml of M9 Casamino acid medium [Molecular 
Cloning, 440-441, Cold Spring Harbor Laboratory (1982)] supplemented with 
thiamine (10 .mu.g/ml) at a temperature of 37.degree. C. for 10 hours. 
After collecting the cells in a conventional manner, the cells were 
suspended in 200 .mu.l of sample buffer for SDS-PAGE described in Item 2. 
The suspension was boiled at a temperature of 100.degree. C. for 5 
minutes. 
This suspension, 40 .mu.l, was subjected to electrophoresis in a 
conventional manner using 7.5% (W/V) polyacrylamide gel. After completion 
of the electrophoresis, the protein developed on the gel was transferred 
onto a nitrocellulose filter by the western blot method [Anal. Biochem., 
112, 195 (1981)]. This nitrocellulose filter was stained with 
anti-luciferase serum using immune blot assay kit (manufactured by Biorad 
Co.). The method was performed in accordance with the operation of Biorad 
Co. 
That is, the nitrocellulose filter was shaken in 100 ml of blocking 
solution [TBS buffer [20 mM Tris-hydrochloride buffer/500 mM NaCl (pH 
7.5)] containing 3% (W/V) gelatin at a temperature of 25.degree. C. for 30 
minutes. Next, this nitrocellulose filter was transferred into 25 ml of 
primary antibody solution [solution obtained by dissolving 1% (W/V) 
gelatin in TBS buffer and diluting luciferase anti-serum with the 
resulting solution] and shaken at a temperature of 25.degree. C. for 90 
minutes, which was then transferred into 100 ml Tween-20 washing solution 
[solution obtained by dissolving 0.05% (W/V) Tween-20 in TBS buffer] and 
shaken at a temperature of 25.degree. C. for 10 minutes. This operation 
was repeated twice. Then, the thus obtained nitrocellulose filter was 
transferred into 60 ml of secondary antibody solution [solution obtained 
by dissolving anti-rabbit antibody labeled with horse raddish peroxidase 
(manufactured by Biorad Co.) with a solution of 1% (W/V) gelatin in TBS 
buffer in 3000-fold (V/V). After shaking at a temperature of 25.degree. C. 
for 60 minutes, the nitrocellulose filter was washed with 100 ml of 
Tween-20 washing solution. The operation described above was repeated 
twice. The thus obtained nitrocellulose filter was transferred into 120 ml 
of color forming solution [solution obtained by mixing a solution of 60 mg 
of 4-chloro-1-naphthol in 20 ml of cold methanol and a solution of 60 
.mu.l of 30% (V/V) hydrogen peroxide aqueous solution in 100 ml of TBS 
buffer] to form a color at a temperature of 25.degree. C. for 10 minutes. 
As such, similar procedures were performed on 4 groups, one being 96 
colonies. In two groups, protein band stained with luciferase anti-serum 
was recognized. Next, 96 colonies belonging to the two groups were divided 
into 8 groups of 12 colonies each and similar operations were conducted. A 
protein that reacted with anti-luciferase serum was noted in one group. 
Finally, with respect to 12 colonies contained in this group, each colony 
was treated in a similar manner, whereby a protein-producing colony that 
reacted with luciferase anti-serum was identified. By the foregoing 
operations, 2 colonies having luciferase c-DNA were obtained. From the two 
colonies, plasmid DNA was prepared by the method described in Item 5. The 
obtained recombinant plasmid DNAs were named pALf2B8 and pALf3A6, 
respectively. 
9. Survey of large luciferase c-DNA - Preparation of c-DNA probe 
In 330 .mu.l of TE buffer was dissolved 100 .mu.g of recombinant plasmid, 
pALf3A6 DNA. To the solution were added 40 .mu.l of Low buffer [100 mM 
Tris-hydrochloride buffer (pH 7.5)/100 mM MgCl.sub.2 /10 mM 
dithiothreitol], 130 units of Pst I (manufactured by Takara Shuzo Co., 
Ltd.) and 120 units of Sac I (manufactured by Boehringer Mannheim Co.) to 
effect cleavage at a temperature of 37.degree. C. for 1.5 hours. 
The whole amount of DNA was separated by electrophoresis using 0.7% (W/V) 
agarose gel. The agarose gel electrophoresis was carried out in accordance 
with the method of T. Maniatis et al., Molecular Cloning, pages 156-161, 
Cold Spring Harbor Laboratory (1984)]. DNA band containing luciferase 
c-DNA was excised and put in a dialysis tube. After 2 ml of TE buffer was 
supplemented, the dialysis tube was sealed and DNA was eluted from the gel 
into the buffer by electrophoresis. An equivalent volume of 
water-saturated phenol was added to this solution. After agitation, the 
aqueous phase was recovered and DNA was recovered by precipitation with 
ethanol in a conventional manner. 
10 .mu.g of the obtained DNA fragment was dissolved in TE buffer and 16 
.mu.l of Med buffer and 64 units of Sau 3 AI (manufactured by Takara Shuzo 
Co., Ltd.) were added to the solution. After reacting at a temperature of 
37.degree. C. for 2 hours, the whole amount was subjected to 
electrophoresis using 5% (W/V) polyacrylamide gel thereby to isolate DNA 
fragments. The polyacrylamide gel electrophoresis was carried out in 
accordance with the method of A. Maxam [Methods in Enzymology, 65, 506 
(1980)]. DNA fragment of 190 bp was isolated by the method as described 
above to give 1 .mu.g of Sau3 AI luciferase c-DNA fragment. 
Using [.alpha.-.sup.32 P] dCTP (manufactured by Amersham Co.), 1 .mu.g of 
this luciferase c-DNA was labeled according to the nick translation 
method. The nick translation method was performed using a kit manufactured 
by Takara Shuzo Co., Ltd. in accordance with the method described in J. 
Mol. Biol., 113, 237-251 (1977) and Molecular Cloning, pages 109-112, Cold 
Spring Harbor Laboratory (1982). 
10. Survey of large luciferase c-DNA - Colony hybridization 
Using as a probe the luciferase c-DNA fragment labelled with .sup.32 P 
prepared by the method described above, c-DNA bank of the tail of Photinus 
pyralis wherein recombinant plasmid pUC 19 DNA was a vector was surveyed 
by colony hybridization [(Protein, Nucleic Acid and Enzyme, 26, 575-579 
(1981)] to give colonies having luciferase c-DNA. Recombinant plasmid DNA 
possessed by one of the colonies was named pALf3 and plasmid DNA was 
prepared by the method described in Item 5. Escherichia coli containing 
the recombinant plasmid DNA was named Escherichia coli DH 1 (pALf3). The 
transformant has been deposited as ATCC 67462. 
The recombinant plasmid pALf3 DNA described above was subjected to single 
digestion and double digestion using Xba I, Hind III, BamH I, EcoR I and 
Pst I (all manufactured by Takara Shuzo Co., Ltd.). The obtained DNA 
fragments were analyzed by agarose gel electrophoresis on mobility 
pattern. By comparing the obtained mobility pattern with standard mobility 
pattern of DNA fragment obtained by digesting .lambda.DNA (manufactured by 
Takara Shuzo Co., Ltd.) with Hind III, the size of the c-DNA inserted in 
pALf3 was turned out to be 1,700 bp. A restriction enzyme map of the 
plasmid described above is shown in FIG. 1. 
11. Preparation of m-RNA of Luciola cruciata 
Ten grams of living Luciola cruciata (purchased from Seibu Department 
Store) were put in a ultra-low temperature freezer box and frozen. Each 
tail was cut off with scissors. To 2 g of the obtained tail was added 18 
ml of guanidine isothiocyanate solution. According to the method described 
in Item 1, 1.1 mg of RNA was prepared. In accordance with the method 
described in Item 1, 1.1 mg of this RNA was subjected to column 
chromatography of oligo (dT)-cellulose to prepare 30 .mu.g of Luciola 
cruciata tail m-RNA. 
12. Preparation of c-DNA bank of Luciola cruciata tail 
Synthesis of c-DNA was performed using a kit purchased from Amersham Co. in 
accordance with the method indicated by Amersham Co. which is described in 
Mol. Cell Biol., 2, 161 (1982) and Gene, 25, 263 (1983). 
From 2 .mu.g of the Luciola cruciata tail RNA, 0.9 .mu.g of double stranded 
c-DNA was synthesized. Using the method described in Item 4, a tail of 
polydeoxycytidine was added to 0.3 .mu.g of this c-DNA. 
This c-DNA, 20 ng, and 500 ng of pUC 19 plasmid prepared in Item 6, wherein 
a polyguanosine tail had been added to the Pst I site thereof, were 
annealed in accordance with the method described in Item 7. Escherichia 
coli DH 1 strain (ATCC 3849) was transformed by annealed DNA by the method 
of Hanahan DNA Cloning, 1, 109-135 (1985)] thereby to prepare c-DNA bank 
of Luciola cruciata tail. 
13. Survey of luciferase c-DNA derived from Luciola cruciata 
In 90 .mu.l of TE buffer was dissolved 10 .mu.g of recombinant plasmid 
pALf3 DNA and, 10 .mu.l of Med buffer, 25 units of restriction enzyme EcoR 
I and 25 units of restriction enzyme Cla I (both manufactured by Takara 
Shuzo Co., Ltd.) were added to the solution. The reaction was performed at 
a temperature of 37.degree. C. for 2 hours to cleave DNA. From the cleaved 
recombinant plasmid pALf3 DNA, 800 bp of EcoR I/Cla I DNA fragment 
containing luciferase c-DNA derived from Photinus pyralis (American 
firefly) was isolated in accordance with the method described in Item 9 
using agarose gel electrophoresis. Thus, 1 .mu.g of EcoR I/Cla I DNA 
fragment was obtained. Using [.alpha.-.sup.32 P] dCTP triphosphate 
(manufactured by Amersham Co.], 1 .mu.g of this DNA was labelled with 
.sup.32 P in accordance with the nick translation method described in Item 
9. Using as a probe the EcoR I/Cla I DNA fragment labeled with .sup.32 P, 
c-DNA bank of the Luciola cruciata tail was surveyed by the colony 
hybridization described in Item 10 thereby to select Escherichia coli 
having luciferase c-DNA derived from Luciola cruciata. Several strains of 
Escherichia coli capable of hybridizing with the probe were obtained. 
Recombinant plasmid DNA possessed by one of these colonies was named 
pGLf1. The recombinant plasmid DNA was isolated in accordance with the 
method described in Item 5. Escherichia coli containing the recombinant 
plasmid DNA was named Escherichia coli DH 1 (pGLf1). The transformant has 
been deposited as ATCC 67482. 
The recombinant plasmid pGLf1 DNA described above was subjected to single 
digestion and double digestion using Hpa I, Hind III, EcoR V, Dra I, Afl 
II, Hinc II, Pst I (all manufactured by Takara Shuzo Co., Ltd.) and Ssp I 
(manufactured by New England Biolab Co.). The obtained DNA fragments were 
analyzed by agarose gel electrophoresis on mobility pattern. By comparing 
the obtained mobility pattern with standard mobility pattern of DNA 
fragment obtained by digesting .lambda.DNA (manufactured by Takara Shuzo 
Co., Ltd.) with Hind III, the size of the c-DNA inserted in pGLF1 was 
turned out ot be 2,000 bp. A restriction enzyme map of the plasmid 
described above is shown in FIG. 2. 
14. Analysis of base sequence of luciferase c-DNA derived from Luciola 
cruciata 
Recombinant plasmid pGLf1 DNA, 10 .mu.g, was cleaved with restriction 
enzyme Pst I (manufactured by Takara Shuzo Co., Ltd.) to give 2.5 .mu.g of 
2.0 Kb DNA fragment containing luciferase c-DNA. This DNA fragment was 
cloned at the Pst I site of plasmid pUC 119 DNA (manufactured by Takara 
Shuzo Co., Ltd.). The obtained plasmid DNAs were named pGLf2 and pGLf3, 
respectively, depending upon difference in the direction of inserting 
c-DNA. A cleavage treatment of recombinant plasmid pGLf1 DNA and plasmid 
pUC 119 DNA with Pst I (method described in Item 6), isolation of the 
luciferase c-DNA fragments using agarose gel electrophoresis (described in 
Item 9), ligation of plasmid pUC 119 DNA and luciferase c-DNA fragment 
(described in Item 5), transformation of Escherichia coli JM 101 strain 
(ATCC 33876) using the ligation reaction solution (described in Item 5) 
and preparation of recombinant plasmid pGLf2 and pGlf3 DNAs (described in 
Item 5) followed the methods described within parentheses. 
Next, using the recombinant plasmid pGLf2 and pGlf3 DNAs, plasmid DNAs 
wherein various deletions were introduced into luciferase c-DNA were 
prepared using a deletion kit for killosequence (manufactured by Takara 
Shuzo Co., Ltd.) in accordance with the method of Henikoff [Gene, 28, 
351-359 (1984)], followed by introducing into Escherichia coli JM 101 
strain (ATCC 33876) described in Item 5. By infecting the thus obtained 
Escherichia coli with helper phage M13K07 (manufactured by Takara Shuzo 
Co., Ltd.), single strand DNA was prepared in accordance with the method 
of Messing [Methods in Enzymology, 101, 20-78 (1983)]. Sequencing with the 
obtained single strand DNA was carried out by the method of Messing 
described above, using M13 sequencing kit (manufactured by Takara Shuzo 
Co., Ltd.). Gel electrophoresis for analyzing a base sequence was carried 
out using 8% (W/V) polyacrylamide gel (manufactured by Fuji Photo Film 
Co., Ltd.). 
The base sequence of the luciferase-coding region of Luciola 
cruciata-derived luciferase c-DNA alone is shown in FIG. 3. An amino acid 
sequence of the protein translated from the c-DNA is shown in FIG. 4. 
15. Construction of recombinant plasmid pGLf15 DNA 
To a solution of 5 .mu.g of recombinant plasmid pGLf1 DNA in 90 .mu.l of TE 
buffer were added 10 .mu.l of Med buffer and 25 units of Ssp I 
(manufactured by New England Biolab Co., Ltd.)]. After digesting at a 
temperature of 37.degree. C. for 2 hours, an equivalent volume of 
water-saturated phenol was added thereto, whereby operation of removing 
protein was conducted in a conventional manner. From the digested 
recombinant plasmid pGLf15 DNA, 1.6 Kb DNA fragment coding for Luciola 
cruciata-derived luciferase c-DNA was isolated by utilizing the method 
using agarose gel electrophoresis described in Item 9. Thus, 1 .mu.g of 
1.6 Kb Ssp I fragment was obtained. 
On the other hand, 1 .mu.g of plasmid pUC 18 DNA (manufactured by Takara 
Shuzo Co., Ltd.) was dissolved in 18 .mu.l of TE buffer and, 2 .mu.l of 
Sma I buffer [100 mM Tris-hydrochloride buffer (pH 8.0)/70 mM magnesium 
chloride/200 mM potassium chloride/70 mM 2-mercaptoethanol/0.1% bovine 
serum albumin] and 5 units of Sma I (manufactured by Takara Shuzo Co., 
Ltd.) were added to the solution. After digesting at a temperature of 
37.degree. C. for an hour, extraction with phenol and precipitation with 
ethanol were performed in a conventional manner to give precipitates. 
In 7 .mu.l of water were dissolved 0.5 .mu.g of the Sma I-digested plasmid 
pUC 18 DNA and 0.5 .mu.g of 1.6 Kb Luciola cruciata-derived luciferase 
c-DNA fragment. To the solution was added 13 .mu.l of a mixture [77 mM 
Tris-hydrochloride buffer (pH 7.4)/15 mM magnesium chloride/15 mM 
dithiothreitol/0.15 mM adenosine triphosphate] and 1 unit of T4 ligase 
(manufactured by Boehringer Mannheim AG). The mixture was subjected to 
ligation at a temperature of 8.degree. C. for 18 hours. Using the reaction 
solution, Escherichia coli JM 101 strain (ATCC 33876) was transformed as 
described in Item 7. From the obtained transformants, plasmid DNA was 
isolated as described in Item 5. The isolated plasmid DNA was subjected 
to single digestion with Hind III (manufactured by Takara Shuzo Co., Ltd.) 
and a plasmid DNA forming 1.5 Kb and 2.9 Kb DNA fragments was selected. 
This recombinant plasmid DNA was named pGLf15 and Escherichia coli bearing 
this plasmid DNA was named Escherichia coli JM 101 (pGLf15). Escherichia 
coli JM 101 (pGLf15) has been deposited as ATCC 67461. 
Escherichia coli JM 101 (pGLf15) was cultured by the method described in 
Item 5. By isolating the recombinant plasmid DNA, 1.2 mg of purified 
recombinant pGLf15 DNA was obtained from 1 liter of the culture solution. 
16. Cultivation of Escherichia coli JM 101 (pGLf15) (ATCC 67461) and 
preparation of crude enzyme solution 
Escherichia coli JM 101 (pGLf15) (ATCC 67461) was shake cultured in 3 ml of 
LB-amp medium [1% (W/V) bactotripton, 0.5% (W/V) yeast extract, 0.5% (W/V) 
NaCl and ampicillin (50 .mu.g/ml)] at a temperature of 37.degree. C. for 
18 hours. This culture solution, 0.5 ml, was inoculated on 10 ml of the 
aforesaid LB-amp medium and 1 mM isopropyl-.beta.-D-thiogalactoside was 
added thereto. After shake culture at a temperature of 37.degree. C. for 4 
hours, the culture was subjected to a centrifuging operation at 8,000 
r.p.m. for 10 minutes to give 20 mg of wet cells. 
The recovered cells were suspended in 0.9 ml of buffer composed of 0.1 M 
KH.sub.2 PO.sub.4 (pH 7.8), 2 mM EDTA, 1 mM dithiothreitol and 0.2 mg/ml 
protamine sulfate. Further 100 .mu.l of 10 mg/ml lysozyme solution was 
supplemented to the suspension. The mixture was allowed to stand in ice 
for 15 minutes. Next, the suspension was frozen in methanol-dry ice bath 
and then allowed to stand at a temperature of 25.degree. C. to completely 
freeze. Further by performing a centrifuging operation at 12,000 r.p.m. 
for 5 minutes, 1 ml of crude enzyme solution was obtained as the 
supernatant. 
The luciferase activity in the thus obtained crude enzyme solution was 
performed by the method described below. The results are shown in the 
table below. 
The measurement of luciferase activity in the crude enzyme solution 
obtained was performed by counting the number of photon formed in 
accordance with the method of Kricka [Archives of Biochemistry and 
Biophysics, 217, 674 (1982)]. 
That is, 260 .mu.l of 25 mM glycylglycine buffer (pH 7.8), 16 .mu.l of 0.1 
M magnesium sulfate and 24 .mu.l of 1 mM luciferine (manufactured by Sigma 
Co.) and 10 .mu.l of the crude enzyme solution were mixed. Then 100 .mu.l 
of 20 mM ATP was added to the mixture. The number of photon formed was 
integrated for 20 seconds. The integrated values were shown in the table 
below. 
For purpose of comparison, luciferase activity was measured also with 
Escherichia coli JM 101 strain bearing plasmid pUC 18 DNA [Escherichia 
coli JM 101 (pUC 18)]. The results are shown in the table below. 
TABLE 
______________________________________ 
Item 
Sample Number of Photon/ml Culture Solution 
______________________________________ 
Escherichia coli 
8.3 .times. 10.sup.6 
JM 101 (pGLf15) 
(invention) 
Escherichia coli 
9.8 .times. 10.sup.4 
JM 101 (pUC 18) 
(control) 
______________________________________ 
As is clear from the table above, it is noted that the count of photon 
increased in the present invention as compared to the comparison and 
therefore, luciferase is produced in the cells of Escherichia coli used in 
the present invention. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it is apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and the scope of the present invention.