JP2834187B2 - DNA compound encoding luciferase and expression vector containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a marine crustacean, Vargula
hilgendorfii, formerly classified as Cypridina hilgendorfii), a DNA compound encoding sea urchin luciferase, which is an enzyme that catalyzes the luminescence phenomenon, an expression vector containing the DNA compound, and an appropriate host using the expression vector. The present invention relates to a method for producing Cypridina luciferase, which comprises transforming cells and culturing the obtained transformant.

[Prior Art and Problems to be Solved by the Invention] Luciferase is an enzyme that catalyzes a chemiluminescent reaction observed in various biological species. This luminescence reaction is a reaction in which substrate luciferin is oxidized to oxyluciferin by the enzymatic action of luciferase in the presence of oxygen, and is represented by the following formula.

Luciferin + O ₂ → oxyluciferin + CO ₂ + photo The reaction mechanism differs depending on the species, and some require nucleotides such as ATP as cofactors.
It is known that closely related species can react with each other,
It is said that substrate specificity is extremely high, and luciferin obtained from the same species of organism reacts with luciferase, but the enzyme derived from a different species and the substrate do not cross-react in principle.

As described above, the substrate (luciferin) specificity of luciferase is extremely high, and its luminescence is sharp, so that various substances can be detected and / or quantified by utilizing the chemiluminescence reaction of this enzyme-substrate. it is conceivable that. In general, enzymatic reactions are highly sensitive due to their substrate specificity and are mild in reaction conditions, so that they are applied in various fields. For example, horseradish peroxidase, which catalyzes the oxidation reaction in the presence of hydrogen peroxide, is one of the most widely used enzymes. In particular, this enzyme is important for the analysis of substances detectable through reactions involving the generation of hydrogen peroxide. Other useful enzymes have been extracted and isolated. However, in various fields, more and more available enzymes are required for diversifying purposes. Therefore, if a method capable of stably supplying luciferase, which is an enzyme that catalyzes a luminescence reaction, is established, a new use of the enzyme will be developed.

By the way, luciferase has an auxiliary substance other than the enzyme and luciferin (a trace amount of AT) to express its catalytic activity.
Some require nucleotides such as P) and others do not. Luciferase belonging to the former is used for detection of auxiliary substances (trace ATP). On the other hand, enzymes belonging to the latter, such as Cypridina luciferase, are simple in reaction because they do not require oxygen and a substrate (in this case, substances other than Cypridina luciferin, and therefore have a wide range of applications and high utility. Research and development of application fields of sea firefly luciferase,
In order to promote practical use, high-purity sea firefly luciferase must be supplied stably. However, as with enzymes from other organisms, extracting, isolating and purifying the enzyme from the organism requires a lot of time and money, making it difficult to satisfy the above demand. Therefore, establishment of a simple and efficient method for producing Cypridina luciferase is desired.

Means for Solving the Problems In view of such a situation, the present inventors focused on producing Cypridina luciferase by genetic engineering, cloned cDNA encoding the enzyme, and obtained the DNA
The sequence was determined. Next, an expression vector containing the DNA compound thus obtained was constructed, the expression vector was expressed in mammalian cells, and secretion of Cypridina luciferase into the medium was successful.

That is, the present invention provides a DNA compound encoding Cypridina luciferase.

The present invention also provides an expression vector containing the DNA compound.

The present invention also provides a method for producing sea urchin luciferase, which comprises transfecting a host cell with the expression vector, culturing, and recovering sea firefly luciferase from a culture medium.

The chemiluminescent reaction between sea firefly luciferase and the substrate sea firefly luciferin has been well studied [Harvey, EN, Am. J. Physiol. 42 318-341, 1917 and Johnson, FH, et al., Methods Enzymol. . 57
331-364, 1978]. The reaction is shown by the following reaction formula.

(Where R ₃ = —CH (CH ₃ ) CH ₂ CH ₃ . Note that λ _max = 460 nm.

Screening of the Cypridina luciferase cDNA, determination of the nucleotide sequence, construction of the expression vector, transfection and cultivation of the host cells were performed using methods known in the art. The outline is shown below.

Literature [Tsuji (FI) Methods Enzymol. ( 57 , 36)
4-372, 1978)] The sea urchin luciferase partially purified by the method described above was completely purified using an affinity column. When this sample was directly subjected to the Edman degradation method, no amino acid could be assigned. This means that the amino group of the N-terminal amino acid of the peptide is blocked. Thus, the purified sea firefly luciferase was digested with endopeptidase, and the amino acid sequence was determined by subjecting the resulting peptide fragment to the Edman degradation method. Next, an oligonucleotide probe is designed using an appropriate part, and a DNA synthesizer (Applied
Biosystems Inc., Model 381A) using the phosphoramidite method [Carousers (Ca
ruthers, MH), Synthesis and Applications of DNA a
nd RNA, Editing: Narang (SA) (Academic Pre
ss, New York), pp. 47-94, 1987].

On the other hand, sea firefly (azalea, supra) collected in Chiba Prefecture is ground into fine powder in liquid nitrogen, and this powder is subjected to a guanidine thiocyanate / cesium chloride method [Chigwin, J. et al.
M.) et al., Biochemistry 18 , 5294-5299, 1979] to extract total cellular RNA. Then, oligo (dT) cellulose column chromatography [Aviv, H.)
USA, 69 , 1406-1412, 1972] to purify the poly (A) RAN, and obtain Morishita (Morishita,
K) et al., A cDNA library was constructed according to the description in J. Biol. Chem. ( 262 , 3844-3851, 1987).

This cDNA library was subjected to plaque hybridization [Benton and WDs (Benton, WD & Davis, RW), Sc]
ience 196 , 180-182, 1977].
Two clones VL16 and VL18 were selected from the eight positive clones, and a restriction map was prepared. Clone VL
16 encodes the N-terminal side of luciferase, and VL18 encodes the C-terminal side, and has an overlapping portion of 830 nucleotides with each other (FIGS. 2b and 2c). So these two
The fragment was digested with Eco R1, the obtained fragment was subcloned, and 7-Deaza DNA sequencing kit (Takara Shuzo) and [α- ³² P] dCTP (222 TBq / mmol) (New England Nucleic Acid)
ar) [Sanger, F. et al., Proc. Natl. Acad. Sci. USA 7
4 , 5463-5467, 1977 and Chen, EY et al., DN
A 4, 165-170, the nucleotide sequence was determined by 1985. The restriction map of the full-length cDNA is shown in FIG. 2a, and the nucleotide sequence and deduced amino acid sequence are shown in FIG. This deduced amino acid sequence was completely consistent with the amino acid sequence determined by the Edman degradation method described above.

Next, the full-length cD constructed from clones VL16 and VL18
An expression vector was constructed using the NA clone (see FIG. 2d). Expression vectors may be prokaryotic or eukaryotic. It is well known to those skilled in the art how to select an appropriate vector as a starting material, insert a DNA compound encoding a desired peptide into the vector, and construct an expression vector for the peptide. In the present specification, an expression vector that can be expressed in mammalian cells is exemplified because the product is secreted into the medium and processing is easy. Plasmid pRSVVL, which is an exemplary vector of the present invention, contains a cDNA encoding Cypridina luciferase downstream of the Rous sarcoma virus long terminal repeat promoter. Using this plasmid pRSVVL, host cells are transfected as usual. Although the transfection method and host cell can be appropriately selected, in the present invention, the calcium phosphate method (Graham, FL et al., Virolog
y 52 , 456-467, 1973 and Wigler, M. et al., Cell 14 , 72.
Monkey COS cells [Glutzman (Glu
zman, Y.), Cell 23 , 175-182, 1981, (7 × 10 ⁶ )]. After incubation with a medium suitable for expression of Cypridina luciferase, the medium was collected and the cells were harvested to prepare a cell extract.

Cypridina luciferase activity of the medium and cell extracts thus obtained was measured with a Mitchell-Hastings photometer [Mitchell, GW et al., Ana.
l, Biochem. 39 , 243-250]. As a result, although the firefly luciferase activity detected from the cell extract was slight, clear transferase activity was detected from the medium (FIG. 4). This luminescence is extremely sensitive,
Using 5μ in 10ml of medium, a signal clearly higher than background was detected.

As described above, according to the present invention, Cypridina luciferase can be easily produced by genetic optics.

DNA encoding firefly luciferase according to the present invention
Now that the compounds have been nucleotide sequenced, those skilled in the art can readily produce firefly luciferase using known means in genetic engineering. Many starting materials (expression vectors including a promoter and the like) and host cells for the construction of an expression vector suitable for expressing a DNA compound encoding a sea firefly luciferase of the present invention are known, and the promoter exemplified in the present specification is known. -A system other than the host system can be selected to achieve the same effect as the method of the present invention. Those skilled in the art know how to select an appropriate promoter-host system, construct an expression vector, transform host cells with the resulting expression vector, culture and isolate the product. Therefore, those skilled in the art will recognize that the present invention is not limited to the exemplified plasmid, pRSVVL, but encodes the sea urchin luciferase of the present invention.
It will be readily understood that any expression vector suitable for expressing a DNA compound is encompassed.
Suitable promoters for such other expression vectors include the following.

That is, promoters for expression in animal cells include simian SV40 virus promoter, cytomegalovirus promoter, adenovirus promoter, AIDS virus promoter, silkworm polygon virus promoter and the like. At this time, mouse NIH3T3 cells, C127 cells, L cells,
Hamster CHO cells, human HeLa cells, silkworms and the like can be used as host cells. In addition, as expression promoters in prokaryotic cells, λ phage PL promoter, Tac
Promoters, T7 promoters, lac promoters and the like are known, and can be expressed using Escherichia coli as a host. Furthermore, it is also possible to express Cypridina luciferase using yeast or Bacillus subtilis as a host and using a suitable promoter.

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

The method of Example 1 Cloning of Umibotaru cDNA 1. sea firefly luciferase purified and sequenced Tsuji (Tsuji, FI) [Methods Enzymol . (57,
364-372, 1978)]. A tryptamine affinity column (Pierce) equilibrated with 2.0 M NaCl / 0.07 M Tris-HCl (pH 7.2) with sea urchin luciferase partially purified according to
30% ethylene glycol / 0.17M Na
Elution was performed stepwise with Cl / 0.07M Tris-HCl (pH 7.2). Then, after concentration by ultrafiltration, purification was carried out using a p-aminobenzamidine affinity column (Pierce Chemical) under the same chromatography conditions until homogeneity.

Purified sea firefly luciferase was subjected to sodium dodecyl sulfate / gradient polyacrylamide gel electrophoresis. Gradient 10-15% fast gel (PhastGel) (Pharmaci
Analyze the sample by a) and use Fast Gel Silver Staining Kit (P
harmacia) showed a single band of Mr 68,000. The results are shown in FIG. In the figure, lane 1 is a low molecular weight marker manufactured by Pharmacia, and lane 2 is sea urchin luciferase (50 ng) purified by affinity chromatography. The molecular weight of the marker protein is shown in kilodaltons.

120 μg of the purified protein was added to trypsin (Boehrin
ger-Mannheim), lysyl endopeptidase (Wako Pure Chemical Industries) or arginyl endopeptidase (Takara Shuzo). C ₁₈
-Each peptide fragment was isolated by reverse phase chromatography using a protein-peptide HPLC column (Vydac). Next, the gas phase protein sequenator (Appl
The N-terminal amino acid sequence of undigested luciferase and purified peptide was determined by Edman degradation using ied Biosystems Inc., Model 477A).

2. Preparation of mRNA and Construction of cDNA Library A sea firefly (azalea, supra) collected in Chiba Prefecture was immediately ligated in liquid nitrogen. This sea firefly (ostacods, ostacods) (weight 5g) is transferred to an ultraturrax homogenizer (Janke &
Using Kunkel) in liquid nitrogen. This fine powder was subjected to a guanidine thiocyanate / cesium chloride method [Chigwin, JM et al., Biochemistry 18 , 5294-5299, 1
979] to extract total cellular RNA. Then, oligo (dT) cellulose column chromatography [Aviv, H. et al., Proc. Natl. Acad. Sci. USA 69 , 1408-14].
12, 1972]. This mRN
A was used to construct a cDNA library, which was purified by double-strand purification using low melting agarose (BioRad).
Except for size selection of Eco R1 digested cDNA,
hita, K.) et al. [J. Biol. Chem. ( 262 , 3844-3851,
1987].

3. Screening of cDNA clone A part of the peptide fragment prepared in the above 1 is a peptide sequence with minimal codon ambiguity, Thr
-Met-Glu-Asn-Leu-Asp-Gly-Gln-Lys. Using this, three sites with high codon degeneracy include deoxyinosine [Ohtsuka, E., et al., J. Bio.
260 , 2605-2608, 1985], the following complementary oligonucleotide probes were designed: 5 '(T / C) TT (T / C) TGICC (A / G) TCIAGGTT (T / C) T
CCATIGT3 'In addition, the second having A at position 15 instead of G
Was synthesized. Oligonucleotide probes were synthesized using a DNA synthesizer (Applied Biosystems In
c., Model 381A) by the phosphoramidite method [Caruthers, MH], Synthesis and Applications
of DNA and RNA, Narang, SA, (Academic Press, New
York), pp. 47-94, 1987]. This oligonucleotide probe was used for T4 polynucleotide kinase (Takara Shuzo) and [γ- ³² P] ATP (222 TBq / mmol, New Englan
d Nuclear) (specific activity: 5-6 × 1)
0 ⁶ cpm / pmol) and a plaque hybrid method [Benton and Divis (Bent
on, WD & Davis, RW), Science 196 , 180-182, 197
According to 7], the sea urchin firefly cDNA library prepared in 2. above was screened. That is, 1 × 10 ⁶ recombinant phages were screened using this probe.
For hybridization, the temperature was lowered to 28 ° C., and the filters were washed with SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) at 37 ° C., except for the wall (Wahl, G. et al.).
M.) [Proc. Natl. Acad. Sci. USA ( 76 , 3683-36).
87, 1979])]. 8 from library
Two positive clones were isolated, of which clones VL16 and VL18 with insert lengths of 1.2 kb and 1.5 kb were selected for further analysis to create a control enzyme map. FIG. 2 shows a restriction map of these clones.

4. Determination of Nucleotide Sequence of Cypridina Luciferase As can be seen from FIGS. 2b and 2c, clones VL16 and VL18 each have a single internal Eco R1 site and an overlap of 830 nucleotide sequences. . The Eco R1 fragment of both these clones was subcloned with pUC8. 7-Deaza DNA sequencing kit (Takara Shuzo)
[Α- ³² P] dCTP (222 TBq / mmol) (New Eng
land Nuclear) using a dideoxynucleotide chain [Sanger, F. et al., Proc. Natl. Acad.
Sci. USA 74 , 5463-5467, 1977 and Chen, E.
Y. et al., DNA 4 , 165-170, 1985)], and the nucleotide sequence was analyzed. As a result, clone VL16 encoded the N-terminal part of luciferase, and clone VL18 encoded the C-terminal part. I knew it was there. Full-length sea firefly luciferase cD constructed from these clones
The restriction map of NA is shown in FIG. 2a, and the nucleotide sequence and deduced amino acid sequence are shown in FIG. In FIG. 2a,
The hatched part is the putative signal sequence. In FIG. 3, the numbers above each column are nucleotide positions,
The numbers below each column are amino acid positions. Horizontal arrows indicate regions having the same amino acid sequence as the amino acid sequence obtained by endopeptidase digestion.
Sequences compatible with N-glycosylation are boxed and the polyadenylation signal AATAAA is underlined.

The molecular weight of Cypridina luciferase calculated from this sequence is 62,171 daltons and contains an open reading frame of 1665 nucleotides encoding a protein of 555 amino acids. This open reading frame contains the amino acid sequence used to construct the oligonucleotide probe. In addition, the amino acid sequences of the other seven peptides determined by the Edman degradation method completely matched the amino acid sequences deduced from the nucleotide sequences.

AT starting translation start codon from nucleotide 16
Applied to G codon. The nucleotide sequence around this putative start codon is consistent with the consensus sequence CC (A / G) CCAUGG characteristic of many eukaryotic mRNAs [Kozak, M., Cell 15 , 1109]. -1123, 1978]. The amino acid sequence at the N-terminus has many features of a signal sequence in order to play a biological role for luciferase as a secreted protein [von Heijne,
G.), Eur. J. Biochem. 133 , 17-21, 1983]. In addition, the blocked N-terminal was predicted by the method described later.

There are two N-glycosylation sites (Asn-X-Ser / Thr) at amino acid residues 186 and 408, as also shown by luciferase positive staining by the periodate-Schiff reaction. At residue 258, the sequence Asn-Pro-Ser is present, but this sequence is generally not efficiently glycosylated [Marshall, RD, Ann. Rev. Biochem. 41 ,
673-702, 1972]. When N-glycosidase F is acted on, the size of the luciferase molecule is reduced by 2000-3000 daltons, but such a reduction is not observed in digestive enzymes specific to O-glycosylation sites. These results indicate that Cypridina luciferase is N-glycosylated, and the theoretical molecular weight of the polypeptide deduced from the nucleotide sequence, gel electrophoresis (FIG. 1) and gel filtration and sedimentation equilibrium analysis (Tsuji et al. Biochemistry 13 , 520
4-5209, 1974), indicating that the difference from the molecular weight of the native protein is related to the absence or presence of the carbohydrate moiety.

The amino terminus was deduced from the structure of the enzyme that catalyzes the bioluminescence reaction of other marine organisms with similar luciferin structures and mechanisms. We chose jellyfish (Aequorea victoria) as such an organism and compared it directly with its bioluminescence reaction [Johnson, FH et al., Methods
Enzymol. 57 , 271-291, 1978].

The jellyfish emits light from a calcium-binding protein, aequorin, which emits light when excited in the presence of calcium ions. Aequorin,
A complex of apoaequorin (apoprotein), coelenterazine and oxygen molecules. When aequorin binds to calcium ions, a conformational change occurs, the protein is converted to oxygenase, and then coelenterazine is oxidized by an intramolecular reaction. The illuminant in this reaction is excited coelenterazine bound to apoaequorin [Shimomura, O. et al., Tetrahed.
ron Lett. No. 31 , 2963-1966, 1973].

The substrate structure and mechanism of the bioluminescent reaction of sea firefly and jellyfish are similar to each other but hardly cross-react. However, comparison of the amino acid sequences of the two showed that two regions of Cypridina luciferase (residues 97 to 154 and 353 to 353).
411) was found to exhibit significant similarity to the amino acid sequence of one region of apoaequorin (residues 82 to 144) (see FIG. 5). FIG. 5 is a diagram showing the amino acid sequence homology between Cypridina luciferase (a) and jellyfish (Aequrea) aequorin (b).
yhoff mutation data matrix [Dayhoff (D
ayhoff, MO), Schwartz, RM, et al., Atlas of Protein Se
quence and Structure (National Biochemical Researc
h Foundation, Washington, DC), Vol. 5, pp. 345-352,
1978] are indicated by two dots (:), and similar amino acids are indicated by one dot (•). The numbers represent the position from the N-terminus of each protein. The size of the apoaequorin molecule is about 1 /
3, and consists of 189 amino acid residues, and it is predicted that one or both of similar regions of Cypridina luciferase are involved in luminescence.

The region of Cypridina luciferase corresponding to residues 87-144 of apoaequorin is residues 97-154, clearly 1
A shift of 0 amino acid residues is observed. This suggests that the first 11 amino acid residues are leader peptides required for secretion, and that the N-terminus of Cypridina luciferase is the 12th tyrosine (see FIG. 3). Furthermore, the relationship between alanine and adjacent amino acid residues is adapted to the cleavage site in the signal sequence [von Heijne, G. et al., Eur. J. Biochem. 133 , 17-2.
1, 1983]. From the above, it is expected that the cleavage site of the peptide is at position 11 and the amino terminus is tyrosine.

Another feature of the amino acid sequence of Cypridina luciferase is that a highly cysteine-rich region is present in the N-terminal portion. In this region, amino acid residues 39 to
Nine cysteine residues are found between 82. However, since no free sulfhydryl group was detected in Cypridina luciferase (Tsuji et al., Supra),
The cysteine residue probably forms an intramolecular disulfide bridge.

Example 2 Construction of Expression Vector Containing Cypridina Luciferase An expression vector was constructed by placing the full-length Cypridina luciferase cDNA under the promoter of Rous sarcoma virus long terminal repeat. That is, the 5 'and 3' ends of the sea firefly luciferase cDNA, were ligated their <br/> respectively Hin d III and Bgl II linker (Takara Shuzo). Plasmid pRSVL encoding firefly luciferase obtained from Dr. S. Subramani [deWet, J. et al.
R.), et al., Mol.Cell.Biol. 7, 725-737, 1987 ] the Sma I
And ligated with Bgl II linker. Then, Hin d
A linear plasmid obtained by cutting with III and Bgl II, by coupling with Hin d III and Bgl II fragments of the above cDNA, Hin d III- Bgl II containing sea firefly luciferase cDNA instead of firefly luciferase cDNA An expression plasmid pRSVVL containing the fragment was obtained. Escherichia coli pRSVVL transformed with this plasmid has been deposited with the Research Institute of Microbial Industry and Technology (Department date: June 15, 1989, accession number: FERMP-10782).

Example 3 Transfection of COS Cells with Plasmid pRSVVL and Expression of Cypridina Luciferase 1. Transfection 10% fetal calf serum (Hyclon) in a 10 cm Petri dish
Monkey COS cells [Gluzma (Gluzma) in 10 ml of Dulbecco's modified Eagle's medium (Nissui) containing e)
n, Y., Cell 23 , 175-182, 1981: ATCC CRL 1650) (7 × 10 ⁶ ). The cells were subjected to the calcium phosphate method [Graham, FL, et al., Virology 52 , 456-467, 19].
73 and Wigler (M) et al., Cell 14 , 725-73.
1, 1978] and the plasmid pRSVV prepared in Example 2.
Transfected with 10 μg of L DNA. After incubation for 48 hours, the medium was collected and the cells were harvested. The cell extract was then prepared by repeatedly freezing and thawing the cells, followed by centrifugation (deWet, JR et al.,
Supra).

2. Measurement of Cypridina luciferase activity The Cypridina luciferase activity of the cell extract and the medium prepared in 1. was measured using Cypridina luciferin as a substrate.

In a scintillation vial having a capacity of 20 ml, the medium or the cell extract obtained in the above 1. was added to 200 mM Tris-HCl (pH 7.
6) Dilute to a total volume of 1.5 ml with Mitchell-Hastin
Mitchell, GW, et al., A in gs photometer
nal. Biochem. 39 , 243-250, 1971 ". On the other hand, Cypridina luciferin prepared according to the azalea method [Methods Enzymol. ( 57 , 364-372, 1978)] was dissolved (at a concentration of 50 nM) in a 200 mM sodium phosphate buffer (pH 6.8), and the 1.5
ml was injected into scintillation vials. Immediately before the injection of luciferin, the shutter of the photometer was opened, the injection point was set to 0 min, and after 1.5 min, the shutter was closed, and the light emission during that time was recorded.

^The luminous intensity was measured using ¹⁴ C-hexadecane light as a standard, and the luminous intensity was converted into photons per second [Hasting (Hast
ings JW) et al., J. Opt. Soc. Am. 53 , 1410-1415, 196
3]. As a result, slight luciferase activity was detected in the cell extract of the transfected COS cells. However, a large amount of luciferase activity was detected in the culture medium of COS cells (see FIG. 4). The luminescence from this culture medium was directly proportional to the volume of the test medium, and the luminescence was extremely sharp. That is, a signal clearly higher than the background was detected from a sample of only 5 μl obtained from 10 ml of the culture medium of COS cells transfected with the Cypridina luciferase expression vector. In contrast, DN
Culture medium of COS cells not transfected with A or pSVOCAT [Gorman, CM, et al., Mol. Cell. Bio.
2 , 1044-1051, 1982] or pRSVL [deWet, JR, et al., Mol. Cell. Biol. 7 , 725-737, 19].
87], the luminescence of the COS cells transfected in the medium and the cell extract was at least two orders of magnitude lower than the luminescence described in FIG.

The above results indicate that the reconstructed cDNA is a full-length sea firefly luciferase cDNA, and that the cDNA also encodes a signal sequence necessary for secreting the protein.

[Action] DNA encoding sea firefly luciferase of the present invention
The compound can be used to express firefly luciferase in the desired host. The chemiluminescence reaction between Cypridina luciferase and Cypridina luciferin is a simple reaction requiring only oxygen molecules. However, the substrate specificity is high, and the luminescence measurement sensitivity is sharp. Moreover, as shown in the Examples, COS cells transfected with the expression vector of the present invention secrete Cypridina luciferase into the culture medium, so that the product can be recovered extremely easily. These facts clearly demonstrate the usefulness of the DNA compound encoding sea urchin luciferase of the present invention. Therefore, the DNA compound of the present invention is considered to be useful not only in the field of biomedicine but also in various fields including the environment.

[Brief description of the drawings]

FIG. 1 is a schematic drawing of a photograph showing the results of purified sodium firefly luciferase subjected to sodium dodecyl sulfate / gradient polyacrylamide gel electrophoresis. FIG. 2 is a restriction map of Cypridina luciferase cDNA and a restriction map of a clone used for construction of a full-length clone.
(A) is full-length sea firefly luciferase cDNA,
(B) is a restriction map showing the restriction enzyme sites of clone VL16 and (c), and (d) is a restriction map showing the restriction enzyme sites of clone VL18.
It is a restriction map of the full length cDNA constructed from 16 and VL18. FIG. 3 is a schematic diagram showing the nucleotide sequence and deduced amino acid sequence of Cypridina luciferase cDNA. FIG. 4 is a graph showing the results of measuring the activity of Cypridina luciferase synthesized and secreted by COS cells. FIG. 5 shows sea firefly luciferase (a)
FIG. 4 is a schematic diagram showing the homology between the amino acid sequences of aequorin (b) and jellyfish.

Continued on the front page (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/53 C12N 9/02 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL (Senez eq)

Claims (5)

(57) [Claims] 1. A DNA compound encoding sea urchin luciferase represented by the amino acid sequence from the 1st to the 555th amino acid in FIG. 2. The DNA compound according to claim 1, which is represented by the nucleotide sequence from the 16th position to the 1680th position in FIG. [3] An expression vector containing the DNA compound according to [1] or [2]. 4. The expression vector according to claim 3, which is a plasmid pRSVVL. 5. A method for producing sea firefly luciferase, comprising transfecting a host cell with the expression vector according to claim 3 or 4, and culturing the host cell.