JP2022139948A - Mutant Beetle Luciferase, Gene, Recombinant Vector, Transformant, and Method for Producing Mutant Beetle Luciferase - Google Patents

Abstract

To provide a mutant beetle luciferase that is less susceptible to luminescence inhibition by NaCl.SOLUTION: Provided is a mutant beetle luciferase that has a mutation in which the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is valine, or a mutation in which the amino acid corresponding to aspartic acid at position 533 is valine, in the amino acid sequence encoding wild-type beetle luciferase, the luminescence intensity of the luciferin-luciferase luminescence reaction in 0.9 mass% of a NaCl solution being 50% or more of that in a NaCl-free solution.SELECTED DRAWING: None

Description

The present invention relates to mutant beetle luciferase, genes, recombinant vectors, transformants, and methods for producing mutant beetle luciferase.

Beetle luciferase is an enzyme that catalyzes the oxidation of firefly luciferin in the presence of adenosine triphosphate (ATP), magnesium ions and oxygen, causing it to emit light. Therefore, beetle luciferase and luciferin-luciferase luminescence reaction using the same are widely used in tests aimed at detecting microorganisms in specimens using ATP as an index, such as microbial contamination ATP tests and endotoxin luminescence tests.

To increase the utility of beetle luciferase in the detection of ATP, various mutant beetle luciferases have been generated. Such mutant beetle luciferases include those with improved thermostability (see, for example, Patent Document 1), those with improved substrate affinity (see, for example, Patent Document 2), and those with altered emission wavelengths (see, for example, , See Patent Document 3), those with improved luminescence sustainability (see, for example, Patent Document 4), those with surfactant resistance (see, for example, Patent Document 5), those with improved luminescence intensity (see, for example, Patent Reference 6) and the like are known.

The enzymatic reaction of the wild-type beetle luciferase is inhibited in an aqueous sodium chloride solution of about 0.9% by mass, and the luminescence intensity is reduced to about 20 to 50% of that in an aqueous solution containing no sodium chloride. . For this reason, when performing luciferin-luciferase luminescence reaction using these beetle luciferases on specimens containing sodium chloride at the level of physiological saline such as dialysate and infusion, luminescence inhibition by sodium chloride causes The measurement sensitivity of ATP detection is lowered. Therefore, a mutant beetle luciferase (Patent Document 7), which is less susceptible to luminescence inhibition by sodium chloride and is suitable for detecting ATP in dialysates, infusions, etc., has also been developed.

Japanese Patent No. 3048466 Japanese Patent Publication No. 2001-518799 Japanese Patent No. 2666561 JP-A-2000-197484 JP-A-11-239493 JP 2007-97577 A JP 2017-225372 A

An object of the present invention is to provide a novel mutant beetle luciferase that is less susceptible to luminescence inhibition by sodium chloride than wild-type beetle luciferase.

The present inventors have made intensive studies to achieve the above object, and found that by substituting an amino acid at a specific position in the amino acid sequence of the wild-type Heike firefly luciferase with a specific different amino acid, the effect of luminescence inhibition by sodium chloride is suppressed. The present inventors have found that it is possible to achieve the present invention.

That is, the mutant beetle luciferase, gene, recombinant vector, transformant, and method for producing the mutant beetle luciferase according to the present invention are the following [1] to [7].
[1] A mutant beetle luciferase in which a mutation is introduced into a wild-type beetle luciferase,
The amino acid sequence encoding the wild-type beetle luciferase has at least one mutation selected from the group consisting of (a) and (b) below,
A mutant beetle luciferase, wherein the luminescence intensity of luciferin-luciferase luminescence reaction in a 0.9% by mass sodium chloride solution is 50% or more of the luminescence intensity in a sodium chloride-free solution.
(a) a mutation in which the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is valine;
(b) A mutation in which the amino acid corresponding to aspartic acid at position 533 in the wild-type Heike firefly luciferase amino acid sequence is valine.
[2] The mutant beetle luciferase of [1] above, wherein the wild-type beetle luciferase is a wild-type Heike firefly luciferase.
[3] The mutant beetle luciferase of [1] or [2], wherein the luminescence intensity of the luciferin-luciferase luminescence reaction in a 0.9% by mass sodium chloride solution is higher than the luminescence intensity of the wild-type beetle luciferase.
[4] A gene encoding the mutant beetle luciferase of any one of [1] to [3].
[5] A recombinant vector containing the gene of [4] above.
[6] A transformant carrying the recombinant vector of [5] above.
[7] A mutant having a culturing step of culturing the transformant of [6] to obtain a culture, and a collecting step of collecting the mutant beetle luciferase from the culture obtained by the culturing step. A method for producing a beetle luciferase.

INDUSTRIAL APPLICABILITY According to the present invention, mutant beetle luciferase is provided that is less susceptible to luminescence inhibition by sodium chloride than wild-type beetle luciferase. By carrying out the luciferin-luciferase luminescence reaction using the mutant beetle luciferase, ATP detection in a specimen whose sodium chloride concentration is equivalent to that of physiological saline can be performed with high sensitivity.
In addition, the mutant beetle luciferase of the present invention can be efficiently produced by using the gene, recombinant vector, transformant, and mutant beetle luciferase production method of the present invention.

In the present invention and the specification of the present application, the “luminescence intensity” of beetle luciferase means, unless otherwise specified, the peak luminescence intensity in the luciferin-luciferase luminescence reaction, that is, the beetle luciferase, ATP, divalent metal ions and oxygen. means the peak luminescence intensity when reacted with firefly luciferin in the presence of It can be judged that the higher the "luminescence intensity", the higher the luciferase activity of the beetle luciferase.

In the present specification, the "residual activity (%)" of beetle luciferase is the relative luminescence intensity in a 0.9% by mass sodium chloride solution when the luminescence intensity in a sodium chloride-free solution is 100%. value ([luminescence intensity in 0.9% by weight sodium chloride solution]/[luminescence intensity in sodium chloride-free solution]×100) (%). In addition, the fact that the reaction solution for the luciferin-luciferase luminescence reaction is a “sodium chloride-free solution” means that the reaction solution is prepared without adding sodium chloride. The "sodium chloride-free solution" and the "0.9% by mass sodium chloride solution" in performing the luciferin-luciferase luminescence reaction for calculating the residual activity are all the same in composition other than sodium chloride, and the reaction temperature is and the reaction conditions such as time.

[Mutant beetle luciferase]
The mutant beetle luciferase according to the present invention is a mutant beetle luciferase in which a mutation is introduced into the wild-type beetle luciferase, and the luminescence intensity in a 0.9% by mass sodium chloride solution is Mutant beetle luciferase with 50% or more of luminescence intensity. The luciferase activity of the mutant beetle luciferase according to the present invention may have a residual activity of 50% or more, preferably 60% or more, more preferably 70% or more, and 80% or more. More preferably, it is more preferably 90% or more.

Like wild-type beetle luciferase, the mutant beetle luciferase according to the present invention can be used in luciferin-luciferase luminescence reaction, and is less susceptible to luminescence inhibition by sodium chloride. It is particularly suitable as a beetle luciferase to be used when performing a luciferin-luciferase luminescence reaction. For example, when performing a luciferin-luciferase luminescence reaction for measuring the amount of ATP for the purpose of detecting microorganisms in specimens collected from animals such as humans, or infusions or dialysates administered to humans, By using the mutant beetle luciferase according to the present invention, ATP can be detected with higher sensitivity.

The mutant beetle luciferase according to the present invention has at least one mutation selected from the group consisting of (a) and (b) below in the amino acid sequence encoding the wild-type beetle luciferase.
(a) A mutation in which the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is valine (b) A mutation in which the amino acid corresponding to aspartic acid at position 533 in the amino acid sequence of wild-type Heike firefly luciferase is valine

In the present invention and herein, the term "wild-type beetle luciferase" refers to North American firefly (Photinus pyralis) luciferase (SEQ ID NO: 1), Heike firefly (Luciola lateralis) luciferase (SEQ ID NO: 2), Genji firefly (Luciola cruciata) luciferase (SEQ ID NO: 2) No. 3), East European firefly (Luciola mingrelica) luciferase, firefly (Lampyris noctiluca) luciferase, click beetle (Pyrophorus plagiophthalamus) luciferase and the like. Amino acid sequences of various wild-type beetle luciferases can be retrieved from databases (eg, EMBL-EBI Database (http://www.ebi.ac.uk/queries/)).

If the wild-type beetle luciferase is not Heike firefly luciferase, the "amino acid corresponding to the amino acid at position X in the amino acid sequence of Heike firefly luciferase" in the amino acid sequence of the wild-type beetle luciferase can be obtained using homology analysis software for amino acid sequences (e.g., Micro Genie (registered trademark) (manufactured by Beckman Coulter)) and the like are used to align the amino acid sequences of the wild-type beetle luciferase and Heike firefly luciferase so that the homology is maximized. means an amino acid at a position corresponding to "the amino acid of".

Specifically, the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is phenylalanine at position 292 in the amino acid sequence of wild-type North American firefly luciferase, and phenylalanine at position 294 in the amino acid sequence of wild-type Genji firefly luciferase. correspond respectively.
The aspartic acid at position 533 in the amino acid sequence of wild-type Heike firefly luciferase corresponds to the aspartic acid at position 531 in the amino acid sequence of wild-type North American firefly luciferase and the aspartic acid at position 533 in the amino acid sequence of wild-type Genji firefly luciferase, respectively.

The mutant beetle luciferase according to the present invention may have only one of the mutations (a) and (b), or may have two of them. Among them, since the luminescence intensity of the luciferin-luciferase luminescence reaction in a 0.9% by mass sodium chloride solution is greater than the luminescence intensity of wild-type beetle luciferase, both mutations (a) and (b) Mutant beetle luciferases with

The mutant beetle luciferase according to the present invention includes, for example, a mutant beetle luciferase in which the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is substituted with valine, and position 533 in the amino acid sequence of wild-type Heike firefly luciferase. The amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of the mutant beetle luciferase in which the amino acid corresponding to aspartic acid is substituted with valine, and the amino acid corresponding to phenylalanine at position 533 in the amino acid sequence of wild-type Heike firefly luciferase is replaced with valine. , respectively substituted mutant beetle luciferases.

The mutant beetle luciferase according to the present invention has improved salt tolerance by having at least one of the mutations (a) and (b), and the luciferase activity is higher than that of the wild-type beetle luciferase. Not easily inhibited by sodium. It is not clear why these specific mutations improve salt tolerance, but the introduction of amino acid substitutions at these specific positions that alter the molecular size and charge of the beetle luciferase enzymatic reaction It is presumed that this is because the three-dimensional structure in the vicinity changed and the entry of sodium ions, which inhibits the oxidation reaction of luciferin, into the reaction site was suppressed.

The mutant beetle luciferase according to the present invention may have the same amino acid sequence as the wild-type beetle luciferase, except for the mutations (a) and (b) above, and the amino acid sequence of the wild-type beetle luciferase It may have mutations other than mutations such as (a). The other mutations are not particularly limited as long as they do not impair the luciferase activity and the effect of improving salt tolerance due to the mutation (a) or the like. For example, the mutations described in Patent Documents 1 to 7 etc.

Examples of mutant beetle luciferases according to the present invention include proteins (1) to (3) below.
(1) A protein consisting of an amino acid sequence in which one or more mutations selected from the group consisting of (a) and (b) have been introduced into the amino acid sequence of wild-type beetle luciferase.
(2) The amino acid sequence of the wild-type beetle luciferase has one or more mutations selected from the group consisting of (a) and (b) and mutations that delete, substitute, or add one or more amino acids. A protein consisting of an amino acid sequence and having luciferase activity.
(3) consists of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence of a wild-type beetle luciferase and having one or more mutations selected from the group consisting of (a) and (b); A protein with luciferase activity.

In the protein (2), the number of amino acids deleted, substituted, or added to the amino acid sequence of the wild-type beetle luciferase other than the mutations (a) and (b) is preferably 1 to 20, 1 to 10 are more preferred, and 1 to 5 are even more preferred.

In the protein (3), the sequence identity with the wild-type beetle luciferase amino acid sequence is not particularly limited as long as it is 80% or more and less than 100%, but it is preferably 85% or more and less than 100%, and 90% It is more preferably 95% or more and less than 100%, and particularly preferably 98% or more and less than 100%.

The sequence identity (homology) between amino acid sequences is determined by aligning the two amino acid sequences so that the corresponding amino acids match the most, while inserting gaps in the portions corresponding to insertions and deletions. It is calculated as the ratio of matching amino acids to the entire amino acid sequence excluding gaps in the middle. Sequence identity between amino acid sequences can be determined using various homology search software known in the art.

In the mutant beetle luciferase according to the present invention, various tags may be added to the N-terminus and C-terminus of the region having luciferase activity. As the tag, for example, a histidine tag, a hemagglutinin (HA) tag, a Myc tag, and a Flag tag, which are widely used in the expression and purification of recombinant proteins, can be used.

[Mutant beetle luciferase gene]
A gene encoding a mutant beetle luciferase (mutant beetle luciferase gene) according to the present invention can be obtained by appropriately modifying a wild-type beetle luciferase gene. The mutant beetle luciferase gene according to the present invention may be one in which codons encoding corresponding amino acids are modified so that mutations are introduced into the wild-type beetle luciferase gene, and degenerate codons are used by the host codons. It may be modified to have a high frequency.

In the present invention and herein, a "gene" is a polynucleotide consisting of DNA or RNA and encoding a protein. In addition, genetic modification can be performed by methods well known to those skilled in the art, such as site-directed mutagenesis, random mutagenesis, and organic synthesis.

Site-directed mutagenesis or random mutagenesis is performed using a wild-type beetle luciferase gene or a recombinant vector containing this as a template. A wild-type beetle luciferase gene or a recombinant vector containing it can be obtained by a method known to those skilled in the art (e.g., "Genetic Engineering Experimental Note" (Yodosha), JP-A-1-51086, JP-A-3048466, etc.). can be prepared by the method described in ). Moreover, you may use a commercial thing.

Site-directed mutagenesis can be carried out by methods well known to those skilled in the art, such as synthesis with T4 DNA polymerase using selection primers and mutagenesis primers. When site-directed mutagenesis is performed using a recombinant vector containing a wild-type beetle luciferase gene as a template and selection primers and mutagenesis primers are used, for example, restriction enzyme recognition sequences present in the recombinant vector When a DNA fragment containing a sequence with a different base is used as a selection primer, the restriction enzyme recognition sequence is present as it is in the recombinant vector into which the mutation was not introduced. Unintroduced recombinant vectors can be selected out.

Random mutagenesis is performed, for example, by adding manganese and dGTP to reduce fidelity, polymerase chain reaction (PCR), agents (hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, etc.) It can be carried out by methods well known to those skilled in the art, such as a method of contact and a method of irradiating with ultraviolet rays. When random mutagenesis is performed, the target mutant beetle luciferase gene or a recombinant vector containing the same can be selected by determining the nucleotide sequence of the mutagenized gene.

Determination of the nucleotide sequence of the mutated gene can be performed by methods well known to those skilled in the art, such as the dideoxy method. Nucleotide sequences of various wild-type beetle luciferase genes (cDNA) can be searched from databases (eg, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)).

[Recombinant vector]
The recombinant vector of the present invention contains the mutant beetle luciferase gene of the present invention.

The recombinant vector can be obtained by inserting the mutant beetle luciferase gene of the present invention into a vector replicable in host cells, such as a plasmid or bacteriophage, according to methods well known to those skilled in the art. Insertion of the mutant beetle luciferase gene into a vector is performed by digesting a DNA fragment in which an appropriate restriction enzyme recognition sequence is added to the mutant beetle luciferase gene with the corresponding restriction enzyme, and inserting the resulting gene fragment into the vector corresponding to the vector. It can be performed by inserting it into a restriction enzyme recognition sequence or a multicloning site and ligating it to a vector.

The recombinant vector can also be obtained by introducing a mutation into a recombinant vector containing a wild-type beetle luciferase gene, as described above.

Plasmids include Escherichia coli-derived plasmids (pET-28a(+), pGL2, pBR322, pUC18, pTrcHis, pBlueBacHis, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50 , pYE52, etc.), and examples of bacteriophage include λ phage.

The recombinant vector according to the present invention is preferably a vector that, when introduced into a host cell, can express the mutant beetle luciferase using the expression system of the host cell. For this reason, the recombinant vector of the present invention preferably incorporates an expression cassette in which an appropriate promoter that functions in host cells is placed upstream of the mutant beetle luciferase gene of the present invention. An enhancer, terminator, splicing signal, poly A addition signal, ribosome binding sequence (SD sequence), etc. can be placed in the expression cassette, if necessary.

In the mutant beetle luciferase expression cassette, the promoter sequence located upstream of the mutant beetle luciferase gene may be a promoter derived from the same species as the wild-type beetle luciferase gene before mutation is introduced, or a heterologous promoter sequence. It may be a promoter derived from the species of the organism, or an artificially synthesized promoter. For example, when Escherichia coli is used as a host cell, promoters that control the expression of the mutant beetle luciferase gene include trp promoter, lac promoter, T7 promoter, PL promoter, PR promoter and the like.

The recombinant vector according to the present invention preferably contains, in addition to the mutant beetle luciferase expression cassette, a selection marker gene for selecting transformed cells from untransformed cells. . Examples of the selectable marker gene include drug resistance genes such as kanamycin resistance gene, hygromycin resistance gene and bialaphos resistance gene.

By using the mutant beetle luciferase gene according to the present invention as a reporter gene, the reporter assay can be performed with sufficiently high sensitivity. Such a reporter assay is made possible by the recombinant vector of the present invention containing the mutant beetle luciferase gene.

[Transformant]
A transformant according to the present invention carries a recombinant vector according to the present invention.

A transformant according to the present invention can be obtained by introducing the above recombinant vector into a host cell according to a method well known to those skilled in the art. Introduction of the recombinant vector into host cells can be performed by methods well known to those skilled in the art, such as the calcium chloride method, electroporation method, polyethylene glycol method, lipofection method, and particle gun method.

Examples of host cells include bacteria such as Escherichia coli and Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, filamentous fungi such as Aspergillus, Sf9 cells, Sf21 cells, and the like. mammalian cells such as insect cells, COS cells, CHO cells, and the like; As the transformant according to the present invention, a transformant obtained using E. coli as a host is preferable because it grows quickly and is easy to handle.

[Method for producing mutant beetle luciferase]
A method for producing a mutant beetle luciferase according to the present invention comprises a culturing step of culturing a transformant according to the present invention to obtain a culture, and recovering a mutant beetle luciferase from the culture obtained by the culturing step. and a recovery step. The mutant beetle luciferase according to the present invention can be obtained by the production method.

The culturing step is a step of culturing the transformant according to the present invention to obtain a culture. Here, the "culture" may be any of culture supernatant, cultured cells, and cell lysates.

The transformant can be cultured by a method well known to those skilled in the art. For example, when the host cell is a microorganism such as Escherichia coli or yeast, the medium used for culturing the transformant includes a carbon source (glucose, sucrose, lactose, etc.), a nitrogen source (peptone, meat extract, etc.) that can be assimilated by the microorganism. , yeast extract, etc.), inorganic salts (phosphates, carbonates, sulfates, etc.), etc., and can be either a natural medium or a synthetic medium as long as it is a medium that can efficiently culture host cells. , and may be either a liquid medium or a solid medium. Which of shaking culture, agitation culture, static culture, etc. is performed, and other culture conditions (culture temperature, medium pH, culture time, etc.) can be appropriately determined according to the host cells, medium, etc. used. . For example, when the host cell is E. coli, the culture temperature is usually 30-42°C, preferably 37°C. The pH of the medium is usually 6.4-8.0, preferably 7.0-7.4. When the culture temperature is 37° C., the culture time is usually 8 to 20 hours, preferably 12 to 16 hours, for the preculture, and 2 to 8 hours, preferably 2 to 4 hours, for the main culture before induction of expression. be. However, the optimal culture time is determined according to the culture temperature and medium pH.

An expression inducer can be added to the medium, if necessary. Examples of such an expression inducer include isopropyl β-thiogalactoside (IPTG) when the recombinant vector contains the lac promoter, and indole acrylic acid (IAA) when the recombinant vector contains the trp promoter. are mentioned.

When the recombinant vector is prepared using a vector having resistance to antibiotics (kanamycin, ampicillin, etc.), the antibiotic resistance is added to the medium to make the selection marker of the transformant can be used as

The recovery step is a step of recovering the mutant beetle luciferase according to the present invention from the culture obtained in the culture step. The mutant beetle luciferase is obtained by a method well known to those skilled in the art, for example, by centrifugation, transformants are recovered from the culture, and freeze-thaw treatment, ultrasonic disruption treatment, or treatment with a lytic enzyme such as lysozyme is performed. can be recovered from the transformant. The mutant beetle luciferase may be recovered in the form of a solution.

In the production method, a purification step of purifying the mutant beetle luciferase (crude enzyme) obtained in the collection step may be further performed after the collection step. The crude enzyme can be purified by, for example, ammonium sulfate precipitation, SDS-PAGE, gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like, either alone or in combination.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
A mutant Heike firefly luciferase (mutant (F294V)) in which phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is substituted with valine, and a mutation in which aspartic acid at position 533 in the amino acid sequence of wild-type Heike firefly luciferase is substituted with valine Production of Heike firefly luciferase (mutant (D533V)) and wild type Heike firefly luciferase (mutant (F294V+D533V)) in which both phenylalanine at position 294 and aspartic acid at position 533 in the amino acid sequence of wild-type Heike firefly luciferase are substituted with valine. and measured its luciferase activity (luminescence intensity).
For comparison, a mutant Heike firefly luciferase (mutant (mutant ( 290I+378P+490V)) was prepared and its luciferase activity (luminescence intensity) was measured.

<Preparation of Recombinant Vector Containing Mutant Heike Firefly Luciferase Gene>
First, using the wild-type Heike firefly luciferase gene (cDNA) (SEQ ID NO: 4) as a template, a primer containing a restriction enzyme NcoI recognition sequence, a primer containing a restriction enzyme XhoI recognition sequence, and TITANIUM Taq DNA polymerase (manufactured by Clontech) were used. A DNA fragment containing a wild-type Heike firefly luciferase gene having restriction enzyme recognition sequences added to both ends was obtained. The nucleotide sequence of the obtained DNA fragment was determined using a DTCS Quick Start Master Mix kit and electrophoresis analyzer CEQ8000 (both manufactured by Beckman Coulter). The DNA fragment was cleaved with restriction enzymes NcoI and XhoI, and a plasmid (pET-28a(+) plasmid DNA (manufactured by Novagen) preliminarily cleaved with NcoI and XhoI was added to DNA Ligation Kit (manufactured by BioDynamics Laboratory). to produce a recombinant vector for expressing wild-type Heike firefly luciferase with a histidine tag added to the C-terminal side. In addition, pET-28a(+) contains a T7 promoter and a T7 terminator, and contains a gene encoding a histidine tag near the cloning site so that the histidine tag is added to the C-terminal side of the target protein to be expressed. is a vector that

Next, using the resulting recombinant vector as a template, site-directed mutagenesis was performed using a Transformer site-directed mutagenesis kit (manufactured by Clontech). As the selection primer, a primer containing a nucleotide sequence different by one nucleotide from the restriction enzyme FspI recognition sequence in pET-28a(+) was used. The selection primer and the mutagenesis primer for obtaining the gene of each mutant were previously phosphorylated at the 5' end with T4 polynucleotide kinase (manufactured by TOYOBO).

Recombinant plasmids were synthesized using T4 DNA polymerase and T4 DNA ligase provided with the Transformer site-directed mutagenesis kit. After the reaction product after ligase reaction was cleaved with restriction enzyme FspI, the recombinant plasmid that was not cleaved with FspI was introduced into E. coli mismatch repair deficient strain BMH71-18mutS, and E. coli was cultured. The resulting recombinant plasmid was further cleaved with FspI, and a recombinant plasmid not cleaved with FspI was selected as a mutated recombinant vector.

The nucleotide sequence of the mutant Heike firefly luciferase gene (DNA) in the recombinant vector into which the mutation was introduced was determined using a DTCS Quick Start Master Mix kit and electrophoresis analyzer CEQ8000 (both manufactured by Beckman Coulter). This produced a recombinant vector into which a gene encoding the desired mutant was integrated. The nucleotide sequence encoding the mutant (F294V) is SEQ ID NO: 5, the nucleotide sequence encoding the mutant (D533V) is SEQ ID NO: 6, the nucleotide sequence encoding the mutant (F294V + D533V) is SEQ ID NO: 7, and the mutant The base sequences encoding (290I+378P+490V) are shown in SEQ ID NO: 8, respectively.

<Preparation of transformed E. coli>
A recombinant vector containing each mutant Heike firefly luciferase gene was introduced into Escherichia coli HMS174 (DE3) strain (manufactured by Novagen) in which the T7 RNA polymerase gene was integrated in the genomic DNA by the calcium chloride method. , and plated on selective agar medium containing 30 μg/mL kanamycin to select for transformed E. coli.

<Collection and purification of mutant Heike firefly luciferase>
Kanamycin-selected transformed E. coli was incubated with a shaker (Takasaki Kagaku Kikai Co., Ltd.) at 37° C. for 2.5 hours in 200 mL of 2×YT medium (containing 30 μg/mL kanamycin). After shaking culture, 200 μL of 100 mM IPTG was added so that the IPTG concentration in the medium was 0.1 mM, and expression was induced at 25° C. for 6 hours. IPTG is an expression inducer that releases the suppression of expression by the lac repressor and induces T7 RNA polymerase.

E. coli cells were collected by centrifuging the culture at 8000 rpm for 5 minutes, frozen at -20°C and stored. Frozen cells were thawed in 5 mL of binding buffer (20 mM NaH ₂ PO ₄ (pH 7.4) containing 500 mM NaCl and 20 mM imidazole), suspended, and sonicated. The resulting cell lysate was centrifuged at 9000 rpm for 30 minutes, and the supernatant was recovered as a solution of mutant Heike firefly luciferase (crude enzyme).

Since a histidine tag was added to the C-terminal side of the expressed mutant Heike firefly luciferase, the crude enzyme was purified using nickel chelate affinity chromatography. First, 0.5 mL of Ni Sepharose 6 Fast Flow (manufactured by Amersham Biosciences) was packed in a column (Disposable Polystyrene Column manufactured by PIERCE) and equilibrated with a binding buffer. Next, 5 mL of the crude enzyme solution was added to the column, washed with binding buffer, and 2.5 mL of elution buffer (20 mM NaH ₂ PO ₄ (pH 7.4) containing 500 mM NaCl and 500 mM imidazole) was used to extract mutant Heike firefly luciferase. eluted. Furthermore, using a PD-10 Desalting column (manufactured by Amersham Biosciences), the elution buffer was replaced with 3.5 mL of a reaction buffer (50 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl ₂ ). Thus, purified mutant Heike firefly luciferase was obtained.

<Measurement of luminescence intensity of mutant Heike firefly luciferase>
Protein quantification of the obtained mutant Heike firefly luciferase was performed using Bio-Rad Protein Assay (manufactured by BIORAD) based on the Bradford method using IgG as a standard. After adding 50 μL of a reaction buffer containing mutant Heike firefly luciferase (20 μg/mL) to a 96-well plate (Luminunc plate manufactured by Nunc), an injector attached to a microplate reader (ARVO MX manufactured by Perkin-Elmer) was used. containing sodium chloride-containing substrate buffer (1.8% by mass sodium chloride, 2×10 ⁻⁶ M D-firefly luciferin (manufactured by Wako Pure Chemical Industries, Ltd.), 2×10 ⁻⁷ M ATP, and 10 mM MgCl ₂ 50 mM Tris-HCl buffer (pH 7.4)) 50 μL or sodium chloride-free substrate buffer (2×10 ⁻⁶ M D-firefly luciferin (manufactured by Wako Pure Chemical Industries, Ltd.), 2×10 ⁻⁷ M ATP, and 10 mM 50 μL of 50 mM Tris-HCl buffer (pH 7.4) containing MgCl ₂ was added. Luminescence intensity was then measured with the microplate reader. The final concentration of sodium chloride in the reaction solution to which the sodium chloride-containing substrate buffer was added was 0.9% by mass. The wild-type Heike firefly luciferase was prepared in the same manner as the mutant Heike firefly luciferase, except that a recombinant vector containing the wild-type Heike firefly luciferase gene (cDNA) was used instead of the recombinant vector containing the mutant Heike firefly luciferase gene. Then, transformed E. coli was prepared, the enzyme was collected and purified, and the luminescence intensity of the enzyme was measured.

The residual activity (%) was calculated from the measured luminescence intensity for the wild-type and each mutant. Table 1 shows the calculation results. In Table 1, the column "No NaCl" shows the measured value of the luminescence intensity of the reaction solution containing no sodium chloride, and the column "+0.9% NaCl" contains 0.9% by mass of sodium chloride. Measured values of luminescence intensity (Relative Light Unit; RLU) of the reacted solution are shown.

As a result, the wild-type had a very low residual activity of about 21% and was greatly inhibited by sodium chloride. In addition, in the mutant (290I + 378P + 490V) introduced with three known mutations, the luminescence intensity in the presence of 0.9% by mass of sodium chloride is stronger than that of the wild type, and the residual luciferase activity is 46%. It was confirmed that the salt tolerance was improved compared to the wild type. On the other hand, the mutant (F294V) and the mutant (D533V) had residual activities of 66% and 55%, respectively, even though only one mutation was introduced. also improved salt tolerance. In addition, the mutant (F294V+D533V) in which these two mutations were introduced together had a high residual activity of 84% and was extremely excellent in salt tolerance.

Claims (7)

A mutant beetle luciferase in which a mutation is introduced into a wild-type beetle luciferase,
The amino acid sequence encoding the wild-type beetle luciferase has at least one mutation selected from the group consisting of (a) and (b) below,
A mutant beetle luciferase, wherein the luminescence intensity of luciferin-luciferase luminescence reaction in a 0.9% by mass sodium chloride solution is 50% or more of the luminescence intensity in a sodium chloride-free solution.
(a) a mutation in which the amino acid corresponding to phenylalanine at position 294 in the amino acid sequence of wild-type Heike firefly luciferase is valine;
(b) A mutation in which the amino acid corresponding to aspartic acid at position 533 in the wild-type Heike firefly luciferase amino acid sequence is valine. 2. The mutant beetle luciferase of claim 1, wherein the wild-type beetle luciferase is wild-type Heike firefly luciferase. 3. The mutant beetle luciferase according to claim 1 or 2, wherein the luminescence intensity of the luciferin-luciferase luminescence reaction in a 0.9% by mass sodium chloride solution is higher than that of the wild-type beetle luciferase. A gene encoding the mutant beetle luciferase according to any one of claims 1 to 3. A recombinant vector containing the gene according to claim 4 . A transformant carrying the recombinant vector according to claim 5 . a culturing step of culturing the transformant according to claim 6 to obtain a culture;
A recovery step of recovering the mutant beetle luciferase from the culture obtained by the culture step;
A method for producing a mutant beetle luciferase.