JP5980608B2 - Firefly luciferase - Google Patents

Description

  The present invention relates to a firefly-derived luciferase.

  In order to examine cell functions such as intracellular signal transduction and gene expression, fluorescent probes such as fluorescent dyes and fluorescent proteins, and chemiluminescent probes using luciferin-luciferase reaction are used. In particular, analysis of gene expression regulation uses luminescence measurement with excellent quantitativeness, which does not cause cell damage due to excitation light and does not cause a problem of self-luminescence. For example, when observing a cell into which a luciferase gene has been introduced, the intensity of luciferase gene expression (specifically, the expression level) can be examined by measuring the intensity of luminescence from the cell due to luciferase activity. . Specifically, cells are first lysed to prepare a cell lysate, and then luciferin, adenosine triphosphate (ATP), etc. are added to the cell lysate, and light is emitted by a luminometer using a photomultiplier tube. The intensity is quantified, and the expression level is specified based on the result. In this method, the luminescence intensity is measured after lysing cells. For this reason, the expression level of the luciferase gene at a certain point in time is obtained as an average value of all cells used for the measurement. As a method for introducing a luminescent gene such as a luciferase gene as a reporter gene, for example, a calcium phosphate method, a lipofectin method, an electroporation method, or the like can be used, and each method is properly used depending on the purpose and the type of cell. By connecting the target DNA fragment upstream or downstream of the luciferase gene to be introduced into the cell and measuring the expression level of the luciferase gene, it becomes possible to examine the influence of the DNA fragment on the transcription of the luciferase gene. In addition, by co-expressing a luciferase gene to be introduced into a cell and a target gene, it becomes possible to examine the influence of the gene product on the expression of the luciferase gene.

  In order to analyze the expression level of a luminescent gene over time, it is necessary to measure the luminescence intensity from living cells over time. Such measurement is performed by culturing cells in an incubator equipped with a luminometer, and measuring the luminescence intensity from the entire cell population at regular intervals. By capturing changes with time in the expression level of the luminescent gene in the entire cell population, it is possible to analyze an expression rhythm having a certain periodicity.

  In recent years, in biological and medical research, there is an increasing need to dynamically capture life phenomena at the molecular level. In the research field using fluorescence observation, time lapse or moving image imaging is performed in order to dynamically grasp protein molecule functions in a sample. In the prior art, temporal observation using a fluorescent sample (for example, moving image observation of one protein molecule with a fluorescent molecule attached) is performed.

On the other hand, when a chemiluminescent sample is used for temporal observation, since the luminescence intensity of the chemiluminescent sample is extremely small, it is necessary to observe using a CCD camera equipped with an image intensifier. Recently, a microscope having an optical system for observing a chemiluminescent sample has also been developed (Patent Documents 1 and 2).
On the other hand, various luciferases derived from various biological species are known, but there is almost no luciferase whose wild type is red. However, red light emission is optically important, and there are merits of combining with other light emission colors or using light transmittance that can be obtained only in red. Up to now, in luciferases of various species, there have been attempts to obtain red mutants through trial and error, such as modification of luminescent substrates, addition of cofactors, modification of peptides and base sequences, etc., but red mutants The emission brightness was much lower.

JP 2006-301599 A International Publication No. 06/088109

  An object of the present invention is to provide a novel luciferase that emits luciferin.

  The luciferase according to one embodiment of the present invention is derived from a Pristrocus sagulatus.

  According to the present invention, a novel luciferase is provided.

It is a figure which shows the emission spectrum in each pH of the light emission induced | guided | derived by the wild-type scallop firefly luciferase when using D-luciferin as a substrate. It is the figure which plotted Km value of each luciferase. It is the figure which compared the emitted light intensity of the light emission induced by various luciferases when D-luciferin was used as a substrate. It is a figure which shows the emission spectrum of the light emission induced | guided | derived by the wild type scallop firefly luciferase when D-luciferin is used as a substrate. It is a figure which shows the luminescence spectrum of the light emission induced by the stripe red mutated red luciferase when D-luciferin is used as a substrate. It is a figure which shows the emission spectrum of the light emission induced by the red luciferase derived from a beetle (Click Beetle Red (CBR) Luciferase) when D-luciferin is used as a substrate. It is the figure which compared the emitted light intensity of the light emission induced by various luciferases when a near-infrared light emission luciferin analog is used as a substrate. It is a figure which shows the emission spectrum of the light emission induced by various luciferases when a near-infrared light emission luciferin analog is used as a substrate. It is a figure which shows the luminescence spectrum of the light emission induced | guided | derived by the stripe luciferase when D-luciferin and a near-infrared light emission luciferin analog are used as a substrate. It is the figure which compared the emitted light intensity of the light emission induced by various luciferases when D-luciferin was used as a substrate. It is a figure which shows the emission spectrum of the light emission induced by the red mutant luciferase when D-luciferin is used as a substrate.

  An embodiment of the present invention relates to a luciferase derived from Pristocycus sagulatus.

  “Luciferase” generally refers to an enzyme that catalyzes a chemical reaction in which luminescence occurs. A substance that is a substrate for the enzyme is called luciferin. Luminescence occurs when luciferin undergoes a chemical change by the catalytic action of luciferase in the presence of ATP. Currently, luciferases derived from fireflies and bacteria are obtained. The luciferase according to the embodiment is also synonymous with the luciferase defined as described above, but is a novel luciferase obtained for the first time from fireflies described later. For embodiments, luciferin refers to luciferin and its analogs that are substrates for firefly luciferase. Luciferin is a substance described as a substrate in WO2009 / 096197 and WO2010 / 106896, for example. Specifically, for example, dimethylaniline-monoene luciferin, dimethylaniline-diene luciferin, dimethylaniline luciferin, D-luciferin, and derivatives thereof.

  The luciferase according to the embodiment is derived from Pristocycus sagulatus. The black-tailed butterfly is a firefly that belongs to the genus Astragalus. In addition, the "luciferase derived from the white squirrel" as used herein means that the mutant luciferase derived from the wild luciferase is also included in addition to the wild luciferase possessed by the white squirrel.

  When a chemiluminescent sample with low emission intensity is imaged with a microscope, the exposure time required to capture a clear image becomes long. Such chemiluminescent samples limit the research applications that can be used. For example, if exposure time of 30 minutes is required due to low emission intensity, it is possible to shoot for 30 minutes over time, but it is not possible to shoot in a shorter time or even in real time. It is. Further, when acquiring an image, it is necessary to acquire and compare a plurality of images in order to focus on the luminescent cells, but when a long exposure time is required due to low emission intensity, 1 It takes time and effort to acquire only one image.

  The luciferase according to the embodiment can induce luminescence having a high luminescence intensity compared to luminescence by a known luciferase. Therefore, the luciferase according to the embodiment has a particularly advantageous effect when used as a reporter for protein imaging. That is, since the luciferase according to the embodiment can provide high luminescence intensity even in a small amount, it enables favorable detection of proteins with low expression levels. The luciferase can reduce the exposure time required for detection due to the high emission intensity. For this reason, if this luciferase is used as a reporter for temporal observation, the exposure time required for imaging can be shortened, and observation near real time becomes possible. That is, observation over time with high time resolution becomes possible.

  An example of the luciferase according to the embodiment is a luciferase comprising the amino acid sequence shown in SEQ ID NO: 1. The luciferase is a wild-type luciferase obtained from a white squirrel firefly. In this specification and claims, the luciferase is referred to as wild-type, wild-type luciferase, wild-type scallop luciferase, luciferase derived from wild-type suji globot or the like.

  FIG. 1 shows an emission spectrum of luciferin obtained when the wild-type scallop firefly luciferase according to the embodiment is used. As shown in this figure, the wild-type scallop luciferase according to the embodiment generates light having a maximum light emission wavelength of around 583 nm at pH 8.0. The maximum emission wavelengths are about 590 nm, about 600 nm, about 609 nm, about 614 nm, and about 613 nm at pH 7.5, pH 7.0, pH 6.5, pH 6.0, and pH 5.5, respectively. There is a tendency for the maximum emission wavelength to become longer. Here, the “maximum emission wavelength” means a wavelength at which the emission intensity is maximum in a spectrum obtained by measuring luminescence of luciferin induced by luciferase.

  The luciferase according to the embodiment is, for example, 1.5 times or more, 2.0 times or more, 2.5 times or more, 3.0 times or more, 3.0 times or more, 4 times or more of the luminescence intensity derived from Photinus pyralis. It can show an emission intensity of 0 times or more. The luciferase according to the embodiment is, for example, 2.0 times or more, 3.0 times or more, 4.0 times or more, 5.0 times or more, luminescence intensity by red beetle-derived red luciferase (CBR luciferase). The emission intensity can be 0 times or more, 7.0 times or more, 8.0 times or more, or 9.0 times or more.

FIG. 3 shows a graph comparing the luminescence intensity of various luciferases in HeLa cells when D-luciferin is used as a substrate. Here, P.I. The relative values are shown when the luminescence intensity by pyralis luciferase (Promega, Luc2 luciferase) is 1. From this figure, under measurement conditions in a specific mammalian cell, It can be seen that the emission intensity of 2.60 times the emission intensity achieved by pyralis luciferase is achieved. In addition, it can be seen that wild-type luciferase achieves 7.87 times the emission intensity achieved by CBR luciferase.
FIG. 7 is induced by each luciferase when a dimethylaniline-diene type luciferin (specifically, Akalumine (registered trademark), manufactured by Wako Pure Chemical Industries, Ltd.), which is a near infrared luciferin analog, is used as a substrate. The emission intensity of emitted light is shown. It can be seen that wild-type luciferase achieves a light emission intensity of about 3.1 times that of CBR luciferase.
3 and 7 show the luminescence intensity when D-luciferin and dimethylaniline-diene luciferin are used as substrates, respectively, in the luminescence reaction using the luciferase according to the embodiment. Other substrates can also be used. For example, the substrates described in WO2009 / 096197 and WO2010 / 106896 can be used. The substrate is, for example, dimethylaniline-monoene type luciferin, dimethylaniline type luciferin, derivatives thereof, dimethylaniline-diene type luciferin derivatives, and D-luciferin derivatives.

  The luciferase according to the embodiment can exhibit high degradation stability compared to known luciferases. “Degradation stability” means the difficulty of causing degradation of the luciferase in the environment where the luciferase is used. Further, “degradation stability” includes not only stability against degradation by proteolytic enzymes but also stability against degradation not involving a proteolytic enzyme, for example, degradation caused by heat or mechanical stimulation. The environment in which luciferase is used means in a solution, in a culture solution, in an extracellular solution, in a cell, or the like. In particular, there may be a large number of proteolytic enzymes in the culture solution, extracellular fluid and cells, and the luciferase according to the embodiment is resistant to degradation even in such an environment. That is, the luciferase according to the embodiment is not decomposed even under such an environment, and can maintain a high emission intensity. The luciferase according to the embodiment is, for example, P.I. Compared to pyralis luciferase, it is more stable against proteolysis.

  The luciferase according to the embodiment includes not only a wild-type luciferase obtained from a white squirrel but also a mutant luciferase in which a part of the amino acid sequence of the wild-type luciferase is mutated. Here, such a luciferase is referred to as a mutant type, a mutant type luciferase, a mutant type sea cucumber luciferase, a mutant type sea cucumber luciferase, or the like.

  The mutant striped luciferase according to the embodiment can induce light emission having higher light emission intensity than light emission by a known luciferase. For example, the luminescence intensity achieved when using the mutant scallop firefly luciferase according to the embodiment is, for example, 1.1 times or more, 1.2 times or more of the luminescence intensity by luciferase derived from Photinus pyralis. The emission intensity can be 5 times or more, 2.0 times or more, or 3.0 times or more. Moreover, the mutant luciferase according to the embodiment is, for example, 2.0 times or more, 3.0 times or more, 4.0 times or more, 5.0 times or more of the light emission intensity by red beetle-derived red luciferase (CBR luciferase), The light emission intensity may be 6.0 times or more or 7.0 times or more. Therefore, if the mutant luciferase according to the embodiment is used as a reporter for temporal observation, the exposure time necessary for imaging can be shortened, and temporal observation with high time resolution becomes possible.

  Another example of a mutation is a mutation that shifts the maximum emission wavelength of the emission spectrum. In the case where such a mutant scallop firefly luciferase is used as the enzyme for the luminescence reaction, an emission spectrum in which the maximum emission wavelength is shifted as compared with the case of using the wild type is obtained. When wild type scallop firefly luciferase is used in HeLa cells, luminescence with a maximum emission wavelength of around 598 nm occurs, but such a mutation shifts the maximum emission wavelength. When this mutant luciferase is expressed in cells together with wild-type luciferase or other mutant luciferases, these luciferases can be distinguished based on the difference in the maximum emission wavelength. Therefore, in studies using these luciferases as markers, the choice of markers can be increased by using mutant luciferases with shifted maximum emission wavelengths.

The aforementioned mutation may cause a change in emission color by shifting the maximum emission wavelength. For example, due to mutation, the emission color of the luminescence reaction changes from green to red.
By observing the luminescence reaction using such a mutant luciferase, it becomes possible to examine the intracellular function in more detail. For example, wild type luciferase and mutant luciferase can be fused to two different proteins and expressed in the same cell, and the behavior of each protein in the cell can be identified based on the difference in luminescent color. it can. In this case, luminescence reactions with different emission colors can be achieved by using only one type of substrate, which is advantageous in terms of experimental simplicity. In addition, even when expressing a target protein in different cells and inducing luminescence showing different luminescent colors in each cell, only one type of substrate is used, which is advantageous from the viewpoint of cost. It is.

  According to the luciferase according to the embodiment, it is expected that the applicable range of the luminescence observation is expanded by variously combining the substrate and the enzyme used for the luminescence observation. Specifically, by selecting a combination of luciferin and luciferase, a plurality of luminescence having different emission wavelengths and / or emission colors can be obtained, and the difference in emission wavelength and / or emission color can be utilized to make intracellular changes. This phenomenon can be observed. By providing such a novel luciferase, the choice of the enzyme of the luminescent reaction utilized for the function analysis of a cell can be increased, and, thereby, the variation of the combination of an enzyme and a substrate can be increased.

  The mutation for obtaining such a mutant squirrel firefly luciferase may be a mutation that does not change the properties of luciferase. For example, it may be a mutation in which there is no change in a sequence or domain having a high contribution rate to the luminescence reaction, and a change occurs in a sequence or domain having a low contribution rate to the luminescence reaction. Specifically, for example, it may be a mutation that deletes a site that is not significantly involved in the luminescence reaction, a mutation that inserts a specific sequence into such a site, a mutation that adds a specific sequence to the terminal, and the like.

In addition, the mutation for obtaining the mutant type white luciferase may be a mutation that changes properties other than the luminescence activity as luciferase. For example, it may be a mutation that improves experimental operability. Specifically, for example, when wild-type luciferase exhibits low solubility in mammalian cells, it may be a mutation that increases its solubility.
Furthermore, the mutation for obtaining mutant-type warrior luciferase is a mutation that improves the properties related to the luminescence reaction, such as a mutation that changes the optimum pH, a mutation that changes the optimum temperature, a mutation that improves the degradation stability, etc. It may be.

  An example of the mutant type scallop firefly luciferase according to the embodiment is a luciferase having the amino acid sequence set forth in SEQ ID NO: 33. SEQ ID NO: 33 gives the Kozak sequence to the amino acid sequence (SEQ ID NO: 1) of wild-type cucumber luciferase, and replaces the 148th isoleucine (I) residue with a valine (V) residue (I148V), And the 283rd serine (S) residue is substituted with a leucine (L) residue (S283L). The nucleic acid encoding this mutant luciferase is, for example, a nucleic acid having the base sequence shown in SEQ ID NO: 34. The base sequence shown in SEQ ID NO: 34 is obtained by subjecting the base sequence of SEQ ID NO: 2 encoding wild-type luciferase to codon optimization for expression in mammalian cells as described below, and further on the corresponding amino acid sequence described above. These are mutations that cause two substitutions.

  The mutant luciferase having the amino acid sequence set forth in SEQ ID NO: 33 has a maximum emission wavelength shift at a specific pH as compared with the wild-type luciferase. Thus, the mutant luciferase that causes the maximum emission wavelength shift in the emission spectrum, for example, emits red light. The mutant luciferase that produces red luminescence is hereinafter referred to as “red mutant luciferase” or “red mutant”. Red light emission refers to light having a maximum emission wavelength of the emission spectrum of 600 to 780 nm, for example.

From FIG. 3, the luminescence intensity by the red mutant when firefly luciferin is used as a substrate is, for example, 0 of the luminescence intensity that can be achieved when wild-type luciferase (SEQ ID NO: 1) is used under specific measurement conditions. .43 times, but from the comparison of the emission spectra obtained with the wild type luciferase and the red mutant luciferase shown in FIGS. 4 and 5, respectively, the maximum emission wavelength was increased from 598 nm to 613 nm by introducing the mutation. It can be seen that it can be shifted. Light having a long wavelength is more excellent in permeability in the living body. Therefore, by using this mutant luciferase, even when there are many shields between the luciferin and the light detection means, as in the case of measuring tissues, embryos and individuals, light emission Luminescence can be detected while suppressing a decrease in intensity.
Further, FIG. 7 shows that the luminescence induced by the red mutant when using the red luminescence luciferin analog shows the luminescence intensity about 2.2 times that of CBR luciferase.
3 and 7 show the luminescence intensity when D-luciferin and dimethylaniline-diene-type luciferin are used as substrates, respectively, in the luminescence reaction using the mutant luciferase according to the embodiment. Other substrates than these can also be used. For example, the substrates described in WO2009 / 096197 and WO2010 / 106896 can be used. The substrate is, for example, dimethylaniline-monoene type luciferin, dimethylaniline type luciferin, derivatives thereof, dimethylaniline-diene type luciferin derivatives, and D-luciferin derivatives.

  The two types of mutations described above can be introduced all at the same time or only one of the amino acid sequences of SEQ ID NO: 1 (SEQ ID NO: 1).

  Alternatively, the two types of mutations are luciferase proteins having an amino acid sequence different from that of SEQ ID NO: 1 (for example, the two types of mutations are 80% of the amino acid sequence shown in SEQ ID NO: 1 but not having the two types of mutations). %, Preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more of luciferase having an amino acid sequence having a mutation showing a degree of homology, or the above two kinds of mutations. It can also be introduced into a luciferase consisting of an amino acid sequence having a mutation of one to several amino acids. For example, a mutation can be introduced in consideration of the correspondence between the amino acid sequence of such a protein and SEQ ID NO: 1. For example, when an I148V mutation is to be introduced into such a protein, the amino acid residue corresponding to the 148th I of the amino acid sequence shown in SEQ ID NO: 1 is compared by comparing SEQ ID NO: 1 with the sequence of the protein to be introduced. The I148V mutation can be introduced by determining the group and replacing the amino acid with V. Here, the “corresponding amino acid residue” can be determined using a known technique such as homology search.

  Embodiments also include improved red mutants that introduce additional mutations to the red mutant. For example, an improved red mutant into which a further mutation is introduced for the purpose of further improving the emission intensity is included. Alternatively, another embodiment is an improved red mutation in which a further mutation is introduced for the purpose of improving the emission intensity of the red mutant, for example, for the purpose of improving the solubility and the resistance to degradation. Including the body. Specifically, the embodiment has a wild-type luciferase having an amino acid sequence having a certain homology with the amino acid sequence of the red mutant (SEQ ID NO: 33) and having the amino acid sequence shown in SEQ ID NO: 1. In comparison, it contains an improved red mutant that causes luciferin to emit light at a higher intensity.

Such an improved red mutant is composed of, for example, an amino acid sequence containing at least one mutation selected from the following eight mutations in wild-type luciferase consisting of the amino acid sequence shown in SEQ ID NO: 1.
(1) a mutation (I148V) that replaces 148th isoleucine of the amino acid sequence of SEQ ID NO: 1 with valine;
(2) a mutation that replaces the 283rd serine of the amino acid sequence of SEQ ID NO: 1 with leucine (S283L);
(3) A mutation (V239M) that replaces the 239th valine of the amino acid sequence of SEQ ID NO: 1 with methionine;
(4) A mutation that replaces the 313th serine of the amino acid sequence of SEQ ID NO: 1 with threonine (S313T);
(5) A mutation (D356Y) for substituting 356th aspartic acid in the amino acid sequence of SEQ ID NO: 1 with tyrosine;
(6) A mutation (V401M) that replaces the 401st valine of the amino acid sequence of SEQ ID NO: 1 with methionine;
(7) a mutation that replaces the 464th phenylalanine of the amino acid sequence of SEQ ID NO: 1 with isoleucine (F464I); and (8) a mutation that replaces the 503rd glycine of the amino acid sequence of SEQ ID NO: 1 with arginine (G503R).

More specifically, such an improved red mutant may be one obtained by introducing a further mutation into a red mutant consisting of the amino acid sequence shown in SEQ ID NO: 33. That is, such an improved red mutant has at least one selected from V239M, S313T, D356Y, V401M, F464I and G503R in addition to the mutations I148V and S283L already possessed by the red mutant consisting of the amino acid sequence shown in SEQ ID NO: 33. It can consist of an amino acid sequence containing one mutation. Examples of such improved red variants include the following three types (see Example 5 below):
(1) A variant having the three mutations I148V, S283L and F464I in the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 36);
(2) a variant having five mutations of I148V, S283L, V401M, F464I and G503R in the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 38); and (3) in the amino acid sequence of SEQ ID NO: 1, I148V, S283L, A mutant having six mutations of V239M, S313T, D356Y and F464I (SEQ ID NO: 40).

The improved red mutant can produce light having a maximum emission wavelength around 610 nm, preferably a maximum emission wavelength of 610 ± 10 nm, when D-luciferin is used as a substrate.
The three types of improved red mutants exhibit red light emission, and thus have the advantage of high transmittance in living tissues when applied to living tissues such as in vivo imaging of small animals (see FIG. 11). The red mutant (SEQ ID NO: 33) and the existing red luminescence luciferase CBR also have an advantage of showing significantly higher luminescence intensity (see FIG. 10).

  Another example of the mutant luciferase according to the embodiment is a mutant luciferase into which a mutation that improves experimental operability is introduced. For example, when the wild-type luciferase exhibits low solubility in mammalian cells, the mutant luciferase according to the embodiment is a luciferase into which a mutation that increases its solubility is introduced.

  The mutant luciferase is an arbitrary variation in the amino acid sequence of the luciferase as long as the characteristics of the luciferase according to the embodiment, that is, the luciferase activity is obtained (preferably as long as high luminescence intensity is obtained as compared with the conventional luciferase). For example, it may be a luciferase in which amino acid substitution, deletion, and / or addition has occurred. This mutation is a mutation of one or more amino acids of the amino acid sequence of wild-type luciferase or a mutation of 1 to several amino acids, preferably a mutation of 1 to 20 amino acids of the amino acid sequence of wild-type luciferase, more preferably 1 to 15 amino acid mutations, more preferably 1 to 10 amino acid mutations, more preferably 1 to 5 amino acid mutations, more preferably 1 to 4 amino acid mutations, more preferably 1 to 3 amino acid mutations. Mutation, more preferably mutation of 1 or 2 amino acids. Preferably, the amino acid sequence of the mutant luciferase is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more with respect to the amino acid sequence of the wild-type luciferase, It has 98% or more or 99% or more homology.

  The introduction of amino acid mutation can be performed according to a known technique, for example, by site-directed mutagenesis based on a general technique such as Inverse PCR method or Kunkel method, or random mutagenesis method.

  Another embodiment of the present invention relates to a polypeptide consisting of a partial sequence of the amino acid sequence shown in SEQ ID NO: 1 (that is, a fragment of luciferase protein), for example, 30 amino acid residues or more of the amino acid sequence shown in SEQ ID NO: 1. A polypeptide comprising 40 amino acid residues or more, a polypeptide comprising 50 amino acid residues or more, a polypeptide comprising 60 amino acid residues or more, a polypeptide comprising 70 amino acid residues or more, 80 amino acid residues or more A polypeptide comprising 90 amino acid residues, a polypeptide comprising 100 amino acid residues or more, a polypeptide comprising 150 amino acid residues or more, a polypeptide comprising 200 amino acid residues or more, 250 amino acid residues or more A polypeptide consisting of 300 amino acids Polypeptide having the above groups, 350 polypeptide having the above amino acid residues, polypeptides consisting of more than 400 amino acid residues, 450 polypeptide having the above amino acid residues or to a polypeptide consisting of more than 500 amino acid residues. In addition, another embodiment of the present invention relates to a polynucleotide (that is, a luciferase gene fragment) comprising a base sequence encoding the above-mentioned polypeptide.

  Such luciferase protein fragments and luciferase gene fragments can be used in split luciferase assays. In the split luciferase assay, two fragments are prepared by cleaving luciferase at a specific position to inactivate bioluminescence, and fusing each fragment with two kinds of test proteins. Based on the principle that the lost luciferase activity is restored when these fusion proteins interact with each other. This assay can detect protein-protein interactions and detect protein translocation to intracellular organelles.

  Other embodiment of this invention is related with the nucleic acid containing the base sequence which codes the luciferase which concerns on the said embodiment. That is, the nucleic acid is a nucleic acid containing a luciferase gene derived from a white squirrel firefly. In embodiments, nucleic acid refers to, for example, DNA or RNA. In the embodiments, the “gene” of luciferase mainly means a region transcribed into mRNA, that is, a structural gene.

  An example of a nucleic acid containing a base sequence encoding luciferase according to an embodiment is a nucleic acid containing the base sequence shown in SEQ ID NO: 2. A gene having this sequence has been cloned from the scorpionate firefly and encodes a wild-type squirrel firefly luciferase.

  Another example of the nucleic acid containing the base sequence encoding the luciferase according to the embodiment is a nucleic acid containing the base sequence shown in SEQ ID NO: 34. The gene having the sequence of SEQ ID NO: 34 encodes a red mutant. SEQ ID NO: 34 is a sequence obtained by introducing a mutation into a wild-type base sequence cloned from Suji Globota.

  The nucleic acid according to the embodiment may be a nucleic acid including a base sequence of a wild type luciferase gene or a nucleic acid including a base sequence of a mutant luciferase gene in which a mutation has occurred. Here, the mutant luciferase gene means that the encoded luciferase can obtain the characteristics of the luciferase according to the embodiment, that is, the luciferase activity (preferably as long as high luminescence intensity is obtained as compared with the conventional luciferase). ), A gene having undergone substitution, deletion and / or addition of a specific base in the base sequence, for example, several bases. Moreover, the variation | mutation which a change does not produce in the encoded amino acid sequence is contained in the variation | mutation of a base sequence. That is, the nucleic acid according to the embodiment includes a nucleic acid containing a mutant luciferase gene encoding a wild type luciferase.

  An example of such a mutation that does not change the encoded amino acid sequence is a mutation that invalidates the recognition sequence of a specific restriction enzyme present in the gene. Although this mutation prevents the nucleic acid containing the gene from being cleaved by the restriction enzyme, the gene can encode a protein having the same amino acid sequence as before the mutation. Such mutation can be achieved by converting the codons constituting the recognition sequence of the restriction enzyme into synonymous codons of different base sequences. Such a mutation is useful when a restriction enzyme recognition sequence used for gene recombination is present in the gene. In this case, by invalidating the recognition sequence in the gene in advance, the nucleic acid containing the gene can be prevented from being fragmented when treated with a restriction enzyme, and as a result, recombination is facilitated. An example in which the recognition sequence of a specific restriction enzyme is invalidated is the base sequence shown in SEQ ID NO: 3. This base sequence is obtained by invalidating the recognition sequence of BamHI and EcoRI from the base sequence (SEQ ID NO: 2) of the Japanese squirrel firefly luciferase gene.

  Another example of a mutation that does not change the encoded amino acid sequence is a mutation that optimizes the codons of the gene for expression in a particular species. The term “optimization” as used herein means that a codon of a gene contained in a nucleic acid is replaced with a codon having a high codon appearance frequency in a specific biological species. When optimization is performed, gene expression in a specific species is increased compared to when optimization is not performed. Since the luciferase gene according to the embodiment is obtained from a firefly, the effect of optimization is considered to be higher as the species to which the gene is introduced is taxonomically far from the firefly. In the embodiment, specific species are, for example, bacterial cells, yeast cells, and mammalian cells. Furthermore, mammalian cells are, for example, mouse cells, monkey cells and human cells.

  An example of a nucleic acid containing a base sequence encoding a luciferase with optimized codons according to an embodiment is a nucleic acid containing the base sequence shown in SEQ ID NO: 4. In the nucleic acid, the BamHI and EcoRI recognition sequences are invalidated from the base sequence (SEQ ID NO: 2) of the streak firefly luciferase gene, and the codon is optimized for expression in mammalian cells. The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 4 is shown in SEQ ID NO: 5.

  The nucleic acid according to the embodiment includes a nucleic acid including a base sequence of a luciferase gene to which a Kozak sequence is added. The Kozak sequence is a sequence composed of a start codon and a plurality of base sequences before and after the start codon. It is known that the presence of the Kozak sequence increases the expression level of the gene. In the Kozak sequence, a common sequence is found for each species or group of organisms. The nucleic acid containing the Kozak sequence according to the embodiment has a Kozak sequence corresponding to the species into which it is introduced. For example, when introduced into a mammalian cell, the nucleic acid comprises the sequence of gccrccatgg (SEQ ID NO: 6) (r means guanine or adenine) as a Kozak sequence. The luciferase gene that imparts the Kozak sequence may be a wild-type gene, or may be a mutant gene that has been subjected to codon optimization as described above.

  Yet another embodiment of the invention relates to vectors comprising these nucleic acids. In addition to the nucleic acid encoding luciferase, the vector may contain a promoter sequence, a sequence for regulating expression, a nucleic acid containing a marker gene sequence, and the like.

  Yet another embodiment of the present invention relates to a kit comprising the aforementioned vector. The kit may contain two or more types of vectors, at least one of which is the vector according to the embodiment. Preferably, the two or more types of vectors each include a base sequence encoding luciferase, and each of these luciferases catalyzes a luminescent reaction that exhibits a different luminescent color and / or luminescent wavelength. By using such a kit, for example, the behavior of a plurality of proteins can be observed simultaneously based on the difference in emission color and / or emission wavelength.

  Yet another embodiment of the present invention relates to a luminescent probe. The luminescent probe may contain wild-type luciferase or may contain mutant luciferase. In particular, those modified by a known technique and suitable for use as a luminescent probe are preferred. Furthermore, the embodiments can be effectively applied to various uses (imaging, photometry, etc.) related to various samples (in vivo, in vitro) using the luminescent probe.

  Yet another embodiment of the present invention relates to a method for analyzing intracellular functions using the luciferase according to the embodiment. The method includes a step of introducing the luciferase according to the embodiment into a cell, and a step of detecting luminescence of luciferin induced by the luciferase with an imaging device. For example, the function of the expression control region is examined by introducing the luciferase gene according to the embodiment downstream of a specific expression control region in DNA and detecting the expression of luciferase by the presence or absence of luciferin luminescence induced by the gene. It is possible.

  Still another embodiment of the present invention relates to a method for analyzing an intracellular protein using the luciferase according to the above embodiment. The method includes a step of introducing a fusion protein comprising the luciferase according to the embodiment and a protein to be analyzed into a cell, and a step of detecting luminescence of luciferin induced by the luciferase using an imaging device.

  The method includes observation of intracellular localization of a protein to be analyzed and observation of temporal change in the localization (time lapse). The method also includes not only localization of the protein, but also confirmation of whether or not the protein is expressed. The cell used is not particularly limited, and may be a cell that can be usually used in the field of cell imaging. The protein to be analyzed is not particularly limited, and a protein can be selected according to the purpose of research. The protein may be a protein that is originally present in the cell to be used, or may be a heterologous or modified protein that is not originally present in the cell.

  When the fusion protein is introduced into the cell, a known introduction method can be used. One method is to introduce a fusion protein purified outside the cell directly into the cell. For example, the fusion protein can be directly injected into the cell by the microinjection method. Alternatively, cells can be incubated in a culture solution containing the fusion protein, and the fusion protein can be taken into the cell by endocytosis. Another method is a method in which a nucleic acid containing a base sequence encoding a fusion protein is first introduced, and then the fusion protein is expressed in cells. For example, an expression vector containing the nucleic acid can be introduced into cells by the calcium phosphate method, lipofection method, electroporation method or the like, and the fusion protein can be expressed from the expression vector. Here, the gene of the fusion protein is a gene including the luciferase gene according to the embodiment and the gene of the protein to be analyzed. Here, the luciferase gene and the protein gene are linked so as to be normally translated. Has been.

A known detection method can be used for the step of detecting luminescence of luciferin with an imaging device. For example, luciferin, ATP, Mg ²⁺ ions, and the like are appropriately given to cells expressing a fusion protein containing luciferase to induce a luciferin luminescence reaction, and the generated luminescence can be detected by an imaging device. An imaging apparatus is a microscope provided with a filter for capturing light emission, for example. By using a microscope, it is possible to specify the position of luminescence in the cell and specify the localization of the protein based on this information. In addition, a microscope having a function capable of imaging over time can be used as the imaging apparatus, and observation over time is also possible with this microscope.

[Example 1: Cloning of a luciferase gene derived from a white-spotted firefly]
1. Materials As a material, larvae of sword-growing fireflies (Pristolyticus sagulatus) collected in Ome City, Tokyo were used.

2. Extraction of Total RNA and Synthesis of cDNA A light emitter was cut out from firefly larvae using scissors. The collected light emitter and 1 mL of total RNA extraction reagent TRIzol Reagent (Invitrogen) were placed in a Lysing Matrix D tube (MP-Biomedicals), which is a tube containing beads for tissue and cell homogenization. This tube was attached to a tissue cell disruption device FastPrep 24 (MP-Biomedicals) or FastPrep FP100A (MP-Biomedicals), and a firefly light emitter was used as a reagent under conditions of a vibration speed of 6.5 m / s and a vibration time of 45 seconds. It was crushed inside. After completion, the tube was removed from the crusher and placed on ice for 30 minutes. Thereafter, crushing was performed again under the same conditions.

  Next, according to the instructions of the total RNA extraction reagent TRIzol Reagent, the total RNA was separated and purified from the disrupted solution. 100 μl of the obtained mRNA solution was concentrated by precipitation by ethanol precipitation. Next, a full-length cDNA was synthesized from the precipitated and concentrated total RNA using a full-length cDNA synthesis reagent GeneRacer (Invitrogen) according to the manual. 20 μl of the obtained cDNA solution was used as a firefly full-length cDNA library for the following gene experiments.

3. 3. Identification of 5 ′ terminal side of firefly luciferase gene 3-1. Preparation of Primer for Rapid Amplification of cDNA End (RACE) Method Cloning of the luciferase gene of the white squirrel was performed by the Polymerase chain reaction (PCR) method. Primers used for this PCR were prepared as follows based on the amino acid sequence of a luciferase gene derived from a known related organism.

  In order to confirm amino acid regions that are well conserved in firefly luciferase, the amino acid sequences of 10 types of firefly luciferases that have already been released were compared using sequence information analysis software DNASIS Pro (Hitachi Software Engineering Co., Ltd.). The relative organisms used in the comparison were Rampyris noctiluca (registration number CAA61668), Luciola cruciata (registration number P13129), Luciola latero15 (Luciola laQal01)・ Mingrelica (registration number Q26304), Hotaria parvula (registration number AAC37253), Photinus pyralis (registration number BAF48390), Photouris pennsylva tyll vs. Q27757), Piroko area Miyako ( yrocoelia miyako) (registration number AAC37254), it is a Piroko area Alpha (Pyrocoelia rufa) (registration number AAG45439) and Rahagofutarumusu-Obai (Rhagophthalmus ohbai) (registration number BAF34360).

As a result, it was found that the amino acid sequence of LI-KY-K-G-Q-V (SEQ ID NO: 7) located near the C-terminal 440 residues of firefly luciferase is well conserved. It was. A nucleotide sequence was predicted from codons encoding these nine amino acid sequences, and 12 kinds of firefly luciferase-specific mixed primers used for 5′-end RACE PCR were designed. The name and sequence of this primer are as follows (Y, R and N in the primer sequence represent mixed bases: Y represents C or T, R represents A or G, and N represents , A, G, C or T):
flexLuc5-ATA (5′-ACY TGR TAN CCY TTA TAT TTA AT-3 ′: SEQ ID NO: 8),
flexLuc5-ATG (5′-ACY TGR TAN CCY TTA TAT TTG AT-3 ′: SEQ ID NO: 9),
flexLuc5-ATT (5′-ACY TGR TAN CCY TTA TAT TTT AT-3 ′: SEQ ID NO: 10),
flexLuc5-ACA (5′-ACY TGR TAN CCY TTA TAC TTA AT-3 ′: SEQ ID NO: 11),
flexLuc5-ACG (5′-ACY TGR TAN CCY TTA TAC TTG AT-3 ′: SEQ ID NO: 12),
flexLuc5-ACT (5′-ACY TGR TAN CCY TTA TAC TTT AT-3 ′: SEQ ID NO: 13),
flexLuc5-GTA (5′-ACY TGR TAN CCY TTG TAT TTA AT-3 ′: SEQ ID NO: 14),
flexLuc5-GTG (5′-ACY TGR TAN CCY TTG TAT TTG AT-3 ′: SEQ ID NO: 15),
flexLuc5-GTT (5′-ACY TGR TAN CCY TTG TAT TTT AT-3 ′: SEQ ID NO: 16),
flexLuc5-GCA (5′-ACY TGR TAN CCY TTG TAC TTA AT-3 ′: SEQ ID NO: 17),
flexLuc5-GCG (5′-ACY TGR TAN CCY TTG TAC TTG AT-3 ′: SEQ ID NO: 18),
flexLuc5-GCT (5′-ACY TGR TAN CCY TTG TAC TTT AT-3 ′: SEQ ID NO: 19). The synthesis of these primers was outsourced to Life Technologies Japan.

3-2.5′-RACE PCR cloning of the 5 ′ end of the firefly luciferase gene Using the firefly full-length cDNA library prepared as described above as a template, 12 kinds of specific mixed primers prepared as described above and GeneRacer 5 ′ Primer (5′-CGA CTG GAG CAC GAG GAC ACT GA-3 ′: SEQ ID NO: 20) and GeneRacer 5 ′ Nested Primer (5′-GGA CAC TGA CAT GGA CTG AAG GAG TA 3 ′: 5′-RACE PCR was performed using SEQ ID NO: 21). The GeneRacer 5 ′ Primer and the GeneRacer 5 ′ Nested Primer included in the full-length cDNA synthesis reagent GeneRacer kit (Invitrogen) were used. In order to efficiently amplify the luciferase gene by 5′-RACE PCR, nested PCR was carried out in which the gene amplified once by PCR was used as a template and the gene was amplified more specifically by the inner primer pair. PCR was performed using polymerase Ex-Taq (Takara Bio Inc.) according to the manual.

As the first PCR, the luciferase gene was amplified using 12 kinds of primer pairs consisting of any of the 12 kinds of specific mixed primers prepared as described above and GeneRacer 5 ′ Primer. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl), 10 μl of a PCR reaction solution containing one of 12 primers with a final concentration of 1.0 μM and GeneRacer 3 ′ Primer with a final concentration of 0.3 μM was prepared. 2 μl was added. The concentration of the firefly full-length cDNA library solution was not quantified. In the PCR reaction, heat denaturation at 94 ° C. for 2 minutes was first performed, and then 94 ° C. for 30 seconds, 45 ° C. for 30 seconds and 72 ° C. for 90 seconds were repeated for 30 cycles, and finally an extension reaction at 72 ° C. for 5 minutes was performed. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% Tris acetate buffer (TAE) agarose gel, and after ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation. Since gene amplification was slightly observed in all 12 reaction solutions, nested PCR reactions were performed as follows using these PCR reaction solutions as templates.

As nested PCR, the luciferase gene was amplified using 4 types of primer pairs consisting of 4 out of 12 kinds of primers used in the first PCR and GeneRacer 3 ′ Nested Primer. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl), 10 μl of a PCR reaction solution containing one of 12 kinds of primers with a final concentration of 1.0 μM and GeneRacer 3 ′ Primer with a final concentration of 0.3 μM was prepared, and the first PCR reaction solution was used as a template there. 1.0 μl of a solution diluted 10-fold with sterilized water was added. In the PCR reaction, heat denaturation at 94 ° C. for 2 minutes was first performed, and then 94 ° C. for 30 seconds, 45 ° C. for 30 seconds and 72 ° C. for 90 seconds were repeated for 30 cycles, and finally an extension reaction at 72 ° C. for 5 minutes was performed. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation. The combination conditions of primers that efficiently amplified the gene in the vicinity of about 1.4 kbp were confirmed.

Determination of the base sequence of the gene amplified by 3-3.5′-RACE In order to read the base sequence of the gene amplified by 5′-RACE, the PCR product was purified by gel extraction, subcloning and direct sequencing. Details are shown below.

PCR (final volume 20 μl) was performed with a combination of primers that efficiently amplified the gene in the vicinity of about 1.4 kbp, and the target gene fragment was recovered using a gel extraction technique. Gel extraction was performed according to the manual using Wizard SV Gel and PCR Clean-Up System (Promega Corporation). Subcloning of the PCR product extracted from the gel was performed using the TA cloning technique. TA cloning was performed using pGEM-T Easy Vector System (Promega Corporation) according to the manual. Thereafter, this vector DNA was transformed into E. coli (TOP10 strain or DH5α strain), and insert positive colonies were selected using a blue / white screening technique. The selected colony was subjected to direct colony PCR to confirm that the gene was introduced. For direct colony PCR, M13-F (−29) Primer (5′-CAC GAC GTT GTA AAA CGA C-3 ′: SEQ ID NO: 22) and M13 Reverse (5′-GGA TAA CAA TTT CAC AGG-3 ′: A primer pair consisting of SEQ ID NO: 23) was used. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl), a 10 μl PCR reaction solution containing a primer pair each having a final concentration of 0.2 μM was prepared, and a small amount of E. coli colony was added thereto as a template. The PCR reaction was first heat-denatured at 94 ° C. for 1 minute, then repeated 25 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 2 minutes. After the PCR reaction, 2 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation.

  For the PCR reaction solution in which amplification was confirmed, the base sequence of the gene was determined using the direct sequencing method. Excess dNTPs and primers contained in the PCR reaction solution were removed using a PCR product purification kit ExoSAP-IT (GE Healthcare Bioscience) to prepare a template for PCR direct sequencing. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), a sequencing reaction solution containing this template was prepared, and a sequencing reaction was performed using a thermal cycler. PCR product purification and sequencing were performed according to the respective manuals. After the sequencing reaction, the reaction product was purified as follows. 2.5 times volume of 100% ethanol was added to the reaction solution, and nucleic acid was precipitated using a centrifuge. Next, after removing the supernatant, 70% ethanol was added to wash the precipitate, and the nucleic acid was precipitated using a centrifuge. Finally, after removing the supernatant, the precipitate was dried. To the purified precipitate, 15 μl of Hi-Di Formatide (Applied Biosystems) was added and dissolved. This solution was heat denatured at 94 ° C. for 2 minutes and further rapidly cooled on ice to prepare a sample for determining the base sequence. The base sequence of this sample was read using an Applied Biosystems 3130xl genetic analyzer (Applied Biosystems). The base sequence analysis method was performed according to the manual. The obtained base sequence is shown in SEQ ID NO: 24 as the base sequence of the untranslated region at the 5 'end side of the Japanese scallop firefly luciferase gene.

  The gene sequence obtained by sequencing was analyzed using the “sequence ligation” function of the sequence information analysis software DNASIS Pro. This sequence was subjected to a homology search using a blastx search provided by National Center for Biotechnology Information (NCBI), and confirmed to show high homology with the base sequence of known firefly luciferase. The base sequence obtained by the above experiment and analysis was determined to be on the 5 'end side of the novel firefly luciferase gene.

4). Obtaining 3 'Race PCR and full-length cDNA of firefly luciferase gene 4-1.3' Design of primers used for 'Race PCR' Sequence of 5 'terminal untranslated region of firefly luciferase gene obtained in 5' Race PCR experiment Based on the above, a primer (SEQ ID NO: 27) used for 3 ′ RACE and a primer (SEQ ID NO: 28) used for Nested PCR were prepared. Primer synthesis was outsourced to Life Technologies Japan.

4-2. 3 'Race PCR for obtaining full-length cDNA of firefly luciferase gene
Primer JP-Sujiguro-Full-F1 (CTTTAACTACTTCGTGTATTCAATAAAG) prepared from the base sequence of the 5′-terminal untranslated region of the target firefly luciferase using the firefly full-length cDNA library prepared as described above as a template: SEQ ID NO: 25 ), GeneRacer 3 ′ Primer (5′-GCT GTC AAC GAT ACG CTA CGT AAC G-3 ′: SEQ ID NO: 27) and GeneRacer 3 ′ Nested Primer (5′-CGC TAC GTA ACG GCA TGA CAG28-3 ) Was used for 3′-RACE PCR. GeneRacer 3 ′ Primer and GeneRacer 3 ′ Nested Primer used were those included in the full-length cDNA synthesis reagent GeneRacer kit (Invitrogen). In order to efficiently amplify the luciferase gene by 3′-RACE PCR, a nested PCR was carried out in which the gene amplified once by PCR was used as a template and the gene was amplified more specifically by the inner primer pair. PCR was performed using polymerase Ex-Taq (Takara Bio Inc.) according to the manual.

As the first PCR, the luciferase gene was amplified using a primer pair consisting of a primer prepared from the base sequence of the 5 ′ end side untranslated region and GeneRacer 3 ′ Primer. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl) and 20 μl of a PCR reaction solution containing primers each having a final concentration of 0.3 μM were prepared, and 0.4 μl of a firefly full-length cDNA library solution was added thereto. The concentration of the firefly full-length cDNA library solution was not quantified. The PCR reaction was first heat-denatured at 94 ° C. for 2 minutes, then repeated 30 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 5 minutes. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation. Since gene amplification was slightly observed, nested PCR reactions were performed using this PCR reaction solution as a template.

As a nested PCR, a luciferase gene amplification was performed using a primer pair consisting of a primer JP-Sujiguro-Full-F2 (CTTTAACTACTTCGTGATTCAATAAAGTAATTTAG: SEQ ID NO: 26) and a GeneRacer 3 ′ Nested Primer for nested PCR. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl) and 20 μl of a nested PCR reaction solution containing primers each having a final concentration of 0.3 μM were prepared, and 1.0 μl of a solution obtained by diluting the first PCR reaction solution 10-fold with sterile water was added thereto as a template. The PCR reaction was first heat-denatured at 94 ° C. for 2 minutes, then repeated 30 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 5 minutes. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation. It was confirmed that the gene was efficiently amplified in the vicinity of about 2 kbp.

Determination of the base sequence of the gene amplified with 4-3.3′-Race In order to read the base sequence of the gene amplified with 3′-RACE, purification of the PCR product by gel extraction, subcloning and direct sequencing were performed. Details are shown below.

PCR (final volume 20 μl) was performed with a combination of primers in which the gene was efficiently amplified in the vicinity of about 2 kbp, and the target gene fragment was recovered using a gel extraction technique. Gel extraction was performed according to the manual using Wizard SV Gel and PCR Clean-Up System (Promega Corporation). Subcloning of the PCR product extracted from the gel was performed using the TA cloning technique. TA cloning was performed using pGEM-T Easy Vector System (Promega Corporation) according to the manual. Thereafter, this vector DNA was transformed into E. coli (TOP10 strain or DH5α strain), and insert positive colonies were selected using a blue / white screening technique. The selected colony was subjected to direct colony PCR to confirm that the gene was introduced. For direct colony PCR, M13-F (−29) Primer (5′-CAC GAC GTT GTA AAA CGA C-3 ′: SEQ ID NO: 21) and M13 Reverse (5′-GGA TAA CAA TTT CAC AGG-3 ′: A primer pair consisting of SEQ ID NO: 22) was used. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl), a 10 μl PCR reaction solution containing a primer pair each having a final concentration of 0.2 μM was prepared, and a small amount of E. coli colony was added thereto as a template. The PCR reaction was first heat-denatured at 94 ° C. for 1 minute, then repeated 25 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 2 minutes. After the PCR reaction, 2 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation.

  For the PCR reaction solution in which amplification was confirmed, the base sequence of the gene was determined using the direct sequencing method. Excess dNTPs and primers contained in the PCR reaction solution were removed using a PCR product purification kit ExoSAP-IT (GE Healthcare Bioscience) to prepare a template for PCR direct sequencing. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), a sequencing reaction solution containing this template was prepared, and a sequencing reaction was performed using a thermal cycler. Primers used for sequencing were vector primers or gene-specific primers. PCR product purification and sequencing were performed according to the respective manuals. After the sequencing reaction, the reaction product was purified as follows. 2.5 times volume of 100% ethanol was added to the reaction solution, and nucleic acid was precipitated using a centrifuge. Next, after removing the supernatant, 70% ethanol was added to wash the precipitate, and the nucleic acid was precipitated using a centrifuge. Finally, after removing the supernatant, the precipitate was dried. To the purified precipitate, 15 μl of Hi-Di Formatide (Applied Biosystems) was added and dissolved. This solution was heat-denatured at 94 ° C. for 2 minutes and further rapidly cooled on ice to prepare a sample for determining the base sequence. The base sequence of this sample was read using an Applied Biosystems 3130xl genetic analyzer (Applied Biosystems). The base sequence analysis method was performed according to the manual. The obtained base sequence is shown in SEQ ID NO: 2 as the base sequence of the wild-type scallop luciferase luciferase gene.

  The full-length firefly luciferase gene was obtained by sequencing. The base sequence (SEQ ID NO: 2) or the sequence translated into amino acids (SEQ ID NO: 1) was subjected to homology search using blastx or blastp search provided by NCBI. Each search confirmed high homology with the base sequence of known firefly luciferase. The base sequence obtained by the above experiment and analysis was determined to be the full-length cDNA sequence of the novel firefly luciferase gene.

The base sequence (SEQ ID NO: 2) is as follows.
ATGGATAACGATAAAAATATTGTATATGGTCCCGAGCCATTTTACCCTGTTGATAAAGGATCCGCAGGAGCACAAATACACAAATATTTGCATCAATACTCAAAACTTGGAGCAATCGCGTTTACCAACGCGCTTACTGGTGTTGATATTACTTACGCCGAATACTTTGATATAACTTGCCGTTTAGGAGAAGCAATGAAAAGATATGGTATTAAAGTAGATGGAAGAATTGCTTTATGCAGTGAAAACTGTGAAGAATTTTTTTATCCTGTAATTGCTGGACTTTACATAGGTGCAGGCGTTGCTCCCACAAACGAGATTTACACCTTACGCGAGTTAGTCCACAGTTTAGGTATCTCAAAACCAACAATTGTATTTAGCTCTAAAAAAGGTTTAGATAAAGTTTTGCACGTACAAAAAACAGTGACATGCATTAAAACGATTGTTATATTAGACAGCAAAGTGGATTATAAAGGATACGACTGTGTGGAAAGTTTTATAAAAAGATACAATCCGCCAGGTTTCcAAGCTGCGAGTTTCAAAACTGTAGAGGTTGACCGCAAAGAGCAAATTGCTCTCATTATGAACTCTTCCGGTTCTACCGGTTTACCAAAAGGTGTACAACTTACTCACGAATGCGCGGTTACTAGATTCTCTCATGCCAAGGATCCCATTTACGGCAACCAAGTTTCACCAGGCACAGCCGTTTTAACTGTTGTTCCATTCCATCATGGTTTTGGTATGTTTACCACTTTAGGATATTTAGCTTGTGGATTTCGCGTCGTTATGCTAACAAGATTTGACGAAGAGATATTTTTAAAAACTATACAAGACTATAAATGTACAAGTGTCATTCTTGTACCGACTTTGTTCGGAATCCTCTCTAAAAGTAAGTTATTGGACAAATACGATCTGTCGAATCTAGTTGAAATTGCGTCTGGCGGAGCTCCATTAGCAAAAGAAGTTAGCGAAGCCGTAGCCAGACGGTTCAACCTTCCCG GAGTCCGTCAAGGTTACGGTTTGACGGAAACAACGTCTGCTATTATTATCACTCCAGAAGGTGACGATAAACCGGGTGCATCTGGAAAAGTTGTACCGTTGTTCAAAGCAAAAGTTGTTGACCTTGACACCAAAAAAACGTTGGGTCCTAACCAACGAGGTGAAGTATGCGTTAAAGGCCCTATGCTTATGAAAGGATACGTAGATAATCCGGAAGCAACCAGAGAAATTATTGACGAAGATGGCTGGTTGCACACTGGGGATATTGGATATTACGATGAGGACCAACACTTCTTTATTGTTGATCGTTTGAAGTCGTTAATAAAATACAAGGGATACCAAGTACCACCTGCAGAATTAGAATCTGTTCTTTTGCAACATCCATACATATTTGATGCTGGAGTAGCTGGTGTTCCTGATGCTGAAGCTGGTGAACTTCCTGGAGCAGTTGTTGTACTTGAAAAAGGAAAGCACGTGACTGAAAAAGAAATAATGGATTACGTTGCTGGCCAAGTCTCAAATGCAAAACGTTTGCGTGCTGGTGTTCGTTTTGTGGACGAAGTACCTAAAGGTCTTACTGGAAAAATTGATGGCAAAGCTATTAGAGAAATTCTTATGAAACCAAAATCTAAAATGTAA

The sequence translated into amino acids (SEQ ID NO: 1) is as follows.
MDNDKNIVYGPEPFYPVDKGSAGAQIHKYLHQYSKLGAIAFTNALTGVDITYAEYFDITCRLGEAMKRYGIKVDGRIALCSENCEEFFYPVIAGLYIGAGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVLHVQKTVTCIKTIVILDSKVDYKGYDCVESFIKRYNPPGFQAASFKTVEVDRKEQIALIMNSSGSTGLPKGVQLTHECAVTRFSHAKDPIYGNQVSPGTAVLTVVPFHHGFGMFTTLGYLACGFRVVMLTRFDEEIFLKTIQDYKCTSVILVPTLFGILSKSKLLDKYDLSNLVEIASGGAPLAKEVSEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDKPGASGKVVPLFKAKVVDLDTKKTLGPNQRGEVCVKGPMLMKGYVDNPEATREIIDEDGWLHTGDIGYYDEDQHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPYIFDAGVAGVPDAEAGELPGAVVVLEKGKHVTEKEIMDYVAGQVSNAKRLRAGVRFVDEVPKGLTGKIDGKAIREILMKPKSKM

  Hereinafter, the protein encoded by the novel luciferase gene is referred to as wild-type swordfish luciferase.

[Example 2: Measurement of enzymological parameters of wild-type scallop firefly luciferase]
1. Protein expression of wild-type Japanese cypress firefly luciferase gene The wild-type Japanese cypress firefly luciferase gene was introduced into pRSET-B vector (Invitrogen) for protein expression in E. coli. The gene expression vector was constructed according to a standard method as follows.

1-1. Modification of restriction enzyme recognition site of wild-type scallop luciferase gene According to the base sequence determined as described above, the wild-type squirrel luciferase gene contains restriction enzyme recognition sequences of BamHI and EcoRI. While maintaining the amino acid sequence of luciferase, genetic modification was performed to remove these recognition sequences in these base sequences. This treatment was performed to facilitate the introduction of the luciferase gene described later into an expression vector. The introduction of gene mutation is shown in Taichi Yoshikazu's “Methods for Gene Function Inhibition—From Simple and Reliable Gene Function Analysis to Application to Gene Therapy” (Yodosha, 2001, p. 17-25) Followed the method. The sequence after mutagenesis is the base sequence shown in SEQ ID NO: 3.

The sequence after mutagenesis (SEQ ID NO: 3) is as follows.
ATGGATAACGATAAAAATATTGTATATGGTCCCGAGCCATTTTACCCTGTTGATAAAGGTTCCGCAGGAGCACAAATACACAAATATTTGCATCAATACTCAAAACTTGGAGCAATCGCGTTTACCAACGCGCTTACTGGTGTTGATATTACTTACGCCGAATACTTTGATATAACTTGCCGTTTAGGAGAAGCAATGAAAAGATATGGTATTAAAGTAGATGGAAGAATTGCTTTATGCAGTGAAAACTGTGAAGAATTTTTTTATCCTGTAATTGCTGGACTTTACATAGGTGCAGGCGTTGCTCCCACAAACGAGATTTACACCTTACGCGAGTTAGTCCACAGTTTAGGTATCTCAAAACCAACAATTGTATTTAGCTCTAAAAAAGGTTTAGATAAAGTTTTGCACGTACAAAAAACAGTGACATGCATTAAAACGATTGTTATATTAGACAGCAAAGTGGATTATAAAGGATACGACTGTGTGGAAAGTTTTATAAAAAGATACAATCCGCCAGGTTTCCAAGCTGCGAGTTTCAAAACTGTAGAGGTTGACCGCAAAGAGCAAATTGCTCTCATTATGAACTCTTCCGGTTCTACCGGTTTACCAAAAGGTGTACAACTTACTCACGAATGCGCGGTTACTAGATTCTCTCATGCCAAGGACCCCATTTACGGCAACCAAGTTTCACCAGGCACAGCCGTTTTAACTGTTGTTCCATTCCATCATGGTTTTGGTATGTTTACCACTTTAGGATATTTAGCTTGTGGATTTCGCGTCGTTATGCTAACAAGATTTGACGAAGAGATATTTTTAAAAACTATACAAGACTATAAATGTACAAGTGTCATTCTTGTACCGACTTTGTTCGGAATCCTCTCTAAAAGTAAGTTATTGGACAAATACGATCTGTCGAATCTAGTTGAAATTGCGTCTGGCGGAGCTCCATTAGCAAAAGAAGTTAGCGAAGCCGTAGCCAGACGGTTCAACCTTCCCG GAGTCCGTCAAGGTTACGGTTTGACGGAAACAACGTCTGCTATTATTATCACTCCAGAAGGTGACGATAAACCGGGTGCATCTGGAAAAGTTGTACCGTTGTTCAAAGCAAAAGTTGTTGACCTTGACACCAAAAAAACGTTGGGTCCTAACCAACGAGGTGAAGTATGCGTTAAAGGCCCTATGCTTATGAAAGGATACGTAGATAATCCGGAAGCAACCAGAGAAATTATTGACGAAGATGGCTGGTTGCACACTGGGGATATTGGATATTACGATGAGGACCAACACTTCTTTATTGTTGATCGTTTGAAGTCGTTAATAAAATACAAGGGATACCAAGTACCACCTGCAGAATTAGAATCTGTTCTTTTGCAACATCCATACATATTTGATGCTGGAGTAGCTGGTGTTCCTGATGCTGAAGCTGGTGAACTTCCTGGAGCAGTTGTTGTACTTGAAAAAGGAAAGCACGTGACTGAAAAAGAAATAATGGATTACGTTGCTGGCCAAGTCTCAAATGCAAAACGTTTGCGTGCTGGTGTTCGTTTTGTGGACGAAGTACCTAAAGGTCTTACTGGAAAAATTGATGGCAAAGCTATTAGAGAAATTCTTATGAAACCAAAATCTAAAATGTAA

1-2. Introduction of wild-type Japanese cypress firefly luciferase gene into gene expression vector In order to introduce the luciferase gene between restriction enzyme sites BamHI-EcoRI of pRSET-B vector, a primer containing a restriction enzyme BamHI recognition sequence GGATCC in front of it A primer (SEQ ID NO: 30) containing (SEQ ID NO: 29) and a stop codon and a restriction enzyme EcoRI recognition sequence GAATTC behind it was prepared. Using this primer pair, a fragment containing the above-mentioned restriction enzyme recognition sites at both ends of the luciferase gene was amplified. PCR was performed according to the manual using polymerase KOD-Plus- (Toyobo Co., Ltd.).

10 × PCR Buffer with final concentration of 1 ×, final concentration of 0.2 mM dNTP Mixture (2.5 mM each), final concentration of 1.0 mM MgSO ₄ , final concentration of 0.02 U / μl TOYOBO KOD− A PCR reaction solution of 20 μl containing a primer pair of Plus- (1 U / μl) and a final concentration of 0.3 μM is prepared, and 0.4 μl of a solution of luciferase gene having no BamHI and EcoRI recognition sequences is added thereto as a template. It was. In the PCR reaction, after heat denaturation at 94 ° C. for 2 minutes, 94 cycles of 30 ° C., 55 ° C. for 30 seconds and 68 ° C. for 2 minutes were repeated 30 times, and finally, an extension reaction at 68 ° C. for 5 minutes was performed. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation. Since gene amplification was confirmed, this PCR reaction solution was precipitated and concentrated by the usual ethanol precipitation method, 4 μl of 10 × H Buffer for restriction enzyme treatment, restriction enzyme BamHI (Toyobo Co., Ltd.) and restriction enzyme EcoRI (Toyobo Co., Ltd.) 2) each and 32 μl of sterilized deionized water were dissolved, and the mixture was incubated at 37 ° C. for 2 hours for restriction enzyme treatment. Then, this reaction solution was precipitated and concentrated by an ethanol precipitation method and then dissolved in sterilized deionized water. This solution was electrophoresed using 1% TAE agarose gel and stained with ethidium bromide. A gel containing a DNA band confirmed under ultraviolet irradiation was cut out using a knife. DNA was extracted from this excised gel using Wizard (R) SV Gel and PCR Clean-Up System (Promega). These operations were performed according to the manual. Thereafter, using Ligation Pack (Nippon Gene) according to the manual, the extracted DNA was introduced into the pRSET-B vector previously treated with the restriction enzymes BamHI and EcoRI by the same method. This vector DNA was transformed into Escherichia coli JM109 (DE3) strain to form colonies.

Direct colony PCR was performed using the obtained colony as a template to amplify the luciferase gene introduced into pRSET-B. Direct colony PCR was performed using T7 promoter Primer (5′-TAA TAC GAC TCA CTA TAG GG-3 ′: SEQ ID NO: 31) and T7 Reverse Primer (5′-CTA GTT ATT GCT CAG CGG TGG-3 ′: SEQ ID NO: 32). The primer pairs were used. Final concentration of 10 × Ex Taq Buffer (20 mM Mg ²⁺ plus), final concentration of 0.2 mM each dNTP Mixture (2.5 mM each), final concentration 0.05 U / μl TaKaRa Ex Taq (5 U / μl) and 10 μl of a PCR reaction solution containing primers each having a final concentration of 0.2 μM were prepared, and a small amount of E. coli colony was added thereto as a template. The PCR reaction was first heat-denatured at 94 ° C. for 2 minutes, followed by 25 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds and 72 ° C. for 2 minutes, and finally an extension reaction at 72 ° C. for 5 minutes. After the PCR reaction, 1 μl of the PCR reaction solution was electrophoresed using a 1% TAE agarose gel. After ethidium bromide staining, the amplified gene band was observed under ultraviolet irradiation.

  For the PCR reaction solution in which amplification was confirmed, the base sequence of the gene was determined using the direct sequencing method. Using PCR product purification kit ExoSAP-IT, excess dNTPs and primers contained in the PCR reaction solution were removed, and a template for PCR direct sequencing was prepared. Using BigDye Terminator v3.1 Cycle Sequencing Kit, a sequencing reaction solution containing this template was prepared, and a sequencing reaction was performed using a thermal cycler. For the sequencing, vector primers or gene-specific primers were used. PCR product purification and sequencing were performed according to the respective manuals. After the sequencing reaction, the reaction product was purified as follows. 2.5 times volume of 100% ethanol was added to the reaction solution, and nucleic acid was precipitated using a centrifuge. Next, after removing the supernatant, 70% ethanol was added to wash the precipitate, and the nucleic acid was precipitated using a centrifuge. Finally, after removing, the precipitate was dried. To the purified precipitate, 15 μl of Hi-Di Formatide (Applied Biosystems) was added and dissolved. This solution was heat-denatured at 94 ° C. for 2 minutes and further rapidly cooled on ice to prepare a sample for determining the base sequence. The base sequence of this sample was read using an Applied Biosystems 3130xl genetic analyzer, and it was confirmed that the base sequence of SEQ ID NO: 3 was normally introduced into the gene expression vector pRSET-B.

2. Purification of photoprotein 0.5 μl of luciferase expression vector was added to 50 μl of E. coli solution containing JM109 (DE3), and incubated on ice for 10 minutes, then at 42 ° C. for 1 minute, and finally on ice for 2 minutes. Thereafter, 50 μl of E. coli solution was added to 200 μl of SOC medium. The E. coli / SOC medium mixed solution was incubated at 37 ° C. for 20 minutes with shaking. 100 μl of the incubated sample was streaked on an LB medium plate (containing 100 μg / ml ampicillin) and incubated overnight at 37 ° C. The colonies obtained the next day were picked up and cultured in a 500 ml scale LB medium. The culture was performed at 37 ° C. for 24 hours and at 18 ° C. for 24 hours. After a total of 48 hours of culture, the cells were collected by centrifugation, resuspended in a 0.1 M Tris-HCl solution (pH 8.0), and sonicated. The cell disruption solution was centrifuged (15000 rpm, 10 minutes), the sediment was removed, and the supernatant was recovered. To a column with a bed volume of 2 ml, 500 μl of Ni-Agar suspension and 2 ml of 0.1 M Tris-HCl were added to equilibrate the column. The collected supernatant was added to the column and allowed to fall naturally. All operations until the entire amount of the supernatant passed through the column were performed at 4 ° C. The column was washed with 2 ml of 25 mM imidazole / 0.1 M Tris-HCl solution. 2 ml of 500 mM imidazole / 0.1 M Tris-HCl solution was added to the washed column to elute luciferase. The eluted sample was filtered through a gel filtration column PD-10 (GE Healthcare) and desalted. The desalted sample was ultrafiltered with Vivaspin 6 (Sartorius), and glycerin was added to the concentrated sample to obtain a 50% glycerin solution. Storage was performed at -20 ° C.

3. Measurement of emission spectrum LumiFl SpectroCapture (ATTO) was used as an apparatus for measurement, and 0.1 mM citric acid / 0.1 M Na ₂ HPO ₄ buffer (pH 6.0-8.0) was used with 1 mM D-luciferin, 2 mM ATP and 4 mM. The purified enzyme was added to the solution containing MgCl ₂ at a final concentration between 1 μg / ml and 10 μg / ml, and the emission spectrum was measured when 15 seconds had elapsed after the addition of the enzyme. The measurement results are shown in FIG.

  As shown in FIG. 1, the obtained wild-type scallop firefly luciferase has a maximum emission wavelength in the vicinity of 583 nm in an environment of pH 8.0. Moreover, the maximum emission wavelengths are about 590 nm, about 600 nm, about 609 nm, about 614 nm and about 613 nm at pH 7.5, pH 7.0, pH 6.5, pH 6.0 and pH 5.5, respectively. There is a tendency that the maximum wavelength becomes longer as the pH is lowered.

4). Kinetic analysis 4-1. Determination of D-luciferin and ATP concentrations The D-luciferin concentration in the D-luciferin solution and the ATP concentration in the ATP solution were determined as follows.

Using a UV-Visible Spectrometer (Hitachi), the UV-visible absorption spectra of the D-luciferin solution and the ATP solution were measured, and the concentration was calculated from the measurement results and the following ε values.
D-luciferin: λ _max 328 nm, ε18200, pH 5.0
ATP: λ _max 259 nm, ε 15400, pH 7.0.

  Each measurement was performed 10 times, and the average value of absorbance was used for concentration calculation. The following Km value calculation was performed using the D-luciferin solution and the ATP solution thus determined.

4-2. Measurement of Km value for D-luciferin The intensity of luminescence by the obtained luciferase was measured under various D-luciferin concentrations. Based on the measurement results, the Km value for D-luciferin was determined.

D-luciferin was added to 0.1 M Tris-HCl (pH 8.0) to make 12 types of D-luciferin solutions with different concentrations. These solutions contain D-luciferin so that the final concentration of D-luciferin is 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 480 and 640 μM, respectively. Including. 50 μl of each of these D-luciferin solutions was dispensed into a 96-well microplate. A 0.1 M Tris-HCl (pH 8.0) solution containing various purified luciferases, 4 mM ATP and 8 mM MgSO ₄ was connected to a standard pump of a luminometer, and 50 μl of the solution was added to the wells, and measurement was performed. Luminescence (ATTO) was used for the measurement. The measurement was performed three times for each luciferin concentration.

  The peak intensity of the photon count value obtained was plotted against the luciferin concentration S as the initial velocity V. This plot was subjected to Michaelis-Menten type curve fitting to calculate the Km value. Curve fitting was performed by a non-linear least square method, and Newton's method was used for parameter search.

4-3. Measurement of Km value for ATP The intensity of luminescence by the obtained luciferase was measured under various ATP concentration environments. Based on the measurement results, the Km value for ATP was determined.

ATP was added to 0.1 M Tris-HCl (pH 8.0) to prepare 12 types of ATP solutions with different concentrations. These solutions contain ATP such that the final concentrations of ATP are 5, 10, 20, 40, 80, 160, 320, 480, 640, 800, 1280, 1600 and 1920 μM, respectively. 50 μl of each ATP solution was dispensed into a 96-well microplate. A 0.1M Tris-HCl (pH 8.0) solution containing various purified luciferases, 1 mM D-luciferin, and 8 mM MgSO ₄ was connected to a standard pump of a luminometer, and 50 μl of the solution was added to the wells for measurement. The measurement was performed three times for each ATP concentration.

  The peak intensity of the obtained photon count value was plotted against the ATP concentration S as the initial velocity V. This plot was subjected to Michaelis-Menten type curve fitting to calculate the Km value. Curve fitting was performed by a non-linear least square method, and Newton's method was used for parameter search.

  Table 1 shows the Km value for D-luciferin and the Km value for ATP determined as described above. Table 1 also shows the Km values of known luciferases measured in the same manner. P. Pyralis is a known firefly-derived luciferase. ELuc, CBG, and CBR are luciferases derived from known beetles. These known luciferases were used commercially.

  Furthermore, about these results, the figure which plotted the vertical axis | shaft as Km value with respect to ATP and the horizontal axis | shaft as Km value with respect to D-luciferin is shown as FIG.

[Example 3: Production of mutant-type white luciferase luciferase and comparison of emission intensity and emission spectrum in HeLa cells]
1. Production of Mutant Lepidoptera Luciferase As described below, a mutant gene that expresses mutant Lepidoptera luciferase was produced.

  A mutation (S283L) was introduced into the luciferase gene (SEQ ID NO: 4) optimized for expression in mammalian cells, which replaces the 283rd serine in the amino acid sequence encoded by SEQ ID NO: 4 with leucine.

The luciferase gene (SEQ ID NO: 4) optimized for expression in mammalian cells is as follows.
ATGGACAACGACAAGAACATCGTGTACGGCCCCGAGCCCTTCTACCCCGTGGATAAGGGATCTGCCGGCGCTCAGATCCACAAGTACCTGCACCAGTACAGCAAGCTGGGCGCCATTGCCTTCACCAATGCCCTGACCGGCGTGGACATCACCTACGCCGAGTACTTCGACATCACCTGTCGGCTGGGCGAGGCCATGAAGAGATACGGCATCAAGGTGGACGGCCGGATCGCCCTGTGCAGCGAGAACTGCGAAGAGTTCTTCTACCCTGTGATCGCCGGCCTGTACATCGGAGCTGGCGTGGCCCCCACCAACGAGATCTACACCCTGCGGGAACTGGTGCACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAAGGCCTGGACAAGGTGCTGCATGTGCAGAAAACCGTGACCTGCATCAAGACCATCGTGATCCTGGACAGCAAGGTGGACTACAAGGGCTACGACTGCGTGGAATCCTTCATCAAGCGGTACAACCCCCCAGGCTTCCAGGCCGCCAGCTTCAAGACCGTGGAAGTGGACCGGAAAGAGCAGATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCTAAAGGCGTGCAGCTGACACACGAGTGCGCCGTGACCAGATTCAGCCACGCCAAGGACCCCATCTACGGCAACCAGGTGTCCCCTGGAACCGCCGTGCTGACCGTGGTGCCTTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTATCTGGCCTGCGGCTTCAGAGTGGTCATGCTGACCCGCTTCGACGAGGAAATCTTCCTGAAAACCATCCAGGACTACAAGTGCACCTCTGTGATCCTGGTGCCCACCCTGTTCGGCATCCTGAGCAAGAGCAAGCTGCTGGATAAGTACGACCTGAGCAACCTGGTGGAAATCGCCTCTGGCGGAGCCCCCCTGGCCAAAGAAGTGTCTGAAGCCGTCGCCAGACGGTTCAACCTGCCTG GCGTGCGGCAGGGCTACGGACTGACAGAGACAACCAGCGCCATCATCATCACCCCCGAGGGCGACGATAAGCCTGGCGCCTCTGGAAAGGTGGTGCCCCTGTTCAAGGCCAAGGTGGTGGACCTGGACACCAAGAAAACCCTGGGCCCCAACCAGCGGGGCGAAGTCTGTGTGAAGGGCCCCATGCTGATGAAGGGCTACGTGGACAACCCCGAGGCCACCAGAGAGATCATCGACGAGGACGGCTGGCTGCACACAGGCGACATCGGCTACTACGACGAGGACCAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAGTATAAGGGCTACCAGGTGCCCCCAGCCGAGCTGGAATCTGTGCTGCTGCAGCATCCCTACATCTTCGATGCCGGCGTGGCCGGGGTGCCAGATGCTGAAGCAGGCGAACTGCCTGGGGCTGTCGTGGTGCTGGAAAAGGGCAAGCACGTGACCGAGAAAGAAATCATGGACTACGTGGCCGGACAGGTGTCCAACGCCAAGAGACTGAGAGCCGGCGTGCGCTTCGTGGATGAAGTGCCTAAGGGCCTGACCGGCAAGATCGACGGCAAGGCCATCCGCGAGATCCTGATGAAGCCCAAGAGCAAGATGTGA

The following primers were used for introducing the mutations.
S283L mutation: (GATACCAAGTGCACCnnnnGTGATCCCTGGTCCCC: SEQ ID NO: 35) (nnn represents a sequence consisting of an arbitrary base)
Using this primer, a gene encoding a mutant luciferase that has an arbitrary modification at the 283rd amino acid, has an activity of inducing high-intensity luminescence, and has a luminescent color shifted to the long wavelength side did.

  Mutation was introduced based on the description in the literature (Asako Sawano and Atsushi Miyawaki, Nucleic Acids Research, 2000, Vol. 28, No. 16). After introduction of the mutation, the sequence was read using a sequencer, and it was confirmed that the target mutation was introduced into the squirrel luciferase gene and that the I148V mutation was further introduced. The I148V mutation is a mutation that was not originally intended and was accidentally substituted in PCR.

The produced sujiglobotaru mutant luciferase is referred to as red mutant 1. It has I148V and S283L mutations. This red variant 1 further has a mutation due to the addition of the Kozak sequence.
The amino acid sequence of red variant 1 is set forth in SEQ ID NO: 33, and the base sequence of the gene encoding red variant 1 is set forth in SEQ ID NO: 34.

The amino acid sequence of red mutant 1 (SEQ ID NO: 33) is as follows. Mutant amino acids are underlined.
MDNDKNIVYGPEPFYPVDKGSAGAQIHKYLHQYSKLGAIAFTNALTGVDITYAEYFDITCRLGEAMKRYGIKVDGRIALCSENCEEFFYPVIAGLYIGAGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVLHVQKTVTCIKT V VILDSKVDYKGYDCVESFIKRYNPPGFQAASFKTVEVDRKEQIALIMNSSGSTGLPKGVQLTHECAVTRFSHAKDPIYGNQVSPGTAVLTVVPFHHGFGMFTTLGYLACGFRVVMLTRFDEEIFLKTIQDYKCT L VILVPTLFGILSKSKLLDKYDLSNLVEIASGGAPLAKEVSEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDKPGASGKVVPLFKAKVVDLDTKKTLGPNQRGEVCVKGPMLMKGYVDNPEATREIIDEDGWLHTGDIGYYDEDQHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPYIFDAGVAGVPDAEAGELPGAVVVLEKGKHVTEKEIMDYVAGQVSNAKRLRAGVRFVDEVPKGLTGKIDGKAIREILMKPKSKM

The base sequence (SEQ ID NO: 34) of the gene encoding red variant 1 is as follows.
ATGGACAACGACAAGAACATCGTGTACGGCCCCGAGCCCTTCTACCCCGTGGATAAGGGATCTGCCGGCGCTCAGATCCACAAGTACCTGCACCAGTACAGCAAGCTGGGCGCCATTGCCTTCACCAATGCCCTGACCGGCGTGGACATCACCTACGCCGAGTACTTCGACATCACCTGTCGGCTGGGCGAGGCCATGAAGAGATACGGCATCAAGGTGGACGGCCGGATCGCCCTGTGCAGCGAGAACTGCGAAGAGTTCTTCTACCCTGTGATCGCCGGCCTGTACATCGGAGCTGGCGTGGCCCCCACCAACGAGATCTACACCCTGCGGGAACTGGTGCACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAAGGCCTGGACAAGGTGCTGCATGTGCAGAAAACCGTGACCTGCATCAAGACCGTCGTGATCCTGGACAGCAAGGTGGACTACAAGGGCTACGACTGCGTGGAATCCTTCATCAAGCGGTACAACCCCCCAGGCTTCCAGGCCGCCAGCTTCAAGACCGTGGAAGTGGACCGGAAAGAGCAGATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCTAAAGGCGTGCAGCTGACACACGAGTGCGCCGTGACCAGATTCAGCCACGCCAAGGACCCCATCTACGGCAACCAGGTGTCCCCTGGAACCGCCGTGCTGACCGTGGTGCCTTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTATCTGGCCTGCGGCTTCAGAGTGGTCATGCTGACCCGCTTCGACGAGGAAATCTTCCTGAAAACCATCCAGGACTACAAGTGCACCCTAGTGATCCTGGTGCCCACCCTGTTCGGCATCCTGAGCAAGAGCAAGCTGCTGGATAAGTACGACCTGAGCAACCTGGTGGAAATCGCCTCTGGCGGAGCCCCCCTGGCCAAAGAAGTGTCTGAAGCCGTCGCCAGACGGTTCAACCTGCCTG GCGTGCGGCAGGGCTACGGACTGACAGAGACAACCAGCGCCATCATCATCACCCCCGAGGGCGACGATAAGCCTGGCGCCTCTGGAAAGGTGGTGCCCCTGTTCAAGGCCAAGGTGGTGGACCTGGACACCAAGAAAACCCTGGGCCCCAACCAGCGGGGCGAAGTCTGTGTGAAGGGCCCCATGCTGATGAAGGGCTACGTGGACAACCCCGAGGCCACCAGAGAGATCATCGACGAGGACGGCTGGCTGCACACAGGCGACATCGGCTACTACGACGAGGACCAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAGTATAAGGGCTACCAGGTGCCCCCAGCCGAGCTGGAATCTGTGCTGCTGCAGCATCCCTACATCTTCGATGCCGGCGTGGCCGGGGTGCCAGATGCTGAAGCAGGCGAACTGCCTGGGGCTGTCGTGGTGCTGGAAAAGGGCAAGCACGTGACCGAGAAAGAAATCATGGACTACGTGGCCGGACAGGTGTCCAACGCCAAGAGACTGAGAGCCGGCGTGCGCTTCGTGGATGAAGTGCCTAAGGGCCTGACCGGCAAGATCGACGGCAAGGCCATCCGCGAGATCCTGATGAAGCCCAAGAGCAAGATGTGA

2. Comparison of luminescence intensity and luminescence spectrum in HeLa cells Next, the luminescence intensity in HeLa cells by wild squirrel firefly and red mutant 1 luciferase was measured, and these were measured with Photinus pyralis (Promega). Luc2 luciferase) and red luminescence luciferase CBR (Promega CBR luciferase) from beetles.

A Kozak sequence was added to the nucleic acid containing the gene for the wild white luciferase and the nucleic acid containing the gene for red mutant 1. Note that these two sequences (SEQ ID NO: 4 and SEQ ID NO: 34) are also codon optimized for expression in mammalian cells. These sequences were then inserted between the SgfI and PmeI sites of the multicloning site of pF9A CMV hRLuc neo Flexi vector (Promega), respectively. The pF9A vector contains the Renilla luciferase gene (hRLuc) as an internal control in the vector sequence, and the luminescence intensity of the luminescence gene inserted into the multicloning site can be calculated as the ratio of the luminescence intensity of Renilla luciferase. is there.
According to a similar procedure, a nucleic acid containing the genes for Photinus pyralis luciferase and CBR luciferase was inserted into the multicloning site of the pF9A vector.

Comparison of luminescence intensity was performed using Dual-Glo (registered trademark) Luciferase Assay System (product number E2940, manufactured by Promega Corp., USA). The four types of plasmids thus obtained were each transfected into HeLa cells seeded in a 48-well plate by the lipofection method, and after 24 hours, the cells were washed with PBS. 200 μl of ₂ mM D-luciferin / CO ₂ Independent Medium (Invitrogen) was added to each well of a 48-well plate, and the luminescence intensity was measured for 90 minutes using a Luminescence (ATTO) at 37 ° C. for 1 second per well. did. The luminescence intensity at the time when 90 minutes had elapsed was defined as the luminescence intensity of the four luciferases. Thereafter, coelenterazine was added according to the manual, and the luminescence intensity by Renilla luciferase as an internal control was measured at 37 ° C. for 1 second using a Luminescence sensor under the conditions of measurement for 1 second per well. Japanese black luciferase, red mutant 1, P. aeruginosa A value obtained by dividing the luminescence intensity by Pyralis luciferase (Luc2 luciferase, Promega) and CBR luciferase (Promega) by the luminescence intensity by Renilla luciferase was calculated, and the value was graphed as the luminescence intensity of each luciferase. The result is shown in FIG. In FIG. It is expressed as a ratio when the light emission intensity by pyralis luciferase is 1.

As shown in FIG. 3, the luminescence intensity obtained by the wild luciferase in HeLa cells The emission intensity by pyralis luciferase was 2.60 times. The emission intensity obtained with the red mutant 1 is P.P. It was 1.13 times the luminescence intensity by pyralis luciferase.
In addition, the emission spectra of various luciferases expressed in HeLa cells (Suji globot wild type, red mutant 1 and CBR) were measured in the same manner as in Example 2. The luciferin concentration was 2 mM, and the temperature was measured at two points of 25 ° C. and 37 ° C. The results are shown in FIGS. FIG. 4 shows the wild-type measurement results, FIG. 5 shows the red mutant 1 measurement results, and FIG. 6 shows the CBR measurement results.

FIG. 4 shows that as the temperature increases from 25 ° C. to 37 ° C., the maximum emission wavelength of the wild type shifts from 598 nm to 609 nm on the longer wavelength side. This is the same result as the spectrum change when changing from high pH to low pH shown in FIG.
Moreover, in the emission spectrum by the red variant 1 shown in FIG. 5, even if temperature rose from 25 degreeC to 37 degreeC, the clear shift of the maximum light emission wavelength was not seen. According to FIGS. 4, 5 and 6, in HeLa cells, red mutant 1 shows a maximum emission wavelength (616 nm) different from the wild type, and this emission wavelength is the maximum emission wavelength of commercially available red-emitting luciferase CBR. It was shown to be comparable to (620 nm). Further, FIG. 3 shows that the luminescence intensity of this red stripe red mutant 1 is 3.42 times that of CBR.

[Example 4: Comparison of luminescence intensity and spectrum by streak firefly luciferase in HeLa cells when a near-infrared luminescence luciferin analog is used as a substrate]
Four types of plasmids containing luciferase gene prepared in Example 3 (Suji Globotar wild type, red mutant 1, P. pyralis, and click-derived CBR) were introduced into HeLa cells in the same manner as in Example 3, and near-infrared. Luminescent luciferin analogs (dimethylaniline-diene luciferin, manufactured by Wako Co., Ltd., Akalumine (registered trademark)) were used as luminescent substrates, and emission spectra of each luciferase were measured. The concentration of the near-infrared emission luciferin analog was 1 mM, and the temperature was 37 ° C. 7 and 8 show the comparison of the measured emission intensity and the emission spectrum, respectively.

FIG. 8 shows that, when a near-infrared light emitting luciferin analog is used, the maximum emission wavelength of the emission spectrum of both wild-type and red mutant luciferases of sedges is 664 nm.
From FIG. It can be seen that the light intensity of 0.94 times is shown in the wild wild type and 0.65 times that in the red mutant 1 with respect to the pyralis. On the other hand, with respect to red luciferase (CBR) derived from rice, it can be seen that the wild type of Sugigrobottal is 3.13 times, and the red variant 1 shows 2.16 times the emission intensity.

FIG. 9 shows an emission spectrum obtained by a white-spotted firefly luciferase when D-luciferin and a near-infrared emitting luciferin analog are used as substrates.
From FIG. 9, when near-infrared light emitting luciferin analog is used as a substrate, the maximum emission wavelength of wild type s. Globular luciferase is mutated from 609 nm to 664 nm as compared with the case where D-luciferin is used as a substrate. It can be seen that the body 1 is shifted from 613 nm to 664 nm toward the longer wavelength side.

[Example 5: Production of improved red mutant luciferase and comparison of emission intensity and emission spectrum in HeLa cells]
1. Production of improved red mutant luciferase As described below, a mutant gene expressing the improved red mutant luciferase was produced by introducing further mutations into the red mutant 1 produced in Example 3.

(I) Red mutant 2
A mutation (F464I) that replaces the 464th phenylalanine of the amino acid sequence of SEQ ID NO: 33 with isoleucine is further added to the gene (SEQ ID NO: 34) encoding red variant 1 (SEQ ID NO: 33) prepared in Example 3. Introduced. The obtained mutant luciferase encoded by the mutant luciferase gene is referred to as red mutant 2. Red variant 2 has three mutations: I148V, S283L and F464I. The gene encoding this red mutant further has a mutation due to the addition of a Kozak sequence.

(Ii) Red mutant 3
Further, the following mutations (a) to (c) were further introduced into the gene (SEQ ID NO: 34) encoding the red mutant (SEQ ID NO: 33) prepared in Example 3:
(A) a mutation (V401M) that replaces the 401st valine of the amino acid sequence of SEQ ID NO: 33 with methionine;
(B) a mutation that replaces the 464th phenylalanine of the amino acid sequence of SEQ ID NO: 33 with isoleucine (F464I); and (c) a mutation that replaces the 503rd glycine of the amino acid sequence of SEQ ID NO: 33 with arginine (G503R).

  The obtained mutant luciferase encoded by the mutant luciferase gene is referred to as red mutant 3. Red variant 3 has five mutations: I148V, S283L, V401M, F464I and G503R. The gene encoding this red mutant further has a mutation due to the addition of a Kozak sequence.

(Iii) Red mutant 4
Further, the following mutations (d) to (g) were further introduced into the gene (SEQ ID NO: 34) encoding the red mutant (SEQ ID NO: 33) prepared in Example 3:
(D) a mutation (V239M) that substitutes methionine for the 239th valine in the amino acid sequence of SEQ ID NO: 33;
(E) a mutation that substitutes threonine for the 313th serine in the amino acid sequence of SEQ ID NO: 33 (S313T);
(F) a mutation that replaces the 356th aspartic acid of the amino acid sequence of SEQ ID NO: 33 with tyrosine (D356Y); and (g) a mutation that replaces the 464th phenylalanine of the amino acid sequence of SEQ ID NO: 33 with isoleucine (F464I).

  The obtained mutant luciferase encoded by the mutant luciferase gene is referred to as red mutant 4. Red variant 4 has six mutations: I148V, S283L, V239M, S313T, D356Y and F464I. The gene encoding this red mutant further has a mutation due to the addition of a Kozak sequence.

Each mutation was introduced according to the manual using GeneMorph II Random Mutagenesis kit (registered trademark) (Agilent Technology Co., Ltd.).
After mutagenesis, E. coli JM109 (DE3) strain was transformed by electroporation to form colonies on the agar medium. The colony was sprayed with D-Luciferin at a final concentration of 2 mM, and then luminescence was imaged with a CCD camera DP-70 (Olympus). Colonies that were brighter than the other colonies and showed red luminescence were picked up, and the sequence was confirmed using a sequencer.

  The mutations of red mutants 1 to 4 are summarized in Table 2 below. In Table 2, “◯” means that the red mutant has the corresponding mutation.

  The amino acid sequence of red variant 2 is set forth in SEQ ID NO: 36, and the base sequence of the gene encoding red variant 2 is set forth in SEQ ID NO: 37.

The amino acid sequence (SEQ ID NO: 36) of red variant 2 is as follows. Mutant amino acids are underlined.
MDNDKNIVYGPEPFYPVDKGSAGAQIHKYLHQYSKLGAIAFTNALTGVDITYAEYFDITCRLGEAMKRYGIKVDGRIALCSENCEEFFYPVIAGLYIGAGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVLHVQKTVTCIKT V VILDSKVDYKGYDCVESFIKRYNPPGFQAASFKTVEVDRKEQIALIMNSSGSTGLPKGVQLTHECAVTRFSHAKDPIYGNQVSPGTAVLTVVPFHHGFGMFTTLGYLACGFRVVMLTRFDEEIFLKTIQDYKCT L VILVPTLFGILSKSKLLDKYDLSNLVEIASGGAPLAKEVSEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDKPGASGKVVPLFKAKVVDLDTKKTLGPNQRGEVCVKGPMLMKGYVDNPEATREIIDEDGWLHTGDIGYYDEDQHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPYI I DAGVAGVPDAEAGELPGAVVVLEKGKHVTEKEIMDYVAGQVSNAKRLRAGVRFVDEVPKGLTGKIDGKAIREILMKPKSKM

The base sequence (SEQ ID NO: 37) of the gene encoding red variant 2 is as follows.
ATGGACAACGACAAGAACATCGTGTACGGCCCCGAGCCCTTCTACCCCGTGGATAAGGGATCTGCCGGCGCTCAGATCCACAAGTACCTGCACCAGTACAGCAAGCTGGGCGCCATTGCCTTCACCAATGCCCTGACCGGCGTGGACATCACCTACGCCGAGTACTTCGACATCACCTGTCGGCTGGGCGAGGCCATGAAGAGATACGGCATCAAGGTGGACGGCCGGATCGCCCTGTGCAGCGAGAACTGCGAAGAGTTCTTCTACCCTGTGATCGCCGGCCTGTACATCGGAGCTGGCGTGGCCCCCACCAACGAGATCTACACCCTGCGGGAACTGGTGCACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAAGGCCTGGACAAGGTGCTGCATGTGCAGAAAACCGTGACCTGCATCAAGACCGTCGTGATCCTGGACAGCAAGGTGGACTACAAGGGCTACGACTGCGTGGAATCCTTCATCAAGCGGTACAACCCCCCAGGCTTCCAGGCCGCCAGCTTCAAGACCGTGGAAGTGGACCGGAAAGAGCAGATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCTAAAGGCGTGCAGCTGACACACGAGTGCGCCGTGACCAGATTCAGCCACGCCAAGGACCCCATCTACGGCAACCAGGTGTCCCCTGGAACCGCCGTGCTGACCGTGGTGCCTTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTATCTGGCCTGCGGCTTCAGAGTGGTCATGCTGACCCGCTTCGACGAGGAAATCTTCCTGAAAACCATCCAGGACTACAAGTGCACCCTAGTGATCCTGGTGCCCACCCTGTTCGGCATCCTGAGCAAGAGCAAGCTGCTGGATAAGTACGACCTGAGCAACCTGGTGGAAATCGCCTCTGGCGGAGCCCCCCTGGCCAAAGAAGTGTCTGAAGCCGTCGCCAGACGGTTCAACCTGCCTG GCGTGCGGCAGGGCTACGGACTGACAGAGACAACCAGCGCCATCATCATCACCCCCGAGGGCGACGATAAGCCTGGCGCCTCTGGAAAGGTGGTGCCCCTGTTCAAGGCCAAGGTGGTGGACCTGGACACCAAGAAAACCCTGGGCCCCAACCAGCGGGGCGAAGTCTGTGTGAAGGGCCCCATGCTGATGAAGGGCTACGTGGACAACCCCGAGGCCACCAGAGAGATCATCGACGAGGACGGCTGGCTGCACACAGGCGACATCGGCTACTACGACGAGGACCAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAGTATAAGGGGTACCAGGTGCCCCCAGCCGAGCTGGAATCTGTGCTGCTGCAGCATCCCTACATCATCGATGCCGGCGTGGCCGGGGTGCCAGATGCTGAAGCAGGCGAACTGCCTGGGGCTGTCGTGGTGCTGGAAAAAGGCAAGCACGTGACCGAGAAAGAAATCATGGACTACGTGGCCGGACAGGTGTCCAACGCCAAGAGACTGAGAGCCGGCGTGCGCTTCGTGGATGAAGTGCCTAAGGGCCTGACCGGCAAGATCGACGGCAAGGCCATCCGCGAGATCCTGATGAAGCCCAAGAGCAAGATGTGA

  The amino acid sequence of red variant 3 is set forth in SEQ ID NO: 38, and the base sequence of the gene encoding red variant 3 is set forth in SEQ ID NO: 39.

The amino acid sequence of red mutant 3 (SEQ ID NO: 38) is as follows. Mutant amino acids are underlined.
MDNDKNIVYGPEPFYPVDKGSAGAQIHKYLHQYSKLGAIAFTNALTGVDITYAEYFDITCRLGEAMKRYGIKVDGRIALCSENCEEFFYPVIAGLYIGAGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVLHVQKTVTCIKT V VILDSKVDYKGYDCVESFIKRYNPPGFQAASFKTVEVDRKEQIALIMNSSGSTGLPKGVQLTHECAVTRFSHAKDPIYGNQVSPGTAVLTVVPFHHGFGMFTTLGYLACGFRVVMLTRFDEEIFLKTIQDYKCT L VILVPTLFGILSKSKLLDKYDLSNLVEIASGGAPLAKEVSEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDKPGASGKVVPLFKAKVVDLDTKKTLGPNQRGEVCVKGPMLMKGY M DNPEATREIIDEDGWLHTGDIGYYDEDQHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPYI I DAGVAGVPDAEAGELPGAVVVLEKGKHVTEKEIMDYVA R QVSNAKRLRAGVRFVDEVPKGLTGKIDGKAIREILMKPKSKM

The base sequence (SEQ ID NO: 39) of the gene encoding red variant 3 is as follows.
ATGGACAACGACAAGAACATCGTGTACGGCCCCGAGCCCTTCTACCCCGTGGATAAGGGATCTGCCGGCGCTCAGATCCACAAGTACCTGCACCAGTACAGCAAGCTGGGCGCCATTGCCTTCACCAATGCCCTGACCGGCGTGGACATCACCTACGCCGAGTACTTCGACATCACCTGTCGGCTGGGCGAGGCCATGAAGAGATACGGCATCAAGGTGGACGGCCGGATCGCCCTGTGCAGCGAGAACTGCGAAGAGTTCTTCTACCCTGTGATCGCCGGCCTGTACATCGGAGCTGGCGTGGCCCCCACCAACGAGATCTACACCCTGCGGGAACTGGTGCACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAAGGCCTGGACAAGGTGCTGCATGTGCAGAAAACCGTGACCTGCATCAAGACCGTCGTGATCCTGGACAGCAAGGTGGACTACAAGGGCTACGACTGCGTGGAATCCTTCATCAAGCGGTACAACCCCCCAGGCTTCCAGGCCGCCAGCTTCAAGACCGTGGAAGTGGACCGGAAAGAGCAGATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCTAAAGGCGTGCAGCTGACACACGAGTGCGCCGTGACCAGATTCAGCCACGCCAAGGACCCCATCTACGGCAACCAGGTGTCCCCTGGAACCGCCGTGCTGACCGTGGTGCCTTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTATCTGGCCTGCGGCTTCAGAGTGGTCATGCTGACCCGCTTCGACGAGGAAATCTTCCTGAAAACCATCCAGGACTACAAGTGCACCCTAGTGATCCTGGTGCCCACCCTGTTCGGCATCCTGAGCAAGAGCAAGCTGCTGGATAAGTACGACCTGAGCAACCTGGTGGAAATCGCCTCTGGCGGAGCCCCCCTGGCCAAAGAAGTGTCTGAAGCCGTCGCCAGACGGTTCAACCTGCCTG GCGTGCGGCAGGGCTACGGACTGACAGAGACAACCAGCGCCATCATCATCACCCCCGAGGGCGACGATAAGCCTGGCGCCTCTGGAAAGGTGGTGCCCCTGTTCAAGGCCAAGGTGGTGGACCTGGACACCAAGAAAACCCTGGGCCCCAACCAGCGGGGCGAGGTCTGTGTGAAGGGCCCCATGCTGATGAAGGGCTACATGGACAACCCCGAGGCCACCAGAGAGATCATCGACGAGGACGGCTGGCTGCACACAGGCGACATCGGCTACTACGACGAGGACCAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAGTATAAGGGCTACCAGGTGCCCCCAGCCGAGCTGGAATCTGTGCTGCTGCAGCATCCCTACATCATCGATGCCGGCGTGGCCGGGGTGCCAGATGCTGAAGCAGGCGAACTGCCTGGGGCTGTCGTGGTGCTGGAAAAGGGCAAGCACGTGACCGAGAAAGAAATCATGGACTACGTGGCCAGACAGGTGTCCAACGCCAAGAGACTGAGAGCCGGCGTGCGCTTCGTGGATGAAGTGCCTAAGGGCCTGACCGGCAAGATCGACGGCAAGGCCATCCGCGAGATCCTGATGAAGCCCAAGAGCAAGATGTGA

  The amino acid sequence of red variant 4 is set forth in SEQ ID NO: 40, and the base sequence of the gene encoding red variant 4 is set forth in SEQ ID NO: 41.

The amino acid sequence of red mutant 4 (SEQ ID NO: 40) is as follows. Mutant amino acids are underlined.
MDNDKNIVYGPEPFYPVDKGSAGAQIHKYLHQYSKLGAIAFTNALTGVDITYAEYFDITCRLGEAMKRYGIKVDGRIALCSENCEEFFYPVIAGLYIGAGVAPTNEIYTLRELVHSLGISKPTIVFSSKKGLDKVLHVQKTVTCIKT V VILDSKVDYKGYDCVESFIKRYNPPGFQAASFKTVEVDRKEQIALIMNSSGSTGLPKGVQLTHECAVTRFSHAKDPIYGNQVSPGTAVLT M VPFHHGFGMFTTLGYLACGFRVVMLTRFDEEIFLRTIQDYKCT L VILVPTLFGILSKSKLLDKYDLSNLVEIA T GGAPLAKEVSEAVARRFNLPGVRQGYGLTETTSAIIITPEGD Y KPGASGKVVPLFKAKVVDLDTKKTLGPNQRGEVCVKGPMLMKGYVDNPEATREIIDEDGWLHTGDIGYYDEDQHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPYI I DAGVAGVPDAEAGELPGAVVVLEKGKHVTEKEIMDYVAGQVSNAKRLRAGVRFVDEVPKGLTGKIDGKAIREILMKPKSKM

  The base sequence (SEQ ID NO: 41) of the gene encoding red variant 4 is as follows. ATGGACAACGACAAGAACATCGTGTACGGCCCCGAGCCCTTCTACCCCGTGGATAAGGGATCTGCCGGCGCTCAGATCCACAAGTACCTGCACCAGTACAGCAAGCTGGGCGCCATTGCCTTCACCAATGCCCTGACCGGCGTGGACATCACCTACGCCGAGTACTTCGACATCACCTGTCGGCTGGGCGAGGCCATGAAGAGATACGGCATCAAGGTGGACGGCCGGATCGCCCTGTGCAGCGAGAACTGCGAAGAGTTCTTCTACCCTGTGATCGCCGGCCTGTACATCGGAGCTGGCGTGGCCCCCACCAACGAGATCTACACCCTGCGGGAACTGGTGCACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAAGGCCTGGACAAGGTGCTGCATGTGCAGAAAACCGTGACCTGCATCAAGACCGTCGTGATCCTGGACAGCAAGGTGGACTACAAGGGCTACGACTGCGTGGAATCCTTCATCAAGCGGTACAACCCCCCAGGCTTCCAGGCCGCCAGCTTCAAGACCGTGGAAGTGGACCGGAAAGAGCAGATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCTAAAGGCGTGCAGCTGACACATGAGTGCGCCGTGACCAGATTCAGCCACGCCAAGGACCCCATCTACGGCAACCAGGTGTCCCCTGGAACCGCCGTGCTGACCATGGTGCCTTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTATCTGGCCTGCGGCTTCAGAGTGGTCATGCTGACCCGCTTCGACGAGGAAATCTTCCTGAGAACCATCCAGGATTACAAGTGCACCCTAGTGATCCTTGTGCCCACCCTGTTCGGCATCCTGAGCAAGAGCAAGCTGTTGGATAAGTACGACCTGAGCAACCTGGTGGAAATTGCCACTGGTGGAGCCCCCCTGGCCAAAGAAGTGTCTGAAGCCGTCGCCAGACGGTTCAACCTGCCTG GCGTGCGGCAGGGCTACGGACTGACAGAGACAACCAGCGCCATCATCATCACCCCCGAGGGCGACTATAAGCCTGGCGCCTCTGGAAAGGTGGTGCCCCTGTTCAAGGCCAAGGTGGTGGACCTGGACACCAAGAAAACCCTGGGCCCCAACCAGCGGGGCGAAGTCTGTGTGAAGGGCCCCATGCTGATGAAGGGCTACGTGGACAACCCCGAGGCCACCAGAGAGATCATCGACGAGGACGGCTGGCTGCACACAGGCGACATCGGCTACTACGACGAGGACCAGCACTTCTTCATCGTGGACCGGCTGAAGTCCCTGATCAAGTATAAGGGGTACCAGGTGCCCCCAGCCGAGCTGGAATCTGTGCTGCTGCAGCATCCCTACATCATCGATGCCGGCGTGGCCGGGGTGCCAGATGCTGAAGCAGGCGAACTGCCTGGGGCTGTCGTGGTGCTGGAAAAGGGCAAGCACGTGACCGAGAAAGAAATCATGGACTACGTGGCCGGACAGGTGTCCAACGCCAAGAGACTGAGAGCCGGCGTGCGCTTCGTGGATGAAGTGCCTAAGGGCCTGACCGGCAAGATCGACGGCAAGGCCATCCGCGAGATCCTGATGAAGCCCAAGAGCAAGATGTGA

2. Comparison of luminescence intensity and emission spectrum in HeLa cells Next, the luminescence intensity in HeLa cells by red mutants 1 to 4 was measured, and these were derived from Photinus pyralis-derived luciferase Luc2 (obtained from Promega) and click beetle The red luminescence luciferase CBR (Promega CBR luciferase) was compared.

  Like the red mutant 1, the mutant gene encoding the red mutants 2 to 4 is given a Kozak sequence and is codon-optimized for expression in mammalian cells. Mutant genes encoding red mutants 2 to 4 were inserted between the SgfI and PmeI sites of the multicloning site of pF9A CMV hRLuc neo Flexi vector (Promega), respectively. The pF9A vector contains the Renilla luciferase gene (hRLuc) as an internal control in the vector sequence, and the luminescence intensity of the luminescence gene inserted into the multicloning site can be calculated as the ratio of the luminescence intensity of Renilla luciferase. is there.

  According to the same procedure, the firefly-derived luciferase Luc2 (Promega Corporation) gene and the CBR luciferase (Promega Corporation) gene were each inserted into the multicloning site of the pF9A vector.

Comparison of luminescence intensity was performed using Dual-Glo (registered trademark) Luciferase Assay System (product number E2940, manufactured by Promega Corp., USA). The resulting 6 types of plasmids were each transfected into HeLa cells seeded in a 48-well plate by the lipofection method, and after 24 hours, the cells were washed with PBS. 200 μl of ₂ mM D-luciferin / CO ₂ Independent Medium (Invitrogen) was added to each well of a 48-well plate, and the luminescence intensity was measured for 90 minutes using a Luminescence (ATTO) at 37 ° C. for 1 second per well. did. The luminescence intensity after 90 minutes was defined as the luminescence intensity of 6 luciferases. Thereafter, coelenterazine was added according to the manual, and the luminescence intensity by Renilla luciferase as an internal control was measured at 37 ° C. for 1 second using a Luminescence sensor under the conditions of measurement for 1 second per well.

  Values obtained by dividing the luminescence intensities by the red mutants 1-4, firefly-derived luciferase Luc2 and CBR luciferase by the luminescence intensities by Renilla luciferase were calculated, and the values were graphed as the luminescence intensities of the respective luciferases. The result is shown in FIG. In FIG. 10, the luminescence intensity is expressed as a ratio when the luminescence intensity by firefly-derived luciferase Luc2 (Promega Corporation) is 1.

  As shown in FIG. 10, the luminescence intensities obtained by the red mutants 1 to 4 are about 1.1 times, about 3.2 times, and about 3.3 times the luminescence intensity by the firefly-derived luciferase Luc2, respectively. Times and about 3.2 times. Furthermore, FIG. 10 shows that the luminescence intensity of the red mutants 1 to 4 is about 8.3 times, about 23.1 times, about 24.2 times, and about 23.1 times that of CBR luciferase, respectively. It was done.

  Moreover, the emission spectrum of the red mutants 1-4 expressed in HeLa cells was measured in the same manner as in Example 2. The luciferin concentration was 2 mM and the temperature was measured at 37 ° C. The result is shown in FIG.

  From FIG. 11, red mutants 1 to 4 all have a maximum emission wavelength different from the wild type, red mutant 1 is about 616 nm, red mutant 2 is 610 nm, red mutant 3 is 609 nm, and red mutant. Body 4 exhibited a maximum emission wavelength of about 609 nm. FIG. 11 shows the emission spectrum of each red mutant, where the emission intensity at the maximum emission wavelength is 1. For this reason, the red mutant 1 has large fluctuations in the spectrum data. From the result of FIG. 11, it was shown that the maximum emission wavelengths of the red mutants 1 to 4 were comparable to the maximum emission wavelength (620 nm) of the commercially available red emission luciferase CBR.

Claims (8)

A luciferase comprising the amino acid sequence shown in any of SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40 .   2. The luciferase according to claim 1, which has an activity of inducing luminescence exhibiting a higher luminescence intensity than at least one of luminescence induced by Photinus pyralis firefly luciferase and luminescence induced by click beetle red luciferase. The luciferase according to claim 1, which induces luminescence having a maximum emission wavelength longer than the maximum emission wavelength of luminescence induced by luciferase comprising the amino acid sequence represented by SEQ ID NO: 1. The luciferase according to claim 3 , wherein the luminescence exhibits red luminescence. A luciferase as claimed in claim 1, comprising an activity to induce light emission showing a high emission intensity compared to wild type luciferase comprising an amino acid sequence shown in SEQ ID NO: 1, and shows a red light emitting luciferase. The luciferase according to claim 1 , which induces light emission having a maximum emission wavelength in the vicinity of 610 nm. A nucleic acid comprising a base sequence encoding the luciferase according to any one of claims 1 to 6 . The nucleic acid according to claim 7 , comprising the base sequence represented by SEQ ID NO: 37 , SEQ ID NO: 39 or SEQ ID NO: 41.

Images