JP2012125216A - Coleopteran luciferase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a mutant coleopteran luciferase having high luciferase activity and a proper Kvalue and an excellent intracellular ATP concentration measuring method using the same.SOLUTION: In the mutant coleopteran luciferase, Michaelis constant KM to the ATP is 0.3 mM or more and kis the value of 50% or more of kof a wild type coleopteran luciferase or the same before introducing mutation when analyzing enzymatic reaction by a model of Michaelis Menten.

Description

  The present invention relates to a beetle luciferase and an ATP concentration measurement method using the same.

  The beetle luciferase protein is a photoprotein possessed by beetles such as fireflies, railroad insects, and light clicks. The beetle luciferase protein catalyzes an oxidation reaction that produces oxyluciferin in an electronically excited state using D-luciferin, oxygen molecules, and ATP as substrates. The generated oxyluciferin in the electronically excited state emits energy as visible light when returning to the ground state. In this application, the beetle luciferase protein is sometimes collectively referred to as “luciferase”, and “luciferase activity” is applied to a series of reactions that combine the oxidation reaction catalyzed by luciferase and the subsequent chemiluminescence of oxyluciferin. "Or" luminescent activity "may be used.

  Luciferase is used for luminescence imaging. For example, luminescent imaging devices, including highly sensitive CCD cameras and bright optics, are now commercially available for imaging cells or tissues that express luciferase.

  Luminescence imaging has many advantages over fluorescence imaging with fluorescent proteins such as GFP. Luminescence imaging does not require excitation light for observation, so there is no fading of fluorescent molecules or damage to cells due to phototoxicity that occurs in fluorescence imaging, so quantitative observation is possible over a long period of several days or more. Is possible. In fluorescence imaging, autofluorescence derived from various pigment molecules in a living body becomes an obstacle to observation, but this does not occur in emission imaging. From the above points, the luminescence imaging method is used for various applications in a wide range of cells, tissues, and individuals.

Luciferase is also used for quantitative detection of ATP. ATP is an energy currency in the cell and a substrate for the phosphorylation reaction of the signal transduction system, and plays a very important role in the cell. Therefore, measurement of intracellular ATP concentration is important for cell function analysis. When the concentration of luciferase is constant and the concentration of one of the substrates (D-luciferin and ATP) in the sample is sufficiently high, the luminescence intensity of luciferase depends on the concentration of the other substrate. That is, by expressing luciferase with a promoter that is constantly expressed and sufficiently adding D-luciferin to the medium, it is possible to measure the ATP concentration in the cell over time while keeping the cell alive. Based on such a principle, it is possible to quantitatively detect a small amount of ATP of about 10 ⁻¹² M by using a highly sensitive photodetection device, and a reagent for realizing such measurement Kits are also commercially available.

  Non-Patent Document 1 and Non-Patent Document 2 describe methods for measuring ATP concentration in living cells using existing firefly luciferase. Also by these methods, the luminescence intensity of the firefly luciferase is measured. However, the existing firefly luciferase is already saturated at the intracellular ATP concentration, and a small change in the ATP concentration causes a small change in luminescence intensity, making it difficult to perform a highly reliable measurement. In addition, since the luminescence intensity of the photoprotein is extremely weak compared to the fluorescent protein, these methods have a limitation that a long-term exposure is required to obtain a clear image. For example, using a currently available luminescence microscope system (such as Olympus LV200) equipped with a high-sensitivity CCD camera and a bright optical system, it is typical to acquire weak luminescence of intracellular luciferase as an image. Requires several minutes of exposure time. The response time of changes in intracellular ATP concentration to external stimuli has been reported to be about 2 minutes in extreme cases such as when a respiratory inhibitor is added to the medium (Non-patent Document 3). From this point, it is not desirable to further extend the exposure time from the current order of several minutes in order to accurately measure changes in intracellular ATP concentration over time. In addition, since the quantum efficiency of current commercially available high-sensitivity CCD cameras (ImagEM manufactured by Hamamatsu Photonics Co., Ltd.) has reached 90% or more in the emission wavelength band of luciferase, the present measurement system has been improved as long as there is no major breakthrough. It cannot be expected to significantly reduce the exposure time. Therefore, in order to detect intracellular ATP concentration and its time change with high sensitivity, for example, by luminescence imaging, luciferase having high luciferase activity and high responsiveness to ATP is required.

Luciferase activity and responsiveness to ATP are discussed using enzymological parameters. That is, the activity of luciferase is analyzed based on the Michaelis-Menten model. When the concentration of D-luciferin is sufficiently high and the ATP concentration at the start of the reaction is [ATP] ₀ , the initial reaction rate v ₀ is

と表すことができる。ここで、Ｋ_ＭはＡＴＰに対するミカエリス定数である。また、ｖ_０は、発生する光子数、すなわち発光強度と比例関係にある。Ｖ_ｍａｘは、ルシフェラーゼの濃度［ＬＵＣ］_{ｔｏｔａｌ}に比例する。すなわち、

It can be expressed as. Here, _{K M} is the Michaelis constant for ATP. V ₀ is proportional to the number of generated photons, that is, the emission intensity. V _max is proportional to the luciferase concentration [LUC] _total . That is,

という関係が成り立つ。ここで、［ＬＵＣ］_{ｔｏｔａｌ}と表記したのは、ＡＴＰと結合したルシフェラーゼと結合していないルシフェラーゼとを区別せずに、反応系に存在するすべてのルシフェラーゼの濃度であることを示すためである。ｋ_ｃａｔは、基質が十分に存在する時の、ルシフェラーゼの単位時間および単位濃度あたりの発光活性に対応しており、「ルシフェラーゼの明るさ」と考えることができる。

This relationship holds. Here, the expression [LUC] _total is intended to indicate the concentration of all luciferases present in the reaction system without distinguishing between luciferase bound to ATP and luciferase not bound. k _cat corresponds to the luminescence activity per unit time and unit concentration of luciferase when the substrate is sufficiently present, and can be considered as “brightness of luciferase”.

1, for the two luciferase K _M are different, the relationship between the emission intensity and the ATP concentration is obtained by plotting calculated from Equation 1. Ordinate two dashed lines parallel to that shown in Figure 1 is a line representing the K _M of each curve. That represents a K _M for the curve left of the dashed line is made from the solid line represents the K _M for the curve right broken line consisting of a broken line. As can be seen from FIG. 1, the luminescence intensity of luciferase increases rapidly as the ATP concentration increases. When ATP concentration is a low concentration compared to K _M, it illustrates the relationship is close to proportional to the ATP concentration and the emission intensity. However, the ATP concentration is greater than K _M, the amount of change in emission intensity with respect to the change amount of the ATP concentration decreases. For example, the ATP concentration reaches three times the value of K _M, the emission intensity of the luciferase already reached 75% of the maximum emission intensity, the change in rise even luminous intensity is ATP concentration more small. In such a concentration range, it becomes difficult to measure the ATP concentration.

From the above, preferred conditions of the luciferase of high responsiveness to high and ATP are the luciferase activity, in other words the condition that the value 3 times the value of the k _cat is larger and K _M as possible is greater than ATP concentration range to be measured be able to.

Although the ATP concentration in living tissue varies depending on the organ and animal species from which it is derived, it has been reported that it is about 2.1 mM for humans (Non-Patent Document 4). There is a report of 1.3 mM (Non-Patent Document 2 and Non-Patent Document 5). Therefore, considering the condition of _{K M} described above, the lower limit value of _{K M} which is suitable for the measurement of ATP concentration in these cells is 0.3 mm to 0.7 mm.

However, K _M of the wild-type Coleoptera luciferase has been reported to date is lower than 0.2mM. Therefore, mutant increased the K _M by introducing a mutation into wild-type Coleoptera luciferase has been produced (Non-Patent Document 6 and Patent Document 1). However, in the mutants described in Non-Patent Document 6, mutations were introduced within a range of 5 mm or less from the substrate binding site, and in most of them, the luminescence activity (that is, the value of k _cat ) was greatly reduced. Further, although Patent Document 1 does not specifically describe k _cat , as in Non-Patent Document 6, the mutation introduction site is present in a portion close to the substrate binding site. Therefore, also in the mutant luciferase described in Patent Document 1, it is expected that the luminescence activity is greatly reduced. Therefore, it is difficult to perform an ideal ATP concentration measurement using a wild-type or mutant luciferase known so far.

US Pat. No. 6,265,177

Ainskow, E. K. et al. (2002), Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K + channels, Journal of Physiology, 544.2, 429-445. Kennedy, H. J. et al. (1999), Glucose generates sub-plasma membrane ATP microdomains in single islet β-cells, Journal of Biological Chemistry, 274, 13281-13291. Imamura, H. et al. (2009), Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators, Proceedings of the National Academy of Science of the United States of America, 106, 15651-15656. Traut, T. W. (1994), Physiological concentrations of purines and pyrimidines, Molecular and Cellular Biochemistry, 140, 1-22. Zamaraeva, M. V. et al. (2005), Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase, Cell Death and Differentiation, 12, 1390-1397. Branchini, B. R. et al. (2003), A mutagenesis study of the putative luciferin binding site residues of firefly luciferase, Biochemistry, 42, 10429-10436.

An object of the present invention is to provide a mutant beetle luciferase and superior intracellular ATP concentration measuring method using the with high luciferase activity and a suitable K _M values.

According to an embodiment of the present invention, Michaelis enzyme reaction - when analyzed by model Menten, Michaelis constant K _M for ATP is greater than or equal to 0.3 mM, and k _cat is the wild-type or mutated previous Coleoptera A mutant beetle luciferase is provided that has a value of 50% or more of the k _cat of luciferase.

  According to the present invention, the exposure time can be shortened and the ATP concentration can be measured with high accuracy.

In the catalytic reaction of K _M has two different luciferase view showing the relationship between the emission intensity and ATP concentrations. The figure which shows the three-dimensional structure model of Yaeyamahimebotaruluciferase. In various Yaeyama Himebotaru mutant luciferase, diagram showing the relationship between the K _M and k _cat. The figure which shows the ATP density | concentration dependence of the emitted light intensity of GL3 luciferase and its variant, and the analysis result by the Michaelis-Menten model.

One aspect of the present invention, the enzymatic reaction Michaelis - when analyzed by Menten model, Michaelis constant K _M for ATP is greater than or equal to 0.3 mM, and k _cat is earlier wild-type or mutated beetle luciferases It is a mutant beetle luciferase having a value of 50% or more of k _cat . In such a mutant beetle luciferase, a mutation may be introduced at a site separated by 5 mm or more from the substrate binding site in the three-dimensional structure of the protein. Furthermore, the site into which the mutation has been introduced may be at least one basic amino acid residue before the mutation introduction.

(Background to Invention)
First, the background of the inventor of the present invention will be described.
As described above, Non-Patent Document 6, as a result of introducing a mutation in the vicinity of the substrate binding site, it decreases at the same time k _cat when K _M rises against ATP, or K _M for D- luciferin is rising. The present inventors, for such a phenomenon, is prevented from substrates by introducing a mutation in the vicinity of the substrate binding site takes a preferred arrangement the reaction, at the same time as positive change that increases the K _M for ATP, luminescence activity increase of _{K M} for lowering and D- luciferin _{(k cat)} were considered to have occurred.

On the other hand, the present inventor hypothesized that luciferase has evolved to collect substrates such as ATP and luciferin in the active site in order to emit light with high efficiency, that is, to increase reaction efficiency. For example, it was considered that ATP and luciferin in contact with the luciferase surface were aggregated toward the active site as a reaction field. In order to realize such a mechanism, it was considered that a site (for example, a side chain of an amino acid residue) suitable for binding to ATP was present on the surface of the molecule and at a position away from the active site (that is, the substrate binding site). Then, if correct, this assumption, by introducing a mutation at this site, it is possible to lower the efficiency (ie the value of K _M) for taking the ATP, the progress of the reaction of the substrate to each other reaching the active site at the same time It was considered that it could be maintained at the same level as the wild type (that is, a high k _cat was obtained).

ATP exists as MgATP ^2- in physiological solution and is negatively charged. Therefore, as side chains preferred by ATP, side chains of basic amino acid residues having a positive charge, for example, side chains of arginine and lysine were examined. In addition, the position of such amino acid residues on the protein tertiary structure was predicted to be located on the surface of the protein and in the hemisphere on the side centered on the depression to which the substrate is bound.

  The three-dimensional structure of firefly luciferase is that of North American firefly (Contin E. et al. (1996), Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes, Structure, 4, 287-298) and Genji firefly. (Lucio cruciata) (Nakatsu, T. et al. (2006), Structural basis for the spectral difference in luciferase bioluminescence, Nature, 440, 372-376) has been reported. North American firefly luciferase and Genji firefly luciferase have 68% homology on the amino acid sequence, but the structures of these firefly luciferases are in good agreement except for the positional relationship between two major domains linked by a flexible linker. ing. From this, it is speculated that the overall structure such as the fold and domain structure is common in the beetle luciferase.

  The present inventor created a structural model of Yaeyama firefly luciferase (YaeLUC) using the three-dimensional structure of Geni firefly luciferase with a known crystal structure bound to a substrate analog as a template (FIG. 2), and based on the structural model, The ATP binding site as described above was searched. YaeLUC is a firefly luciferase (SEQ ID NO: 1) cloned by Akiyoshi et al. From Luciola filiformis yayeyamana (WO 2009/075306), 66% between North American firefly luciferase and Genji firefly luciferase There is 81% homology between them. Focusing on this point, it was used to construct a structural model based on homology and to specify the positions of amino acid residues near the substrate.

Based on the strategy described above, the present inventors designed and produced mutants for various sites and examined the affinity for ATP and the luminescence activity. It was possible to obtain a mutant luciferase that decreased and at the same time maintained high luminescence activity. For example, a mutant obtained by substituting lysine at position 447 (K447), lysine at position 535 (K535) or arginine at position 538 (R538) with a neutral or acidic amino acid residue of YaeLUC has a luminescence activity (k _cat value). ) is while maintaining a high value of 50% or more relative to the wild type, _{K M} value for ATP became more 0.3 mM.

These sites into which mutations have been introduced are located at least 5 mm away from the substrate binding site (site where ATP is present) on the structural model (FIG. 2), and the reaction between ATP and luciferin and subsequent luminescence are directly performed. Is reasonably understood not to inhibit. Further, the multiple mutants a combination of these mutations, the effect of lowering the K _M for the high maintaining and ATP light emission activity was found to be obtained synergistically.

(Mutant beetle luciferase)
Next, aspects of the present invention will be described.
One aspect of the present invention, the enzymatic reaction Michaelis - when analyzed by Menten model, Michaelis constant K _M for ATP is greater than or equal to 0.3 mM, and k _cat is earlier wild-type or mutated beetle luciferases It is a mutant beetle luciferase having a value of 50% or more of k _cat .

  The luciferase according to one embodiment of the present invention is derived from a beetle. Coleoptera is a general term for insects classified into the order Coleoptera, for example, insects belonging to the family Lampyridae, the family Phengodidae or the family Elateroidae. Examples of the insects belonging to the firefly family include the firefly (Luciola filformis yayeyamana), the genji firefly (Luciola cruciata), the mosquito firefly (Luciola lateralis), and the North American firefly (Photinus pyrus). Insects belonging to the family Fengodidae are, for example, railway insects (Prixothrix hirtus). Insects belonging to the family Beetleidae are, for example, Hikari Kometsuki (Pyrophorus plagiophthalamus).

  The luciferase according to one embodiment of the present invention is a mutant luciferase. That is, it is a luciferase obtained by introducing a mutation into a known wild-type beetle luciferase or a mutant thereof or a newly identified wild-type beetle luciferase or a mutant thereof. The known wild type beetle luciferase or variant thereof may be a commercially available luciferase. Here, the mutation means, for example, amino acid substitution, deletion or addition in the amino acid sequence, or a combination thereof. This mutation may be a mutation that occurs in one amino acid residue or a mutation that occurs in two amino acid residues in the amino acid sequence of wild-type luciferase. It may be a mutation occurring in 4 or more amino acid residues. Mutations in a plurality of amino acid residues may occur in consecutive amino acid residues or may occur in amino acid residues separated from each other. Mutant luciferase is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the wild type luciferase. There may be homology.

  Such mutations may have been introduced by methods common in the relevant technical field. For example, it may be obtained by introducing a mutation into DNA encoding luciferase protein. An example of such an introduction method is a method in which a mutation is introduced into a nucleic acid corresponding to an amino acid sequence to which a mutation is to be introduced in DNA containing a nucleotide sequence of a luciferase gene into which the mutation is to be introduced. Introduction of mutations in DNA can be easily performed using commercially available kits such as QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The mutant luciferase may correspond to the full length of the wild type luciferase, or may correspond to a protein lacking a part of the wild type luciferase. The mutant luciferase may include a part or all of a protein other than the wild-type luciferase. For example, the mutant luciferase may be a protein in which a protein derived from wild-type luciferase and a protein derived from another protein are fused.

In luciferase aspect of the present invention, the Michaelis constant _{K M} for ATP is at least 0.3 mM, preferably 0.7mM or more. It referred to herein, Michaelis The Michaelis constant K _M for ATP, shown in the reaction of ATP and luciferin being catalysed by luciferase, if luciferin is sufficiently present, the relationship between ATP concentration and the reaction rate - Menten it is a _{K M} in the formula. That is, Michaelis shown in Equation 3 below - a K _M of Menten equation.

この式において、ｖは反応速度または発光強度を意味し、［ＡＴＰ］はＡＴＰ濃度を意味する。また、Ｖ_ｍａｘはＡＴＰが十分量存在するときの反応速度を意味する。図１は、この式から算出されるＡＴＰ濃度と反応速度との関係を、ＡＴＰ濃度を横軸とし発光強度を縦軸としてプロットしたものである。この図には、Ｖ_ｍａｘは同一であるが、Ｋ_Ｍが異なる２つの曲線が示される。すなわち、これらの曲線のＫ_Ｍは、一方が０．１ｍＭであり、他方が１．０ｍＭである。この図からわかるように、ＡＴＰ濃度の増大とともに発光強度が増大するものの、ＡＴＰ濃度が一定値を超えると発光強度が飽和してしまい、ほとんど増大しなくなる。このように発光強度が飽和した範囲では、ＡＴＰ濃度を測定することが困難であり、発光強度が飽和する前の範囲が、ＡＴＰ濃度の測定に適していることがわかる。Ｋ_Ｍの異なる２つの曲線を比較した場合、Ｋ_Ｍが大きいルシフェラーゼの方が測定に適した範囲（飽和前の範囲）が広いことがわかる。図１によれば、Ｋ_Ｍが０．１ｍＭの場合、例えば０．５ｍＭを超えるＡＴＰ濃度の範囲にて測定を行うことは困難であるが、Ｋ_Ｍが１．０ｍＭの場合には、図示される最大のＡＴＰ濃度である２．０ｍＭ付近でも測定可能であることがわかる。上述の通り、ヒトの細胞ではＡＴＰ濃度が約２．１ｍＭと報告され、培養細胞では１〜１．３ｍＭと報告されているが、Ｋ_Ｍが０．３ｍＭ以上であれば１ｍＭ付近の測定に利用でき、０．７ｍＭ以上であれば２．１ｍＭ付近の測定にも利用できる。

In this formula, v means reaction rate or luminescence intensity, and [ATP] means ATP concentration. V _max means the reaction rate when a sufficient amount of ATP is present. FIG. 1 is a plot of the relationship between the ATP concentration calculated from this equation and the reaction rate, with the ATP concentration as the horizontal axis and the luminescence intensity as the vertical axis. In this _{figure, V max} but are identical, _{K M} is two curves different is shown. That, _{K M} of these curves, one is 0.1 mM, the other is 1.0 mM. As can be seen from this figure, although the emission intensity increases as the ATP concentration increases, the emission intensity saturates and hardly increases when the ATP concentration exceeds a certain value. Thus, it can be seen that it is difficult to measure the ATP concentration in the range where the emission intensity is saturated, and the range before the emission intensity is saturated is suitable for the measurement of the ATP concentration. When comparing two curves having different K _M, it is understood that ranges towards K _M is greater luciferase suitable for measurement (range before saturation) is wide. According to FIG. 1, when _{K M} is 0.1 mM, for example it is difficult to perform the measurement within a range of ATP concentrations greater than 0.5 mM, if _{K M} is 1.0mM is illustrated It can be seen that measurement is possible even at around 2.0 mM which is the maximum ATP concentration. As described above, the human cell is reported ATP concentration of about 2.1 mM, it has been reported to 1~1.3mM in cultured cells, used to measure near 1mM if _{K M} is 0.3mM or more If it is 0.7 mM or more, it can be used for measurement in the vicinity of 2.1 mM.

In the mutant beetle luciferase according to one embodiment of the present invention, k _cat is a value that is 50% or more, preferably 60% or more of the wild type or k _cat of the beetle luciferase before mutagenesis. That is, one mode of the mutant beetle luciferase of the invention, when comparing the k _cat between its previous mutagenesis luciferase, k _cat of the mutant beetle luciferase, k _cat luciferase before mutagenesis Of 50% or more. k _cat is the luminescence activity per unit time and unit concentration of luciferase when the substrate is sufficiently present, and satisfies the following formula 4.

ここにおいて、［ＬＵＣ］_{ｔｏｔａｌ}とは、反応系に存在する全てのルシフェラーゼの濃度である。ｋ_ｃａｔが発光活性の最大値Ｖ_ｍａｘと比例することを考えれば、ｋ_ｃａｔが５０％以上であるという特定は、変異型ルシフェラーゼの発光活性が野生型ルシフェラーゼの発光活性の５０％以上であることを意味する。また、変異型ルシフェラーゼのｋ_ｃａｔは、野生型甲虫類ルシフェラーゼのｋ_ｃａｔの１００％以上であってもよい。すなわち、変異型ルシフェラーゼの方が、野生型ルシフェラーゼよりも高い発光活性を示してもよい。例えば、変異型ルシフェラーゼが、ＡＴＰの応答性に関する変異に加えて、発光活性を高めるような変異を含むことで、野生型よりも強く光るルシフェラーゼであってよい。

Here, [LUC] _total is the concentration of all luciferases present in the reaction system. Given that k _cat is proportional to the maximum value V _max of emission activity and identify that k _cat is 50% or more, the luminescent activity of the mutant luciferase is 50% or more of the luminescent activity of the wild type luciferase Means. Also, _{k cat} of the mutant luciferase, may be 100% or more of the _{k cat} of the wild-type Coleoptera luciferase. That is, mutant luciferase may exhibit higher luminescence activity than wild-type luciferase. For example, the mutant luciferase may be a luciferase that shines stronger than the wild type by including a mutation that enhances the luminescence activity in addition to the mutation related to ATP responsiveness.

  Although the mutant beetle luciferase of one embodiment of the present invention has a low affinity for ATP as compared to the wild type, it maintains a high luminescence activity. By using such a luciferase, measurement in a concentration range that cannot be measured by existing luciferase is possible, and even if measurement is possible, measurement is possible in a range that is not practical due to low emission intensity. Furthermore, not only the measurement of ATP concentration but also various applications utilizing the characteristics of mutant beetle luciferase can be expected.

In the mutant beetle luciferase of one embodiment of the present invention, a mutation may be introduced at a site separated by 5 mm or more from the substrate binding site in the three-dimensional structure of the protein. That is, at least one of the mutations in the luciferase may be present at a site separated by 5 mm or more from the substrate binding site. The substrate binding site is a site in luciferase that ATP stably arranges when a reaction between ATP and luciferin occurs. In other words, the substrate binding site is the active site. Mutations that are present at a site distant from the substrate binding site 5Å or more, to minimize adverse effects on luminescence activity (i.e. suppressing a decrease in k _cat), increasing the ATP response (i.e. increasing the K _M) is it can. Further, the mutation may be introduced 5 Å or more away from the substrate binding site and on the surface of the luciferase protein.

  The distance from the substrate binding site can be calculated using, for example, luciferase whose crystal structure of the co-crystal with the substrate or substrate analog is known. For example, since the crystal structure of Genibotaruluciferase is known, this is used (Nakatsu, T. et al. (2006), Structural basis for the spectral difference in luciferase bioluminescence, Nature, 440, 372-376). First, in Genji firefly luciferase, a site separated by 5 mm or more from the substrate binding site is specified. Next, in consideration of amino acid sequence homology between the luciferase according to one embodiment of the present invention and the geniferal luciferase, it is possible to specify a site separated by 5 cm or more in the luciferase according to one embodiment of the present invention.

  Alternatively, a structural model of luciferase according to one embodiment of the present invention can be prepared, and a site separated by 5 cm or more can be identified on the model. Specifically, a structural model is created by structure prediction based on sequence homology with a protein with a known structure, and the distance from the substrate binding site is calculated on the model. For the production of the structural model, for example, an online service such as SWISS-MODEL (http://swissmodel.expasy.org/) can be used. FIG. 2 shows a structural model of wild-type Yaeyama firefly luciferase created using this service. This structural model was created using a template structure of a co-crystal structure (PDB ID: 2D1S) of gentian firefly luciferase and the substrate analog 5′-O- [N- (dehydroluciferyl) -sulfamoyl] adenosine (DLSA). did. By superimposing DLSA in the template structure on this structural model, it is possible to estimate the substrate binding site in the structural model of wild-type Yaeyama luciferase.

  In the mutant beetle luciferase of one embodiment of the present invention, the site where the mutation is introduced may be at least one basic amino acid residue before the mutation is introduced. That is, the mutant beetle luciferase may be a luciferase obtained by introducing a mutation into this basic amino acid residue. The number of basic amino acid residues is not limited to one, but may be plural. The term “before mutation introduction” as used herein means that the protein before mutation introduction includes not only wild-type luciferase but also luciferase in which some mutation has already been introduced. The basic amino acid is an amino acid having basicity by having two or more amino groups, for example, lysine, arginine or histidine. In such a mutant beetle luciferase, mutations may be introduced into other residues in addition to the basic amino acid residues.

  In the mutant beetle luciferase of one embodiment of the present invention, the basic amino acid residue into which the mutation is introduced is an amino acid residue corresponding to the lysine at position 447 of the wild-type Yaeyama luciferase in the sequence comparison based on homology, It may be an amino acid residue corresponding to lysine at position 535 or an amino acid residue corresponding to arginine at position 538. That is, for any beetle luciferase, when the homology of the amino acid sequence was examined with wild-type pine luciferase, it corresponds to the lysine at position 447, 535-position lysine or 538-position arginine of wild-type pine luciferase. Mutations may be introduced into amino acid residues in any beetle luciferase.

  It is known that beetle luciferases generally have high homology. For example, Hikari Kometsuki and Firefly have about 50% homology between luciferases, although the taxonomy is different. There are also many sites that are completely conserved among all beetle luciferases. Therefore, it is technically easy to specify an amino acid residue corresponding to a specific amino acid residue (for example, lysine residue at position 447) of wild-type Yaeyama luciferase in a luciferase derived from another species.

  Such identification can be performed using a known sequence comparison program such as ClustalW (http://www.clustal.org/). In this case, sequence comparison can be performed by inputting the sequences of two luciferases to be compared and executing the program. Here, if the wild-type pine luciferase is input as one of the two sequences, it is possible to easily identify which position in the other luciferase corresponds to the residue at the specific position of the wild-type pine luciferase.

  Alternatively, a luciferase having a known crystal structure of a co-crystal with a substrate or a substrate analog (eg, Genji firefly luciferase (Nakatsu, T. et al. (2006), Structural basis for the spectral difference in luciferase bioluminescence, Nature, 440, 372-376)) makes it possible to identify where a particular residue of any luciferase corresponds on a known luciferase protein conformation.

  FIG. 2 shows a model of the three-dimensional structure of the Yaeyama firefly luciferase. In this figure, lysine at position 447, lysine at position 535 and arginine at position 538 are shown. As can be seen from this figure, these residues are located away from the substrate binding site (site where DLSA is located) and near the surface of the protein. On the other hand, in the mutant North American firefly luciferase disclosed in Patent Document 1, mutations are introduced into residues at positions 245 and 318. According to the homology search, these positions correspond to positions 247 and 320 of the Yaeyama firefly luciferase and are located in the vicinity of the substrate binding site as shown in FIG. That is, the position of lysine at position 447, lysine at position 535, and arginine at position 538 is greatly different from the mutation introduction site in the known mutant luciferase, at least with respect to the distance from the substrate binding site.

  Table 1 below shows residues corresponding to the lysine residue at position 447, the lysine residue at position 535, and the arginine residue at position 538 in a representative beetle luciferase.

既知の各ルシフェラーゼについて、表１に示される残基に変異を導入することで本発明の一態様に係る変異型甲虫類ルシフェラーゼを作製することができる。

For each known luciferase, a mutant beetle luciferase according to one embodiment of the present invention can be produced by introducing mutations into the residues shown in Table 1.

The mutation to be introduced is preferably a mutation that cancels the electrical properties of the original amino acid residue. If the original amino acid residue is a basic residue, a mutation that cancels the property of being positively charged is preferable, that is, a mutation that replaces the basic residue with an acidic residue or a neutral residue. It is preferable that To decrease the affinity of the ATP, that in order to increase the K _M, it is preferable to replace the basic residues in acidic residues. For example, substitution to glutamic acid, aspartic acid or asparagine is preferable. However, K _M values and k _cat values suitable for the measurement, can be appropriately selected depending on the stability of the luciferase.

The mutant beetle luciferase of one embodiment of the present invention may include a plurality of mutations as described above. That is, the basic amino acid residue into which the mutation is introduced is the amino acid residue corresponding to the lysine at position 447 of the wild type wild luciferase in the sequence comparison based on homology, and the amino acid residue corresponding to the lysine at position 535 and Two or more amino acid residues selected from the group consisting of amino acid residues corresponding to arginine at position 538 may be used. Thus, variant luciferase mutation was introduced to the plurality of basic amino acid residues, synergistically K _M will increase.

  Specific examples of the mutant beetle luciferase of one embodiment of the present invention include a K447E mutant (SEQ ID NO: 3), a K535D mutant (SEQ ID NO: 5), a K535E mutant (SEQ ID NO: 7), and a K535N mutant (sequence). No. 9), R538E mutant (SEQ ID NO: 11) and K447E / K535E mutant (SEQ ID NO: 13). These are all mutants obtained by substituting specific amino acid residues in wild-type Yaeyama firefly luciferase. That is, the K447E mutant is a mutant in which the lysine residue at position 447 is substituted with glutamic acid, the K535D mutant is a mutant in which the lysine residue at position 535 is replaced with aspartic acid, and the K535E mutant is A mutant in which the lysine residue at position 535 is substituted with glutamic acid, the K535N mutant is a mutant in which the lysine residue at position 535 is substituted with asparagine, and the mutant R538E is an arginine at position 538 with glutamic acid. The K447E / K535E mutant is a mutant in which the lysine residue at position 447 is replaced with glutamic acid and the lysine residue at position 535 is replaced with glutamic acid. Positions 447, 535, and 538 that have introduced mutations in these mutants are located away from the substrate binding site, as shown in FIG.

(Nucleic acid)
Another aspect of the present invention is a nucleic acid comprising a base sequence encoding a mutant beetle luciferase protein. Such a base sequence may be a base sequence derived from a beetle. “Derived” as used herein means that the base sequence defined herein includes not only the wild-type base sequence inherent in the organism belonging to the beetle but also the base sequence in which the mutation has occurred. . The mutation herein refers to substitution, deletion and / or addition of a specific base in the base sequence. The nucleotide sequence mutation includes a mutation that does not cause a change in the encoded amino acid sequence. The nucleic acid particularly refers to DNA or RNA.

  Preferred examples of such a nucleic acid include a base sequence encoding the K447E mutant (SEQ ID NO: 4), a base sequence encoding the K535D mutant (SEQ ID NO: 6), and a base sequence encoding the K535E mutant (SEQ ID NO: 8). , A nucleic acid comprising a nucleotide sequence encoding a K535N variant (SEQ ID NO: 10), a nucleotide sequence encoding a R538E variant (SEQ ID NO: 12), or a nucleotide sequence encoding a K447E / K535E variant (SEQ ID NO: 14).

  Yet another embodiment of the present invention is a vector containing such a nucleic acid. In addition to the nucleic acid encoding luciferase, the vector may include a nucleic acid containing a sequence for regulating expression or a marker gene sequence. Moreover, you may include the gene of another protein. The vector is, for example, an expression vector.

(Transformants and transductants)
Another aspect of the present invention is a transformant or transductant comprising a nucleic acid as described above. A transformant means an organism (particularly a cell) that has taken in nucleic acid from the outside. The cell is introduced with the nucleic acid containing the mutant beetle luciferase gene according to one embodiment of the present invention, and as a result, retains the nucleic acid containing such a gene. Such a cell may be, for example, an animal cell, a plant cell, or a yeast cell. A transformant can be prepared by a general method in a related technical field. For example, a nucleic acid can be introduced into a cell or the like by a calcium phosphate method, lipofection, electroporation, or the like. The transductant means a bacterium into which a nucleic acid has been introduced from the outside by bacteriophage infection. A transductant can be prepared by a general method in a related technical field, for example, by introducing a bacteriophage carrying a target nucleic acid in advance and infecting it with a bacterium. Can do. Such a bacterium is, for example, E. coli.

(Production method)
Another embodiment of the present invention is a method for producing a mutant beetle luciferase. The production method includes a step of expressing a mutant beetle luciferase in a transformant or a transductant as described above, or a step of synthesizing a mutant beetle luciferase in a cell-free expression system. When transformants or transductants are used, mutant beetle luciferases are expressed by culturing them under conditions suitable for protein expression. Thereafter, the mutant beetle luciferase is purified as necessary. As these specific methods, those common in the related technical field can be used. In addition, cell-free expression systems that are common in related technical fields can be used. For example, RNA encoding mutant luciferase is added to a buffer containing ribosomes, amino acids, and the like necessary for translation. Add to synthesize luciferase.

[Example 1]
Various mutant Yaeyama firefly luciferases were prepared, and responsiveness to ATP and luminescence activity were examined.

(Preparation of expression vector)
Literature (Sambrook, J and Russell, DW (2001), Molecular Cloning:. A laboratory manual, 3 rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) according to the method described in, Yaeyama cloned from a cDNA library Expression of YaeLUC by inserting a DNA containing a nucleotide sequence (SEQ ID NO: 2) (International Publication No. 2009/075306) encoding Japanese firefly luciferase (YaeLUC) between the BamHI and EcoRI sites of the pRSET-B vector (Invitrogen) A vector was prepared. From the expression vector, wild-type Yaeyama firefly luciferase (SEQ ID NO: 1) is expressed.

  Mutation was introduced into the wild-type luciferase DNA to prepare an expression vector for the mutant-type luciferase. Specifically, using a set of complementary oligo DNAs having mutations at the site of interest and QuikChange II Site-Directed Mutagenesis Kit (Stratagene), they were prepared according to the method described in the manual of the kit. Table 2 below summarizes the mutants produced, the set of oligo DNAs used, the introduced mutations, the resulting base sequences and amino acid sequences.

なお、Ｋ４４７Ｅ／Ｋ５３５Ｅ変異体については、Ｋ４４７Ｅ変異体の発現ベクターを作製した後、さらにＫ５３５Ｅの変異を導入することで作製した。

The K447E / K535E mutant was prepared by preparing a K447E mutant expression vector and then introducing a K535E mutation.

  The base sequence of each mutant is confirmed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130XL Genetic Analyzer (Applied Biosystems), and the target mutation is introduced into the region encoding the protein. It was also confirmed that there were no unintended mutations due to PCR errors.

(Purification of luciferase protein)
Next, a mutant luciferase protein was prepared in E. coli using the expression vector prepared as described above.

  First, each expression vector prepared as described above was transformed into E. coli for expression. Add 0.5 μL of luciferase expression vector (approximately 10 ng of DNA) to 50 μL of competent cells of E. coli strain JM109 (DE3), leave it on ice for 30 minutes, and then treat it in a 42 ° C. heat bath for 45 seconds, then again. Let stand on ice for 2 minutes. Then, 0.5 mL of LB medium was added, and after shaking culture at 37 ° C. for 20 minutes, 100 μL of the culture solution was applied to an LB medium plate containing ampicillin. The plate was statically cultured overnight at 37 ° C., and one of the resulting colonies was arbitrarily selected and inoculated into LB medium containing 2 mL of ampicillin, followed by shaking culture at 37 ° C. and 130 rpm. After the growth of Escherichia coli was visually confirmed after 4 to 6 hours, the cells were transferred to LB medium containing 250 mL of ampicillin and cultured at 30 ° C. at 100 rpm overnight (14 to 18 hours). After completion of the culture, E. coli was collected by centrifuging at 4 ° C. and 8000 rpm for 10 minutes. The obtained cell paste is suspended in 25 mL of a washing buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA, pH 7.5), and centrifuged again at 4 ° C. and 8000 rpm for 10 minutes. Went. The obtained microbial cells (wet weight of about 1 g) were stored frozen at −80 ° C. until purification work.

  The frozen cells were suspended in 10 mL of disruption buffer (50 mM Tris-HCl, 150 mM NaCl, 25 mM imidazole, 1 mM DTT, 0.5 mM PMSF, pH 7.5), and the sample was placed on ice for ultrasonic disruption. . For ultrasonic disruption, an ultrasonic homogenizer VP-050 manufactured by Taitec Co., Ltd. was used, and a cycle consisting of a treatment for 5 seconds at an output power of 50% and a rest for 5 seconds thereafter was performed for 15 minutes. After crushing, the suspension was transferred to a 30 mL centrifuge tube and centrifuged at 4 ° C. and 15000 rpm for 30 minutes. The supernatant after centrifugation was suspended in 0.5 mL of Ni-NTA agarose resin (Qiagen) equilibrated with a crushing buffer, followed by inversion stirring (about 6 rpm) at 4 ° C. for 1 hour.

  Next, the Ni-NTA agarose resin suspension was poured into a φ10 mm × 50 mm open column (Bio-rad), and the resin was laminated at the bottom of the column. After all the suspension had passed through the column, 10 mL of disruption buffer was poured into the column in several portions to wash the resin. 1.2 mL of elution buffer (50 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, 1 mM DTT, pH 7.5) was gently added from the top of the column, and the eluate was fractionated by about 180 μL each. The fraction after elution was immediately stored on ice. The protein concentration of each fraction was calculated using the Bradford method protein assay kit (Bio-rad), and the 2-3 fractions with the highest concentration were collected as Ni-NTA elution fractions. Immediately after determining the recovered fraction, desalting was performed by gel filtration. The Ni-NTA elution fraction sample was applied to a NAP-10 column (GE Healthcare) equilibrated with a desalting buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.5). I waited until Desalting buffer was added so that the total amount with the sample was 1 mL, and after waiting until the buffer soaked, 1.5 mL of desalting buffer was added, and the eluate was fractionated by about 200 μL each. The fraction after elution was immediately stored on ice. The protein concentration of each fraction was calculated by the Bradford method protein assay kit, and the fraction with the highest concentration was recovered as the desalted elution fraction. The same amount of storage buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM DTT, 90% v / v glycerol, pH 7.5) as the eluted fraction was added and stored in a −30 ° C. freezer.

(Measurement of luminous activity)
Luminescent activity was measured for each purified luciferase.
The ATP solution was prepared as follows. 0.3 g of ATP disodium salt trihydrate (Roche # 5199179) was dissolved in 3.5 mL of water, and 1 M NaOH solution was added while measuring pH to adjust the pH to 7.0. Absorbance at 259 nm was measured, and finally adjusted by adding water to a concentration of 100 mM using an extinction coefficient ε = 15400 (M ⁻¹ cm ⁻¹ ) of ATP. Place 25 μL of substrate solution (100 mM Tris-HCl, 20 mM MgSO ₄ , 800 μM D-(−)-luciferin, 0-20 mM ATP, pH 8.0) into the well of a black 96-well assay plate (Thermo Fisher Scientific). The luminescence intensity was measured over time from the time when 75 μL of a luciferase solution (2 μg / mL luciferase, 100 mM Tris-HCl, pH 8.0) was added. A 100 μL reaction solution immediately after adding 75 μL luciferase solution to 25 μL substrate solution contains 100 mM Tris-HCl, 5 mM MgSO ₄ , 200 μM D-(−)-luciferin, 1.5 μg / mL (25 nM) luciferase, ATP was prepared so that 8 wells contained 0 mM, 0.001 mM, 0.005 mM, 0.02 mM, 0.1 mM, 0.5 mM, 2.5 mM and 5 mM, respectively. For automatic dispensing of the luciferase solution and measurement of the luminescence intensity, Luminesencer JNR-II manufactured by ATTO, equipped with an automatic dispenser and a photomultiplier tube, was used.

(Analysis of emission intensity data)
In order to calculate the enzymatic parameters of luciferase for ATP, the luminescence activity data was evaluated by the Michaelis-Menten model shown in Equation 3. The derivation of K _M value and V _max values, deriving a value near the peak of the temporal change of the emission intensity at each ATP concentration as v _0, the parameter fitting Equation 3 into data v ₀ values for different ATP concentrations did. For fitting the equations, a non-linear least square method based on the Levenberg-Markert method was used.

(result)
_{K M} value determined in each mutant and summarized relative _{k cat} values in Table 3. Further, in FIG. 3, is plotted as a vertical axis relative _{k cat} was _{K M} and the horizontal axis. In Figure 3, the broken lines parallel to the horizontal axis means a linear relative k _cat value is 50%, the parallel broken lines in the vertical axis means lines K _M value is 0.3 mM.

表３および図３の結果から、各変異体のＫ_Ｍ値は野生型ルシフェラーゼと比較して増大しており、一方で、ｋ_ｃａｔ値は野生型ルシフェラーゼと比較して高い水準に維持されていることがわかる。また、Ｋ４４７Ｅ／Ｋ５３５Ｅ変異体は、Ｋ４４７Ｅ変異体およびＫ５３５変異体と比較してＫ_Ｍ値が増大している。このことは、変異を二重で導入することで、相乗的にＫ_Ｍ値を増大する効果が得られることを意味する。

From the results of Table 3 and Figure 3, K _M values for each mutant is increased compared to the wild-type luciferase, while, k _cat values are maintained at high levels compared to the wild-type luciferase I understand that. Further, K447E / K535E mutant, _{K M} value is increased compared with the K447E mutant and K535 mutants. This means that the effect of synergistically increasing the _KM value can be obtained by double introduction of mutations.

[Example 2]
A mutant of GL3 luciferase was prepared, and responsiveness to ATP and luminescence activity were examined. In addition, since GL3 luciferase is obtained by introducing a mutation of N50D / N119G / S548I / K549A / L550V to North American firefly luciferase, the following examples are based on the North American firefly luciferase mutant. This is also an example in which additional mutations are introduced.

(Preparation of expression vector)
The gene portion (SEQ ID NO: 25) encoding luciferase incorporated in the pGL3-Basic (Promega) vector was inserted between the BamHI and EcoRI sites of the pRSET-B vector (Invitrogen) in the same manner as in Example 1. A GL3 expression vector was prepared. GL3 luciferase (SEQ ID NO: 26) is expressed from the expression vector.

  Mutation was introduced into the GL3 luciferase DNA to prepare a mutant GL3 expression vector. Specifically, it was prepared by the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) using two types of oligo DNAs having mutations at the K445 and R533 sites of GL3 and complementary to the sense strand. The K445E mutagenesis oligo DNA sequence is shown in SEQ ID NO: 27, and the R533E mutagenesis oligo DNA sequence is shown in SEQ ID NO: 28, respectively. The nucleotide sequence of the obtained mutant GL3 is shown in SEQ ID NO: 29, and its amino acid sequence is shown in SEQ ID NO: 30.

  The base sequence of each mutant is confirmed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130XL Genetic Analyzer (Applied Biosystems), and the target mutation is introduced into the region encoding the protein. It was also confirmed that there were no unintended mutations due to PCR errors.

(Purification of luciferase protein)
Using the above GL3 and mutant GL3 expression vector, a luciferase protein was produced by the method described in Example 1 and purified.

(Measurement of luminous activity)
The luminescence activity of the purified GL3 and mutant GL3 protein was measured by the method described in Example 1. A 100 μL reaction solution immediately after adding 75 μL luciferase solution to 25 μL substrate solution contains 100 mM Tris-HCl, 5 mM MgSO ₄ , 200 μM D-(−)-luciferin, 1.5 μg / mL (25 nM) luciferase, ATP is added to 16 adjacent wells in two rows, 0 mM, 0.0002 mM, 0.0005 mM, 0.001 mM, 0.002 mM, 0.005 mM, 0.01 mM, 0.04 mM, 0.1 mM, 0.2 mM, It was prepared to contain 0.5 mM, 1 mM, 2 mM, 4 mM and 5 mM.

(Analysis of emission intensity data)
FIG. 4 summarizes the luminescence intensity of GL3 luciferase and its variants under the above ATP concentration conditions. The result of applying Equation 3 to the measurement result is shown by the solid line in FIG. The parameters obtained by equation fitting are summarized in Table 4.

Claims (11)

The enzymatic reaction Michaelis - when analyzed by Menten model, Michaelis constant _{K M} for ATP is greater than or equal to 0.3 mM, and _{k cat} is the wild-type or prior mutagenesis beetle luciferase _{k cat} 50% or more of Mutant beetle luciferase that is value.   The mutant beetle luciferase according to claim 1, wherein a mutation is introduced into a site separated by 5 mm or more from the substrate binding site in the three-dimensional structure of the protein.   The mutant beetle luciferase according to claim 2, wherein the site into which a mutation has been introduced is at least one basic amino acid residue prior to the introduction of the mutation.   4. The mutant beetle luciferase according to claim 3, wherein the basic amino acid residue is an amino acid residue corresponding to a lysine residue at position 447 of wild-type Yaeyama firefly luciferase in sequence comparison based on homology.   The mutant beetle luciferase according to claim 3, wherein the basic amino acid residue is an amino acid residue corresponding to a lysine residue at position 535 of wild-type Yaeyama firefly luciferase in sequence comparison based on homology.   4. The mutant beetle luciferase according to claim 3, wherein the basic amino acid residue is an amino acid residue corresponding to the arginine residue at position 538 of wild-type Yaeyama firefly luciferase in sequence comparison based on homology.   In the sequence comparison based on homology, the basic amino acid residue is the amino acid residue corresponding to the lysine residue at position 447 of the wild type wild luciferase, the amino acid residue corresponding to the lysine residue at position 535, and the 538 position. The mutant beetle luciferase according to claim 3, which is two or more amino acid residues selected from the group consisting of amino acid residues corresponding to the arginine residues.   A nucleic acid comprising a base sequence encoding the mutant beetle luciferase protein according to any one of claims 1 to 7.   A transformant comprising the nucleic acid according to claim 8.   A transductant comprising the nucleic acid according to claim 8.   A process comprising expressing a mutant beetle luciferase in the transformant according to claim 9 or the transductant according to claim 10, or synthesizing the mutant beetle luciferase in a cell-free expression system. Item 8. A method for producing a mutant beetle luciferase according to any one of Items 1 to 7.

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