JP5382687B2 - Composition and method for suppressing decrease in luminescence intensity in luciferin-luciferase reaction - Google Patents

Description

  The present invention relates to a composition and method for suppressing a decrease in luminescence intensity that occurs with the progress of a reaction between Cypridina luciferase and luciferin. Furthermore, this invention provides the new knowledge regarding suppressing the light emission intensity by Cypridina luciferase with time.

  Luciferase is an enzyme that emits light by catalyzing the oxidation of luciferin. For this reason, luciferase is also known as a luminescent enzyme. Various luciferases from various organisms are known, for example, luciferases derived from firefly (Photinus pyralis), Renilla, railroad worms, worms, copepods, oplophorus, etc. It has been known. Each luciferase has a different structure and characteristics (for example, substrate specificity, thermal stability, luminescence stability, luminescence intensity, etc.).

  The light emitted by the oxidation reaction of luciferin catalyzed by luciferase (hereinafter also referred to as luciferin-luciferase reaction) can be detected using a luminometer, a special optical microscope, or the like. Therefore, luciferase is excellent as a reporter protein, and allows biological phenomena in living cells and organisms to be observed in real time. Thus, luciferase is widely used in biological tests and research. The main application of such a luciferin luciferase system is a reporter assay, which evaluates the influence of foreign factors such as drug candidates on the living body, analyzes signal transduction between cells, Is widely used for observing and measuring the expression of target genes. Reporter assays are also used to quantitatively measure target substances such as proteins in living bodies.

  Since there are many other uses besides the above, many luciferases including recombinant luciferases have been used to date. Among them, Cypridina luciferase has excellent stability near temperature and pH close to the body environment of mammals such as humans, can be produced in E. coli and human cells, and is secreted extracellularly. Therefore, it is one of the most commonly used luciferases at present.

  As described above, luciferase is a very useful luminescent enzyme, but has the following problems. That is, as the luciferin-luciferase reaction proceeds, the luminescence intensity decreases. This problem is particularly noticeable when a reporter assay using luciferase is performed on a large number of samples (for example, when a 96-well plate is used). Dispensing luciferin or luciferase to all wells of a 96-well plate usually takes 10 minutes or more, even for a skilled artisan. Therefore, there is a gap between the time when the reaction starts and the time when the emission intensity decreases. Therefore, the luminescence intensity in each sample at a specific time after the start of the reaction cannot be directly compared. Therefore, it is required to suppress a decrease in luminescence intensity associated with the luciferase reaction.

In relation to such a problem of decrease in luminescence intensity due to luciferase, Patent Document 1 discloses that the luminescence intensity by Gaussia luciferase over time using NP-40 and Triton X-100 which are nonionic surfactants. It is disclosed that a significant decrease was suppressed. On the other hand, Patent Document 1 discloses that an anionic surfactant (sodium dodecyl sulfate) does not suppress a decrease in luminescence intensity but rather inactivates the luciferase (for example, FIG. 2). reference). The action of such a nonionic surfactant and an anionic surfactant is the same for Renilla luciferase (see FIG. 6).
WO2006 / 096735A2

  Under such circumstances, an object of the present invention is to provide a means for suppressing the intensity of light emitted by the reaction between Cypridina luciferase and luciferin over time.

  As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that the anionic surfactant does not deactivate Cypridina luciferase, and the luminescence intensity over time by Cypridina luciferase. It was found that the decrease was suppressed. In addition, as a result of further studies, the present inventors have found that the action of suppressing the luminescence intensity by an anionic surfactant is enhanced by the presence of a nonionic surfactant. The present invention has been completed as a result of further improvements based on these findings.

That is, the present invention provides the following composition and method for suppressing the decrease in the luminescence intensity of Cypridina luciferase over time.
Item 1. A composition for suppressing a decrease in luminescence intensity over time due to sea urchin luciferase, comprising at least one anionic surfactant.
Item 2. Item 2. The composition according to Item 1, further comprising at least one nonionic surfactant.
Item 3. Item 2. The composition according to Item 1, wherein the anionic surfactant is sodium dodecyl sulfate.
Item 4. Item 2. The composition according to Item 1, wherein the nonionic surfactant is polyethylene glycol-p-octylphenyl ether (NP-40) and / or polyoxyethylene octylphenyl ether (Triton X-100).
Item 5. A method for suppressing a decrease in luminescence intensity over time caused by a sea urchin luciferase, which comprises reacting a sea urchin luciferase with luciferin in the presence of at least one anionic surfactant.
Item 6. Item 6. The method according to Item 5, wherein in the reaction between Cypridina luciferase and luciferin, at least one nonionic surfactant is further present.
Item 7. Item 6. The method according to Item 5, wherein the anionic surfactant is sodium dodecyl sulfate.
Item 8. Item 7. The method according to Item 6, wherein the nonionic surfactant is polyethylene glycol-p-octylphenyl ether (NP-40) and / or polyoxyethylene octylphenyl ether (Triton X-100).
Item 9. A suppressant for the temporal decrease in emission intensity by Cypridina luciferase, comprising an anionic surfactant.

  According to the present invention, it is possible to suppress a decrease in luminescence intensity over time due to Cypridina luciferase. In one embodiment, the emission intensity of 50% or more, preferably 60% or more, more preferably 70% or more, and particularly preferably 80% or more compared to the emission intensity at the start of the reaction is 3 minutes after the start of the reaction. Maintained at the time. In another embodiment, the luminescence intensity is 14% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 60% or more compared to the luminescence intensity at the start of the reaction, 15 minutes after the start of the reaction. Has been maintained at the time.

  In one embodiment, the present invention is a composition for suppressing a decrease in luminescence intensity over time caused by a sea urchin luciferase, comprising at least one anionic surfactant. In another embodiment, the present invention is a method for suppressing a decrease in luminescence intensity over time caused by luciferase, which comprises reacting sea urchin luciferase with luciferin in the presence of at least one surfactant.

Wild-type Cypridina luciferase is a monomeric glycoprotein having a molecular weight of about 62 kDa, can be expressed in mammalian cells, and is secreted. Unlike firefly luciferase, which is ATP-requiring, Cypridina luciferase does not require cofactors other than water and O ₂ to oxidize luciferin and emit light. The optimum pH of Cypridina luciferase is about 7.2, and the enzyme works well at salt concentrations in mammalian cells. Due to these properties, Cypridina luciferase is suitable for assays using mammalian cells.

  The amino acid sequence of wild-type Cypridina luciferase is shown in SEQ ID NO: 1. The base sequence of wild-type Cypridina luciferase is registered in the NCBI database as access number AAA86460, AAA30332, or BAD08210. A vector incorporating a gene encoding the amino acid sequence of Cypridina luciferase is commercially available from, for example, Ato Corporation. Therefore, based on such information, the sea urchin luciferase that can be used in the present invention can be easily obtained by a known genetic engineering technique or chemical synthesis method.

  Recombinant Cypridina luciferase can also be used as Cypridina luciferase as long as the recombinant has the ability to catalyze the oxidation of luciferin and emit light. Recombinant luciferase can be obtained using any known genetic engineering technique such as site-directed mutagenesis. The recombinant Cypridina luciferase preferably has 85% or more homology with the amino acid sequence of the wild Cypridina luciferase, more preferably 95% or more, and even more preferably 95% or more. And most preferably has a homology of 98% or more.

  In the present invention, luciferin is a substrate of Cypridina luciferase, and is a substance that emits light as a result of being oxidized. A preferred example of luciferin is Cypridina luciferin produced by Cypridina, which is represented by Formula 1 below.

Cypridina luciferin is commercially available from, for example, Ato Corporation. Cypridina luciferin analogs can also be used as long as they have the ability to emit light as a result of oxidation by the catalysis by Cypridina luciferase. Since such an analog of luciferin has been disclosed by the present inventors (Tetrahedron Letters, vol. 47, p. 753 (2006)), it can be synthesized according to the description.

  The “decrease in luminescence intensity over time” means a decrease in luminescence intensity that occurs as the reaction between Cypridina luciferase and luciferin proceeds. The decrease in luminescence intensity usually begins immediately after Cypridina luciferase and luciferin are mixed in solution, and the rate of decrease is not constant. Therefore, in the present invention, the “decrease with time” is not limited to that proportional to the reaction time, but means any reduction in luminescence intensity that occurs after the reaction between luciferin and luciferase has started. In many cases, as shown in FIG. 1, a relatively rapid decrease occurs immediately after the start of the reaction, and then gradually changes to a more gradual decrease. The cause of such a decrease in emission intensity is not yet fully known, but is probably due to product inhibition in the oxidation reaction. As the oxidation of luciferin proceeds, oxidized luciferin accumulates in the reaction solution, which slows the reaction rate, resulting in a decrease in light intensity. Since the decrease in luminescence intensity is observed even when an excessive amount of luciferin is supplied, it is not considered to be caused by a lack of substrate.

  The decrease in emission intensity over time is suppressed by the composition of the present invention containing at least one anionic surfactant. That is, the decrease in luminescence intensity is suppressed by reacting Cypridina luciferase with luciferin in the presence of at least one anionic surfactant. The anionic surfactant that can be used in the present invention is not particularly limited as long as the decrease in the emission intensity can be suppressed. Such specific anionic surfactants include carboxylates such as sodium cholate, sodium laurate, sodium myristate, sodium N-lauroyl sarcosine, sodium oleate, sodium palmitate, sodium stearate; -Sodium pentadecane sulfonate, sodium 1-dodecane sulfonate, sodium 1-hexadecane sulfonate, sodium 1-octadecane sulfonate, sodium 1-tetradecane sulfonate, sodium 1-tridecane sulfonate, di (2-ethylhexyl) sulfosuccinate Sulfonates such as sodium, dimethyl sodium 5-sulfoisophthalate and sodium dodecylbenzenesulfonate; phosphate esters such as sodium monododecyl phosphate; dodecyl sulfate Contained thorium (SDS), and like sulfuric acid ester salts such as hexadecyl sodium sulfate. These anionic surfactants are commercially available. The composition of the present invention may contain one of these anionic surfactants alone, or may contain a combination of two or more anionic surfactants. Preferred anionic surfactants are those classified as sulfate salts, and more preferably sodium dodecyl sulfate.

  In the present invention, the anionic surfactant can be used alone without being combined with other substances. In such an embodiment, the anionic surfactant can be recognized as a reduction inhibitor of the emission intensity by Cypridina luciferase.

  Cypridina luciferase differs from other luciferases in that it has 17 disulfide bonds in addition to sugar chains. Due to such structural features, this enzyme is believed to have excellent stability. And because of this structure and excellent stability, Cypridina luciferase does not lose enzymatic activity in the presence of relatively strong surfactants such as SDS, but rather in the presence of such surfactants, As a result, it is considered that a decrease in light emission intensity over time is suppressed.

  In one preferred embodiment, the composition of the present invention comprising at least one anionic surfactant further comprises at least one nonionic surfactant. The effect of suppressing the decrease in luminescence intensity over time is enhanced by reacting Cypridina luciferase with luciferin in the presence of at least one anionic surfactant and at least one nonionic surfactant. be able to. Thus, the nonionic surfactant used in combination with the anionic surfactant is not particularly limited as long as the inhibitory effect by the anionic surfactant can be enhanced. Examples of such nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene sorbitan monoester. Ester-ether surfactants such as oleate (Tween 80) and polyoxyethylene sorbitan trioleate (Tween 85); monomyristin, monopalmitin, monostearin, polyethylene glycol monostearate, sorbitan laurate (Span 20), sorbitan mono Palmitate (Span 40), sorbitan monostearate (Span 60), sorbitan monooleate (Span 80), sorbitan sesquiolee (Span 83) and ester surfactants such as sorbitan trioleate (Span 85); diethylene glycol monododecyl ether, ethylene glycol monododecyl ether, polyethylene glycol mono-4-octylphenyl ether, tetraethylene glycol monododecyl ether, triethylene Ether surfactants such as glycol monododecyl ether, polyoxyethylene octyl phenyl ether (Triton X-100) and polyethylene glycol-p-octyl phenyl ether (NP-40) are included. These and other nonionic surfactants that can be used in the present invention are commercially available. Preferred nonionic surfactants are ether surfactants, more preferably Triton-X100 and NP-40. The composition of the present invention may contain one nonionic surfactant or a combination of two or more nonionic surfactants in combination with one or more anionic surfactants. Good.

  When the composition of the present invention comprising at least one anionic surfactant further comprises at least one nonionic surfactant, the anionic surfactant and the nonionic surfactant are cypress firefly luciferase. As long as the decrease in the luminescence intensity due to time can be suppressed, they can be used in any combination, and they can be combined in any ratio. A preferred combination is a combination of one or more anionic surfactants classified into sulfate ester salts and one or more nonionic surfactants selected from ether surfactants. A more preferable combination is one or more nonionic surfactants selected from SDS and ether surfactants, and more preferably a combination of SDS and Triton X-100, or SDS and NP-40. It is a combination.

  The anionic surfactant and the nonionic surfactant are combined at a weight ratio of 1: 1 to 1:10, and more preferably 1: 1 to 1: 5.

  In one preferred embodiment, the composition of the present invention comprising at least one anionic surfactant and optionally at least one nonionic surfactant further comprises at least one calcium compound. Cypridina luciferase by reacting Cypridina luciferase and luciferin in the presence of at least one anionic surfactant, optionally at least one nonionic surfactant, and at least one calcium ion The light emission intensity due to is increased. This is thought to be due to the activation of Cypridina luciferase by the presence of calcium ions. A calcium compound is a calcium-containing compound that can release calcium ions in solution. Therefore, it is considered that the calcium compound can enhance the luminescence intensity due to the sea firefly luciferase and further enhance the action of suppressing the decrease in the luminescence intensity over time due to the anionic sea surface active agent. Calcium compounds that can be used in the present invention are commercially available, for example, inorganic calcium compounds such as calcium oxide, calcium hydroxide, calcium chloride, calcium phosphate, and calcium sulfate; and calcium carbonate, calcium citrate And organic calcium compounds such as calcium malate, calcium maleate, calcium lactate, calcium formate, calcium gluconate, calcium glycerophosphate, calcium levulinate, and calcium tartrate. A preferred calcium compound is an inorganic calcium compound, more preferably calcium hydrochloride. The composition of the present invention comprising at least one anionic surfactant and optionally at least one nonionic surfactant may further comprise one calcium compound, and two or more calcium compounds. May be included in combination.

  The composition of the present invention may appropriately contain substances usually added in the luciferin-luciferase reaction (for example, BSA, preservatives, stabilizers, etc.) in addition to the surfactant and the calcium compound.

  The composition of the present invention containing at least one anionic sea surface active agent can be prepared in any form, and can be prepared, for example, in powder or liquid form. The composition in such a form can be prepared according to a method known in the art. For example, the composition of the present invention comprises at least one anionic surfactant, at least one nonionic surfactant, and at least one calcium compound in powder form or in liquid form (eg, water or various In the buffer))). The powdery composition of the present invention can be used in advance or immediately after the start of the reaction in a reaction solution of Cypridina luciferase and luciferin. Preferably, the composition of the present invention is used by adding to the reaction solution prior to the luciferin-luciferase reaction.

  The composition of the present invention can be added to the reaction solution at any concentration as long as it can suppress a decrease in light emission intensity over time. Preferably, the composition of the present invention has a final concentration of the anionic surfactant of 0.01 to 1.0% by weight, more preferably 0.1 to 0.5% by weight, and the concentration of the nonionic surfactant. However, it is added in an amount such that 0.01 to 1.0% by weight, more preferably 0.1 to 0.5% by weight, and the calcium ion concentration is 1 to 300 mM, more preferably 10 to 50 mM. Such a reaction solution itself is one preferred embodiment of the composition of the present invention.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these.

Example 1
Eight types of Tris-HCl buffer (0.1 M) containing 10 ng / ml Cypridina luciferase were prepared as follows.
Buffer 1: 0.1 M Tris-HCl pH7.4
Buffer 2: 0.1 M Tris-HCl pH7.4, 0.2wt% Triton X-100
Buffer 3: 0.1 M Tris-HCl pH7.4, 0.2wt% NP-40
Buffer 4: 0.1 M Tris-HCl pH7.4, 0.2wt% SDS
Buffer 5: 0.1 M Tris-HCl pH7.4, 0.2wt% SDS, 0.2wt% Triton X-100
Buffer 6: 0.1 M Tris-HCl pH7.4, 0.2wt% SDS, 0.2wt% NP-40
Buffer 7: 0.1 M Tris-HCl pH 7.4, 0.2 wt% SDS, 0.2 wt% Triton X-100, 25 mM CaCl ₂
Buffer 8: 0.1 M Tris-HCl pH7.4 , 0.2wt% SDS, 0.2wt% NP-40, 25mM CaCl 2
Triton X-100, NP-40 and SDS were purchased from Wako Pure Chemical Industries. The luciferin-luciferase reaction was initiated by adding luciferin to each buffer at a final concentration of 0.5 μM and stirring. The luminescence intensity in each buffer was measured using a luminometer. The results are shown in Table 1 below and FIG.

I ₀ : Light intensity immediately after mixing luciferin and Cypridina luciferase I ₃ : Luminous intensity after 3 minutes from the start of reaction I ₁₅ : Luminous intensity after 15 minutes from the start of the reaction

By comparing the results for buffer solutions 1 and 4 shown in Table 1 and FIG. 1, when no anionic surfactant is present (buffer solution 1), the luminescence intensity rapidly decreases immediately after the start of the reaction. On the other hand, it can be seen that the decrease is suppressed or alleviated by the presence of the anionic surfactant (buffer solution 4). This inhibitory effect is enhanced when a nonionic surfactant coexists with an anionic surfactant (buffers 5 and 6).

Example 2
A Tris-HCl buffer solution (0.1 M, pH 7.4) containing 10 ng / ml Cypridina luciferase, 0.2 wt% SDS, and 0.2 wt% NP-40 was prepared as in Example 1. . As a control, a Tris-HCl buffer solution (0.1 M, pH 7.4) containing 10 ng / ml of Cypridina luciferase but no surfactant was prepared. Each buffer was divided into 96 wells, and Cypridina luciferin was added to a final concentration of 0.5 μM. Luciferin was added in order from 1A well to 1H well, then 2H well to 2A well, then 3A well to 3H well, up to 12A well. After adding luciferin to all wells, the luminescence intensity in each well was measured using a luminometer. The integration time for each well at this time was 20 seconds. The result about the buffer solution which added the composition of this invention containing SDS and NP40 is shown in FIG. On the other hand, the results of the control are shown in FIG. Comparing the results of FIGS. 2 and 3, it can be seen that the composition of the present invention remarkably suppresses the decrease in luminescence intensity over time due to the sea urchin luciferase.

FIG. 1 shows the temporal decrease in luminescence intensity from Cypridina luciferase in 8 different buffers prepared in Example 1. The number on the upper left of each figure indicates the buffer number. FIG. 2 shows the time course of luminescence intensity from Cypridina luciferase in the composition of the present invention (buffer) containing 10 ng / ml Cypridina luciferase, 0.2 wt% SDS, and 0.2 wt% NP-40. Shows a significant decrease. FIG. 3 shows a decrease in luminescence intensity over time from the sea urchin luciferase in a control buffer containing no surfactant.

Claims (3)

The following components (A) and (B)
(A) Sodium dodecyl sulfate
(B) Polyethylene glycol-p-octylphenyl ether (NP-40) and / or polyoxyethylene octylphenyl ether (Triton X-100)
The composition for suppressing the time-dependent fall of the emitted light intensity by Cypridina luciferase, Comprising: The density | concentration of sodium dodecyl sulfate is 0.1-0.5 weight% . The following components (A) and (B)
(A) Sodium dodecyl sulfate
(B) Polyethylene glycol-p-octylphenyl ether (NP-40) and / or polyoxyethylene octylphenyl ether (Triton X-100)
A method for suppressing a decrease in luminescence intensity over time caused by a sea urchin luciferase, wherein the concentration of sodium dodecyl sulfate is 0.1 to 0.5% by weight , characterized by reacting the sea urchin luciferase with luciferin in the presence of Is that way . The following components (A) and (B)
(A) Sodium dodecyl sulfate
(B) Polyethylene glycol-p-octylphenyl ether (NP-40) and / or polyoxyethylene octylphenyl ether (Triton X-100)
A chronological decrease inhibitor of luminescence intensity by Cypridina luciferase, wherein the concentration of sodium dodecyl sulfate is 0.1 to 0.5% by weight .

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