JP2009142188A - Method for extracting multi-color luciferase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stable extraction method for detecting luciferases in a sample with the light-emitting reaction of luciferin-luciferase, in more detail, a method for simultaneously extracting luciferases having a plurality of light-emitting spectrum characteristics and originated from beetles with an extraction reagent comprising Nonidet (R) P40 or polyoxyethylene (20) cetyl ether, and glycerol, and to provide a reagent therefor. <P>SOLUTION: This method for extracting the luciferase enzymes from cells for expressing one or more luciferases selected from the group consisting of green light-emitting luciferase originated from Ragophthalmidae Ohbai, mutant type orange color light-emitting luciferase originated from Ragophthalmidae Ohbai, and red color light-emitting luciferase originated from Phrixothrix hirtus comprises (a) a process for mixing polyoxyethylene (20) cetyl ether or Nonidet (R) P40 with glycerol, and (b) a process for bringing the mixture liquid into contact with the cells to extract the luciferase enzymes. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a stable extraction method for detecting luciferase in a sample using a luciferin-luciferase luminescence reaction. More specifically, the present invention relates to a method for simultaneous extraction of beetle-derived luciferase having a plurality of emission spectral characteristics using an extraction reagent containing Nonidet (registered trademark) P40 or polyoxyethylene (20) cetyl ether and glycerol, and a reagent therefor.

  Gene expression control analysis using a reporter gene involves introducing a plasmid containing a cis-acting base sequence element (gene expression control sequence such as a promoter, enhancer, silencer, etc.) linked to the reporter gene into a cell, and under certain conditions This is a technique for evaluating gene expression control using the activity of the expressed reporter enzyme as an index. Many reporter enzymes have been used for this evaluation so far, but systems using the light emission of firefly luciferase are widely used because of their high sensitivity and simple activity measurement.

  However, when the reporter activity is evaluated between samples, there are factors that cause changes in the absolute amount of the reporter enzyme that are not related to gene expression control, such as transfection efficiency, cell number, growth state, and cell death. Therefore, in addition to the reporter gene linked to the test sequence, a reporter molecule with different substrate specificity or reactivity linked to a constant expression (control) promoter must be added as an internal standard, and standardization must be performed between samples. There is. Thus, in order to simultaneously measure the expression of two or more genes, various genes, particularly Renilla luciferase showing a substrate specificity different from that of firefly luciferase, have been cloned and used. However, in this method, since the mechanism of luminescence is different, a plurality of reactions and measurements must be performed, which is complicated.

On the other hand, research to find mutants with different emission colors from the firefly luciferase gene (Non-patent Document 1), isolation of four kinds of Luciferase luciferases with different emission colors (Non-patent Document 2), derived from two-color railway insects Research on luciferase (Non-patent Document 3), research on mutants of Iriomote botulinum-derived luciferase (Non-patent Document 4) and the like have been advanced. These luciferases were noted by showing different emission colors on the same substrate. In particular, since the emission spectrum of firefly luciferase mutants varies with pH, luciferases whose emission spectra do not vary with respect to pH are preferred, such as click beetles, railroad insects, and Iriomote botal luciferases in multicolor measurement. An assay system using three-color luminescent luciferases has been put into practical use (Omiya et al., Multigene Transcription Activity Measurement System, Patent Document 1). However, since the stability of these luciferases is different, it is desirable to develop a detection reagent that can stably and efficiently extract any luciferase from cells and perform a luminescence reaction, particularly when simultaneously detecting multiple luciferases. It was.
Contag C.I. et al, Red Shifted luciferase, United States Patent 6,495,355 (2002) Wood K. V. , Lam Y. et al. A. Seliger H. H. , And McElroy W., et al. D. 1989, Science, 244, 700-702: Complementary DNA Coding Click Beetles Luciferases Can Elicity Bioluminescence of Different Colors Viviani V. R. , Bechara E .; J. et al. H. , Ohmiya Y .; 1999, Biochemistry, 38, 8271-8279. Viviani V. Uchida A., et al. , Suenaga N .; Ryufuku M., et al. , And Ohmiya Y .; 2001, Biochem. Biophys. Res. Commun. 280, 1286-1291 WO2004 / 099421

  An object of the present invention relates to a detection reagent capable of stably and efficiently extracting luciferase from a cell sample and performing a luminescence reaction with respect to a plurality of luminescent enzymes. More particularly, the present invention relates to a detection reagent that can stably and efficiently extract a plurality of luminescent enzymes simultaneously and perform a luminescence reaction.

  In order to solve the above-mentioned problems, the present inventors have stably extracted by mixing glycerol with an extractant containing polyoxyethylene (20) cetyl ether or nonidet (registered trademark) P40 as a surfactant. The inventors have found that a luminescence reaction can be performed and have completed the present invention.

That is, the present invention has the following configuration.
[Claim 1]
Extraction of a fraction containing luciferase enzyme from cells expressing one or more luciferases selected from the group of green luciferase derived from Iriomote butter, mutant orange luciferase derived from Iriomote butter and red luciferase derived from a railworm A way to
(A) a step of mixing polyoxyethylene (20) cetyl ether or Nonidet (registered trademark) P40 and glycerol, and (b) contacting the mixture obtained in the step of (a) with a cell to give a luciferase enzyme. The process of extracting,
Including methods.
[Section 2]
Item 2. The method according to Item 1, wherein the concentration of glycerol in the mixed solution obtained in the step (a) is 20 to 25%.
[Section 3]
Item 3. The method according to Item 1 or 2, wherein the mixed solution further contains 10 to 50 mM ammonium sulfate.
[Claim 4]
The green luminescence luciferase derived from Iriomote botaru, the mutant-type orange luminescence luciferase derived from iriomote botaru, and the railway further comprising the step of measuring luminescence of the fraction containing the luciferase enzyme extracted by the method according to any one of Items 1 to 3 A method for detecting luciferase from a cell that expresses one or more luciferases selected from the group of red luciferases derived from insects.
[Section 5]
The green luciferase derived from Iriomote botaru, the mutant-type orange luminescence luciferase derived from Iriomote botaru, and the railworm further comprising the step of leaving the fraction containing the luciferase enzyme extracted by the method according to any one of Items 1 to 3 A method of retaining one or more luciferases selected from the group of derived red-emitting luciferases.
[Claim 6]
Item 6. A reagent for use in the method according to any one of Items 1 to 5, comprising polyoxyethylene (20) cetyl ether or Nonidet (registered trademark) P40, and glycerol.
[Claim 7]
Item 7. The method according to Item 6, wherein the concentration of glycerol in the reagent is 20 to 25%.
[Section 8]
Item 8. The reagent according to Item 6 or 7, comprising 10 to 50 mM ammonium sulfate in the reagent.

  By the method of the present invention, each luciferase can be stably extracted from a specimen containing multiple colors of luciferase. Therefore, by measuring the amount of luminescence, the transcriptional activity of multiple genes can be monitored simultaneously. It became possible to quantify various life phenomena. Furthermore, the method of the present invention makes it possible to measure multiple specimens more stably at once using a plate reader, and can be widely applied to drug discovery screening, chemical substance toxicity evaluation, and the like.

The present invention is described in detail below.
The luciferase to be extracted by the extraction method of the present invention is a green luminescence luciferase derived from Iriomote butterfly Ragophtalmidae Ohbai (Non-patent Document 5), a mutant-type orange luminescence luciferase derived from Iriomote bottal (Non-patent Document 4), and a railworm Phrixothrix hirtus Red light emitting luciferase (Non-patent Document 3) and the like. These luciferases may have a wild-type sequence, or substitution, insertion or deletion of one or more amino acid residues. These are usually introduced into cells in the form of expressible gene constructs, but natural genes such as cDNA may be used for the luciferase gene, but translation efficiency is improved in the cell type used for the test. Thus, it is more preferable to use a gene modified by genetic engineering as exemplified by Omiya et al. Luciferase (multigene transcription activity measurement system, Patent Document 1).
Ohmiya Y. et al. Mina Y., et al. , Viviani V. R. Ohba N .; 2000, Sci. Rep. Yokosuka City Mus. 47, 31-38

  The luciferase to be extracted by the extraction method of the present invention further has the same amino acid sequence as that of a natural luminescent enzyme, and further includes substitution, addition, deletion or insertion of one or more amino acids. It may be a fusion protein in which a second protein is bound to the N-terminus or C-terminus.

  As another embodiment of the present invention, there is exemplified a method for detecting luciferase extracted by the above method using D-luciferin as a luminescent substrate in the same manner as firefly luciferase. That is, a sample solution containing luciferase is mixed with a luminescence reagent containing D-luciferin, adenosine triphosphate (ATP), magnesium salt, etc. at a concentration sufficient for a luminescence reaction, and the amount of luminescence produced by this reaction is measured. The relative abundance of luciferase in is determined.

  The luciferase detection method can also generate luminescence by allowing D-luciferin to permeate cells, but for more sensitive detection, cells expressing luciferase are physically disrupted or chemically disrupted. Dissolve and prepare a luminescence reaction solution containing Mg ions, ATP, luciferin, etc. at a concentration sufficient for the luminescence reaction with all or part of the treatment solution, and luciferase in each sample based on the luminescence amount of the reaction solution It is preferred to determine the relative abundance of. Examples of a method for physically disrupting cells include an ultrasonic disruption method. A chemical lysis method is a method in which cells are brought into contact with a solution containing a surfactant. The luciferase abundance can be examined by mixing the disrupted / dissolved cell extract with the reagent and measuring luminescence. For detection of luciferase expressed in such cells, the method of performing a luminescence reaction while lysing cells by adding a surfactant to the luminescent reagent is easy to operate and is optimal when the number of samples is large. . However, if you want to analyze the amount of protein to be examined in the same cell sample or the state of modification such as phosphorylation by antigen-antibody reaction, cell lysis and luminescence can be performed in separate steps. preferable.

  Moreover, as another aspect of the present invention, a method of allowing the luciferase extracted by the above method to stand and hold is exemplified.

The surfactant added to the above-described method for lysing cells and extracting luciferase should be of a kind and concentration that does not deactivate luciferase or inhibit its activity as much as possible. For the extraction of luciferase used in the present invention, it is preferable to use a nonionic surfactant. In general, Triton X100 (polyoxyethylene (10) octylphenyl ether) is used as a surfactant that extracts luciferase derived from firefly Photinus Pyralis from cells (Non-patent Document 6). Patent Document 2 reports that Triton X100 is also used for cell extraction of green luminescent luciferase, orange luminescent luciferase, and red luminescent luciferase.
Biomedical series for experimental medicine separate volume (4) Gene transfer and expression / analysis method (issued April 20, 1994, Yodosha) p. 89-98 WO2006 / 061985

In contrast, in the method for extracting luciferase of the present invention, Nonidet P40 (NP40, polyoxyethylene (9) octylphenyl ether) (CAS 9036-19-5) or polyoxyethylene (20) cetyl ether (Brij ( Registered trademark) -58) (CAS 9004-95-9).
The lower limit of the concentration of Nonidet P40 (NP40) is preferably 0.005%, more preferably 0.05%. The upper limit is preferably 1%, and more preferably 0.25%.
Moreover, as a minimum of the use density | concentration of polyoxyethylene (20) cetyl ether, 0.005% is preferable, More preferably, it is 0.05%. The upper limit is preferably 1%, and more preferably 0.25%.

  The extraction method of luciferase of the present invention may be configured to include both polyoxyethylene (20) cetyl ether and nonidet P40. By including both, the risk due to the difference between lots can be dispersed.

In the extraction method of the present invention, glycerol is further added to stabilize luciferase. The lower limit of the preferred concentration is 10%, more preferably 20%. The upper limit of the preferred concentration is 30%, more preferably 25%.
At this time, Triton X100 also shows a glycerol stabilizing effect, but Nonidet P40 and polyoxyethylene (20) cetyl ether can be further stabilized.

  In the extraction method of the present invention, ammonium sulfate can be further added to stabilize luciferase. The lower limit of the preferred concentration is 10 mM. The upper limit of the preferred concentration is 50 mM, more preferably 20 mM.

  The extraction method of the present invention may further contain a buffer component in order to maintain the ionic strength and pH. Examples of the buffer component include HEPES, Tricine, Tris, MOPS, glycylglycine, and the use of 20 to 100 mM is usually preferable. A preferred pH range is 7.0 to 8.5. The maximum light emission can be obtained at pH 7.8 to 8.0, but the light emission persistence can be enhanced by adjusting the pH to 7.8 or less. In the present invention, a proteinaceous material that further enhances the activity of luciferase, for example, mammalian serum albumin such as bovine, lactalbumin, and the like may be present.

  In the extraction method of the present invention, a chelating agent such as EDTA or CDTA or EGTA is added to suppress the activity of metal-containing proteases or phosphatases that are present in the sample and may adversely affect luciferase or ATP. It can be included. A preferred concentration is 1 to 5 mM.

  Furthermore, the extraction method of the present invention may include a reducing agent that is considered to have an effect of protecting the stability of the luciferase protein. Examples of the reducing agent include sulfhydryl compounds such as dithiothreitol and 2-mercaptoethanol.

  In the extraction method of the present invention, all or part of the cell extract of the above method is contacted with a luminescent reagent containing D-luciferin, adenosine triphosphate (ATP), and magnesium ions, and the amount of luminescence is measured. Determine the relative abundance of luciferase contained in the sample. The luminescent reagent may further contain coenzyme A, DTT, and the like.

  One embodiment of the present invention is exemplified by a method for detecting luciferase expressed by linking a transcription regulatory sequence upstream of the gene encoding luciferase and introducing the gene construct into a cell. This makes it possible to analyze the transcriptional control of the transcriptional regulatory sequence in the cell using the expression level of luciferase as an index.

  The cells used in the present invention may be derived from eukaryotes, prokaryotes, etc., but are preferably animal-derived cells, particularly preferably mammal-derived cells, which are human, mouse, rat, hamster, guinea pig. And cells such as rabbits, dogs, cows, horses, sheep, monkeys and pigs. When using prokaryotes such as Escherichia coli, it is preferable to combine the extraction / maintenance method of the present invention with physical cell disruption means.

  As another aspect of the present invention, the luciferase is expressed in the form of a fusion protein with the second protein to be tested, and the abundance thereof is detected (measured) to detect (measure) the luminescence. It is a method of monitoring the amount adjustment. For example, when expressed in a cell as a fusion protein with IκB, IκB is degraded by stimulation with TNFα, IL-1, etc., and the pathway activated by the transcription factor NFκB is activated, but linked to a constant expression promoter. This activation can be monitored quantitatively by measuring the extent of degradation of the fusion protein with the resulting IκB.

Furthermore, another aspect of the present invention is a reagent for use in any one or more of the extraction method, detection method, and retention method.
The reagent may be a reagent containing all of the components necessary for detection, or may be a kit comprising separate parts. It includes a reagent composition prepared each time in order to use the method of the present invention in a laboratory or the like, or a kit in which necessary reagents and the like are prepared in advance so that they can be easily used in the method of the present invention. Furthermore, in addition to an aqueous solution in which all the reagents of the present invention are mixed, a product having a concentration twice or more (diluted when used) is used.
The reagent contains Nonidet (registered trademark) P40 or polyoxyethylene (20) cetyl ether as a surfactant. Furthermore, it is preferable that glycerol is included. Alternatively, it is preferable to further contain ammonium sulfate.
One such composition is an aqueous solution containing 0.05 to 0.25% Nonidet® P40 or polyoxyethylene (20) cetyl ether, and 10 to 25% glycerol. Preferably, the composition further contains 10 to 50 mM ammonium sulfate.
More preferable conditions for each component in the reagent of the present invention or a combination thereof are the same as the contents described in the above-mentioned items described for the method of the present invention.
The reagent is mixed with a cell that expresses one or more luciferases selected from the group consisting of a green luminescence luciferase derived from Iriomote butter, a mutant orange luciferase derived from Iriomote botaru, and a red luminescence luciferase derived from a railworm. Luciferase may be extracted from the cells, or the luciferase sample may be mixed with the reagent and used as a stock solution for the luciferase.

Hereinafter, the effects of the present invention will be further clarified by illustrating examples of the present invention.

Example 1 Confirmation of Effect of Addition of Glycerol / Ammonium Sulfate to Luciferase Extractant CHO cells were seeded in a 24-well plate (1 × 10 ⁵ cells / well), Ham's F12 medium containing 10% FCS (Nissui Pharmaceutical) ) Cultured in 1 ml / well. The next day, green luciferase derived from Iriomote butter, mutant-type orange luciferase derived from Iriomote butterfly, red luciferase derived from railroad insect (respectively, Toyobo Co., Ltd., MultiReporter Assay-Triluc (registered trademark) -SV40 control vector) PSLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control) were transfected using GeneJuice Transfection Reagent (Novagen). After incubation for 24 hours, a mixture consisting of 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1% polyoxyethylene (20) cetyl ether or Nonidet P40, 0, 10, or 20% glycerol, 0 or 10 mM ammonium sulfate After removing the medium from the cells, it was added to the cells to a concentration of 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted, and the amount of luminescence was measured in the same manner.

  Figure 1 shows the luminescence intensity of the extract obtained by adding glycerol and ammonium sulfate to a cell extractant containing polyoxyethylene (20) cetyl ether and Nonidet P40, and the enzyme after standing at room temperature for 2 hours. It is a figure which shows a residual activity. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). . (-) On the horizontal axis indicates no addition of ammonium sulfate, and (+) indicates that 10 mM ammonium sulfate is contained. Under these conditions, there is almost no difference in green luciferase (SLG) derived from Iriomote botal, and polyoxyethylene (20) cetyl ether is used as a surfactant in mutant orange luciferase (SLO) derived from Iriomote botaru. The light emission intensity was higher when selected, but no difference was observed in stability. On the other hand, railway insect red luciferase (SLR) has low stability, and the enzyme is inactivated during the extraction process, but a significant increase in luminescence intensity was observed by adding about 5 to 10% glycerol. A stabilization tendency was also observed by adding 10 mM ammonium sulfate.

Example 2 Optimization of addition amount of glycerol / ammonium sulfate to luciferase extractant As described above, pJLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control) were applied to CHO cells seeded in a 24-well plate as GeneJuice Transfection Regression. (Novagen) was used for transfection. After incubation for 24 hours, a mixture consisting of 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1% polyoxyethylene (20) cetyl ether, 10, 15, 20 or 25% glycerol, 0, 10, or 20 mM ammonium sulfate After preparing the solution and removing the medium from the cells, it was added to the cells at 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted, and the amount of luminescence was measured in the same manner.

  FIG. 2 shows the luminescence intensity of the extract obtained by adding 0 to 25% glycerol and 0 to 20 mM ammonium sulfate to a cell extract containing polyoxyethylene (20) cetyl ether for 2 hours at room temperature. It is a figure which shows the residual activity of the enzyme after leaving to stand. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). . 0, 10, and 20 on the horizontal axis indicate ammonium sulfate concentration (mM), and Gly indicates glycerol. As a result, significant stabilization of SLR was observed with the addition of glycerol, and a further increase was observed with the addition of ammonium sulfate. On the other hand, in SLG and SLO, the emission intensity is slightly decreased by adding ammonium sulfate, but this does not hinder the measurement.

Example 3 Confirmation of Addition Amount of Ammonium Sulfate to Luciferase Extractant As described above, pJLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control were applied to GeneJuice Transfection Reagent (a JU cell seeded in a 24-well plate). ) Was used for transfection. After culturing for 24 hours, a mixed solution consisting of 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1% polyoxyethylene (20) cetyl ether, 20% glycerol, 0 to 50 mM ammonium sulfate is prepared, and the medium is removed from the cells. After removal, cells were added to 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted, and the amount of luminescence was measured in the same manner.

  FIG. 3 shows the luminescence intensity of the extract obtained by adding 20% glycerol and 0-50 mM ammonium sulfate to a cell extract containing polyoxyethylene (20) cetyl ether and leaving it at room temperature for 2 hours. It is a figure which shows the residual activity of the subsequent enzyme. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). . As a result, for SLR, a significant increase in stabilization was observed with the addition of 10 mM to 20 mM ammonium sulfate, and almost no change was observed even when the addition amount was further increased. On the other hand, for SLG and SLO, since the amount of luminescence tends to decrease, it can be said that the addition amount is preferably about 10 to 20 mM.

Example 4 Confirmation of addition amount of polyoxyethylene (20) cetyl ether to luciferase extractant (1)
As described above, pSLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control) were transfected into CHO cells seeded in a 24-well plate using GeneJuice Transfection Reagent (Novagen). After culturing for 24 hours, a mixed solution consisting of 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 0.1 to 3% polyoxyethylene (20) cetyl ether, 20% glycerol, and 10 mM ammonium sulfate is prepared. After removal, cells were added to 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted, and the amount of luminescence was measured in the same manner.

  FIG. 4 shows the luminescence intensity of the extract obtained after extraction with a cell extractant (containing 20% glycerol and 10 mM ammonium sulfate) containing 0.1 to 3% polyoxyethylene (20) cetyl ether for 2 hours at room temperature. It is a figure which shows the residual activity of the enzyme after leaving to stand. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). . As a result, it was confirmed that any luciferase was inactivated by addition of 1% or more of polyoxyethylene (20) cetyl ether.

Example 5 Confirmation of addition amount of polyoxyethylene (20) cetyl ether to luciferase extractant (2)
As described above, pSLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control) were transfected into CHO cells seeded in a 24-well plate using GeneJuice Transfection Reagent (Novagen). After culturing for 24 hours, a mixed solution composed of 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 0.001 to 0.5% polyoxyethylene (20) cetyl ether, 25% glycerol, 10 mM ammonium sulfate was prepared, and from the cells. After removing the medium, it was added to the cells to 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted, and the amount of luminescence was measured in the same manner.

  FIG. 5 shows the luminescence intensity of the extract obtained after extraction with a cell extractant (containing 25% glycerol and 10 mM ammonium sulfate) containing 0.1 to 3% polyoxyethylene (20) cetyl ether for 2 hours at room temperature. It is a figure which shows the residual activity of the enzyme after leaving to stand. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). .

  As a result, it can be seen that sufficient cell extraction cannot be performed by adding 0.001% or less of polyoxyethylene (20) cetyl ether in any luciferase. However, when polyoxyethylene (20) cetyl ether is 0.005% and 0.01%, it takes time until dissolution, but it can be extracted and measured after 2 hours by extending the extraction time. Recognize. Moreover, if it is 0.05% or more, it turns out that it can measure immediately after a process. Accordingly, the lower limit of the use concentration of polyoxyethylene (20) cetyl ether is preferably 0.005%, more preferably 0.05% practically.

  On the other hand, it can be seen that when the use concentration of polyoxyethylene (20) cetyl ether exceeds 0.5%, the residual activity for 2 hours is lowered. Therefore, the upper limit of the use concentration of polyoxyethylene (20) cetyl ether is preferably when combined with the fact that the use concentration exceeds 1% in Example 4 (FIG. 4), the luciferase activity is extremely reduced. 1%, more preferably 0.25%.

Example 6 Comparison of Surfactant as Extractant for Luciferase Similarly to the above, for CHO cells seeded in a 24-well plate, pSLG-SV40 control, pSLO-SV40 control, and pSLR-SV40 control were added to GeneJuice Transfection Reagent (Nova). And transfected. After 24 hours of culture, from 20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM DTT, 20 or 25% glycerol, 10 mM or 20 mM ammonium sulfate, 0.1% polyoxyethylene (20) cetyl ether, Nonidet P40, or Triton X-100 After the medium was removed from the cells, the mixture was added to the cells at 200 μl / well. Ten minutes later, 75 μl of the cell extract was extracted, mixed with 75 μl of the luminescent reagent MultiReporter Assay System-Triluc (registered trademark) -Triluc (registered trademark) Luciferase Assay Reagent (manufactured by Toyobo), and the amount of luminescence was measured. Further, after 2 hours of standing at room temperature, 75 μl of the cell extract was extracted and the amount of luminescence was measured in the same manner.

  FIG. 6 shows an extraction treatment with a cell extractant containing polyoxyethylene (20) cetyl ether (symbol: PEC), nonidet P40 (symbol: NP40), Triton X-100 (symbol: TX100 in the diagram). It is a figure which shows the comparison result of the residual activity of the enzyme after leaving the luminescence intensity of the extract at the time of standing for 2 hours at room temperature. Each bar graph represents the luminescence intensity immediately after the cell extraction treatment (0 hr) and after 2 hours of standing (2 hr), and the line graph shows the change in relative luminescence intensity after 2 hours of standing as 100 immediately after the cell extraction treatment (0 hr). . As a result, in any luciferase, Triton X-100 has lower emission intensity than the other two surfactants. On the other hand, in the case of polyoxyethylene (20) cetyl ether, high luminescence intensity and residual activity were confirmed in any luciferase.

The method for measuring multicolor luciferase in the present invention is used for measurement of an assay system using a multicolor luminescent enzyme that has recently attracted attention. This assay system is a signal that uses complex intracellular transcriptional control analysis and gene transcription as an index. It can be used as a system for analysis of transmission systems, and further for compound screening, and contributes greatly to industries such as drug discovery and medicine.

It is a figure which shows the effect of glycerol and ammonium sulfate in the cell extraction of the green light emission luciferase derived from Iriomote butterfly, the mutant type orange light emission luciferase derived from Iriomote fireflies, and the red light emission luciferase derived from a railroad insect expressed in mammalian cells. It is a figure which shows the optimal density | concentration of glycerol and ammonium sulfate in the cell extraction of the green light emission luciferase derived from Iriomote butterfly, the mutant type orange light emission luciferase derived from Iriomote fireflies, and the red light emission luciferase derived from a railway insect expressed in mammalian cells. It is a figure which shows the effective density | concentration of ammonium sulfate in the cell extraction of the green light emission luciferase derived from Iriomote butterfly derived from the mammalian cell, the mutant type orange light emission luciferase derived from Iriomote firefly, and the red light emission luciferase derived from the railworm It is a figure which shows the effective density | concentration of polyoxyethylene (20) cetyl ether in the cell extraction of the green light emission luciferase derived from Iriomote butterfly, the mutant type orange light emission luciferase derived from Iriomote fireflies, and the red light emission luciferase derived from a railway insect expressed in a mammalian cell. . It is a figure which shows the effective density | concentration of polyoxyethylene (20) cetyl ether in the cell extraction of the green light emission luciferase derived from Iriomote butterfly, the mutant type orange light emission luciferase derived from Iriomote fireflies, and the red light emission luciferase derived from a railway insect expressed in a mammalian cell. . Polyoxyethylene (20) cetyl ether, Nonidet P40, Triton X-100 in cell extraction of Iriomote butterfly-derived green-emitting luciferase expressed in mammalian cells, Iriomote butter-derived mutant orange-emitting luciferase, Railway worm-derived red-emitting luciferase It is a figure which shows the comparison result of.

Claims (8)

Extraction of a fraction containing luciferase enzyme from cells expressing one or more luciferases selected from the group of green luciferase derived from Iriomote butter, mutant orange luciferase derived from Iriomote butter and red luciferase derived from a railworm A way to
(A) a step of mixing polyoxyethylene (20) cetyl ether or Nonidet (registered trademark) P40 and glycerol, and (b) contacting the mixture obtained in the step of (a) with a cell to give a luciferase enzyme. The process of extracting,
Including methods.   The method according to claim 1, wherein the concentration of glycerol in the mixed solution obtained in the step (a) is 20 to 25%.   The method according to claim 1, wherein the mixed solution further contains 10 to 50 mM ammonium sulfate.   A green-emitting luciferase derived from Iriomote butter, a variant orange-emitting luciferase derived from Iriomote buta, further comprising a step of measuring luminescence of a fraction containing the luciferase enzyme extracted by the method according to any one of claims 1 to 3. A method for detecting luciferase from a cell expressing one or more luciferases selected from the group of red-emitting luciferases derived from railroad worms.   A green-emitting luciferase derived from Iriomote butter, a variant orange-emitting luciferase derived from Iriomote butter, and a railway, further comprising a step of standing a fraction containing the luciferase enzyme extracted by the method according to any one of claims 1 to 3. A method of retaining one or more luciferases selected from the group of insect-derived red-emitting luciferases.   A reagent for use in the method according to any one of claims 1 to 5, comprising polyoxyethylene (20) cetyl ether or nonidet (registered trademark) P40, and glycerol.   The method of claim 6, wherein the concentration of glycerol in the reagent is 20-25%. The reagent of Claim 6 or 7 which contains 10-50 mM ammonium sulfate in a reagent.

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