WO2015056762A1 - 人工生物発光酵素に用いるための発光基質 - Google Patents
人工生物発光酵素に用いるための発光基質 Download PDFInfo
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- WO2015056762A1 WO2015056762A1 PCT/JP2014/077618 JP2014077618W WO2015056762A1 WO 2015056762 A1 WO2015056762 A1 WO 2015056762A1 JP 2014077618 W JP2014077618 W JP 2014077618W WO 2015056762 A1 WO2015056762 A1 WO 2015056762A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
- C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
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- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
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- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
Definitions
- the present invention relates to a bioluminescent substrate suitably used for an artificial bioluminescent enzyme (ALuc) created based on common genetic information in bioluminescent enzymes derived from marine animals. Specifically, it relates to the substrate necessary for deriving the high-luminance luminescence characteristics and luminescence persistence, which are the advantages of ALuc, and particularly relates to the design and development of an efficient luminescent substrate based on the three-dimensional structure of ALuc. is there.
- ALuc artificial bioluminescent enzyme
- Non-Patent Document 1 the establishment of many new bioluminescent enzymes related to bioluminescent enzymes (luciferases) has been reported.
- luciferases bioluminescent enzymes
- This enzyme has a molecular weight of 1/2 (19 kD) compared to a conventional Renilla luciferase (RLuc) -derived luminescence enzyme (Renilla luciferase; RLuc).
- RLuc Renilla luciferase
- 11 kinds of bioluminescent enzymes derived from plankton were reported by Mosato et al. (Non-patent Document 20). It was evaluated that some of these luminescent enzymes showed brightness similar to RLuc.
- Non-Patent Document 2 a deep-sea luminescent animal belonging to the Augaptiloidea superfamily has been discovered (Non-Patent Document 2).
- a luminescent enzyme (GLuc) derived from Gaussia princeps belonging to the Metridinidae family, a luminescent enzyme derived from Metridia longa (MLuc), and luminescent enzymes derived from Metridia pacifica (MpLuc1, MpLuc2) have been discovered (Non-patent Documents 15, 19, 21).
- Lucia an artificial bioluminescent enzyme similar to copepod luminescent enzyme, called Lucia from InvivoGen, was established (http://www.invivogen.com/lucia).
- Non-patent Document 14 Loening et al. Established a high-brightness and stable RLuc mutant by introducing an amino acid mutation into RLuc (Non-patent Document 14). In this study, “consensus sequence-driven mutagenesis strategy” was used to identify the site of introduction of mutation (Non-patent Document 13). In addition, the researchers assumed that the active site of the enzyme was based on the distribution map of hydrophilic amino acids and introduced a mutation to increase the brightness and stability of luminescence of GLuc, MpLuc1 and MLuc, luminescent enzymes derived from deep-sea luminescent animals. (Non-patent Document 11).
- the present inventors divided the sequence of a single luminescent enzyme into two, and aligned the front and back sequences around similar amino acids to obtain hints related to their luminescent properties (single).
- a proposal was made to create a thermodynamically stable luminescent enzyme sequence by sequence alignment (SSA) (Non-patent Document 3). This technique is based on the premise that there are two enzyme active sites in a marine animal-derived luminescent enzyme. By aligning these two enzyme active sites around similar amino acids, it is possible to easily compare the similarity of the preceding and succeeding enzyme active sites.
- thermodynamically stable luminescent enzyme sequence by trying to create a thermodynamically stable luminescent enzyme sequence by increasing the similarity between the sequences before and after this (Patent application number: Japanese Patent Application No. 2012-237043).
- reporter gene probes belonging to the class of “activatable” are characterized in that the reporter itself reacts actively to ligand stimulation and emits light.
- PCA Protein-complentation-assay
- PSA protein-splicing-assay
- single-molecule bioluminescent probes single-molecule bioluminescent probes
- luminescent capsules etc.
- Non-patent Document 12 a molecular permutation technique (Non-Patent Document 12) using circular permutation (Non-Patent Document 9) and a low molecular weight luminescent enzyme was developed. All of these studies have been used as a means of efficiently measuring molecular phenomena in cellular and non-cellular systems.
- Fluorescence imaging is a main method for exploring intracellular and extracellular molecular phenomena and a method widely used beyond luminescence imaging.
- fluorescent proteins have a high background due to autofluorescence, require an external light source, and require a large device such as a fluorescence microscope and a precise filter system.
- a fluorescence microscope and a precise filter system.
- a fluorescence microscope there is a limit to the number of cells that can be observed at one time, and there has been a problem in quantitativeness (Non-patent Document 8).
- bioluminescent enzymes the number of enzymes that exhibit bioluminescence of various colors is scarce. Despite the fact that (i) simultaneous measurement of multiple signals and (ii) good biological tissue permeability of long-wavelength light emission are taken up as advantages of multicolor emission, the luminescence principle of bioluminescence enzymes has been discussed so far. There has been almost no systematic multicolor research based on.
- Bioassays that place particular importance on selection of reaction solutions include (1) reporter-gene assay, (2) two-hybrid assay, and (3) enzyme-linked immunosorbent assay (enzyme-). linked (immunosorbent assay), (4) radioimmunoassay (RIA), etc. (Non-patent document 22, Non-patent document 23).
- Non-patent Document 27 a multiple-recognition-type bioluminescent probe combining a reporter gene assay and a single-molecule bioluminescent probe.
- This probe is characterized by sensing twice for one calibration substance.
- a multi-color bioluminescence imaging probe set was developed by combining two-color single-molecule bioluminescence probes (Patent Document 5). This probe is characterized by being capable of multicolor imaging of multifaceted physiological activities of a specimen.
- Bioassays always require a reaction solution, and are roughly classified into (1) a technique using a fluorescent protein and (2) a technique using a bioluminescent enzyme (luciferase) depending on the type of luminescence signal.
- a fluorescent protein When a fluorescent protein is used, an external light source is required in addition to a very high background due to autofluorescence.
- a relatively large light emission detector for example, a fluorescence microscope
- a precise spectral filter is required (Non-patent Document 8).
- the above-mentioned problem does not occur, but there is a problem that the light is weaker than fluorescence and a substrate is always required.
- additives have been used in the reaction solution (assay buffer) in order to improve the assay effect.
- the functions required of additives include (1) preventing protein degradation by proteases, (2) suppressing the influence of interfering substances, and (3) supporting stable signal transmission by having it act as a buffer solution. , (4) homogenous assay conditions such as gently breaking the cell membrane, (5) stabilizing the protein, and (6) not impairing the performance of the probe that is the core of the luminescence reaction. .
- the salts NaCl, KCl, (NH 4 ) was added and 2 SO 4, was added mercaptoethanol, and DTT such as SH reagent, addition of glycerol and sucrose and the like as a polyol EGTA, EDTA or the like is added as a chelating reagent.
- Surfactants include polyoxyethylene (10) octylphenylylether (TritonX-100; TX100), Nonidet P-40 (NP40), polyoxyethylene sorbitan monolaurate (Tween20; TW20), polyoxyethylene sorbitan monooleate (Tween80; TW80), polyoxyethylene (20) Add cetyl ether (Brij58), sodium decyl sulfate (SDS), etc.
- the degree of hydrophilicity is in the order of TW20> Brij58> TW80> TX100> NP40
- the degree of surfactant action is in the order of NP40> TX100> Brij58> TW20> TW80 As appropriate.
- Protease Inhibitors used to inhibit proteolysis include aprotinin (molecular weight 6.5 kD), leupeptin (molecular weight: 427), pepstatin A (pepstatin, molecular weight: 686), phenylmetylsulfonyl fluoride (PMSF, molecular weight: 174), antipine ( Antipain, molecular weight: 605), chymostatin (molecular weight: 608), etc. are used.
- Pefabloc SC AEBSF, 240 Da
- DFP 184 Da
- p-APMSF 216 Da
- STI 20,100 Da
- Leupeptin 460 Da
- N-Tosyl-L-phenylalaninechloromethylketone 3,4- dichloroisocoumarin (215 Da)
- EDTA-Na2 372 Da
- EGTA 372 Da
- 1,10-phenanthroline 198 Da
- phosphoramidon 580 Da
- Dithiobis (2-amino-4-methylpentane
- E-64 357 Da
- cystatin bestatin
- Epibestatin hydrochloride aprotinin
- minocycline ALLN (384 Da) and other proteolytic inhibitors
- the receptor By adding Sodium molybdate, the receptor can be stabilized and protected from degradation. Glycerol can be used as a protein blocking agent, and dithiothreitol (DTT) has been used as a reducing agent.
- DTT dithiothreitol
- the substrate which is another factor that enables light emission luminance, light emission stability, and long wavelength shift, is based on research results of several decades ago, and its technological progress was at a very slow pace.
- the reason for this stagnation is that (1) research on luminescent substrates is based on organic synthesis, so it was a research area that only a limited number of researchers capable of organic synthesis were capable of, and (2) luminescent enzymes Among them, marine organism-derived luminescent enzymes are particularly susceptible to oxidation by oxygen in the air, and therefore it was necessary to prepare a synthetic environment.
- Non-patent Document 5 The definition of the substrate is not precisely defined, and its meaning has changed with the times.
- the first definition of luciferin was a substance essential for a luminescent reaction and extracted from a living organ. Around 1960, Cypridina luciferin was discovered from luminescent fish parapriacanthus, etc. Coelenterazine was discovered around 1970-1980, and was isolated from various luminescent organisms. It was found to be a substrate (Non-patent Document 5).
- the present invention focuses on coelenterazine as a substrate that contributes to the luminescence reaction of an artificial bioluminescent enzyme, and clarifies the chemical structure and reaction mechanism of the optimal substrate for the artificial bioluminescent enzyme.
- Coelenterazine was first separated from Renilla in the mid 1960s, and its chemical structure was confirmed by Inoue et al. In 1977 (Non-patent Document 10). It was named coelenterazine in 1975 by Shimomura (2008, Nobel Prize in Chemistry).
- Non-patent Documents 30 and 31 Coelenterazine derivatives themselves have a history of more than 30 years, and many non-patent literatures have been published.To date, it has been reported that coelenterazine derivatives and their derivatives are not patentable. This relates to the development of a new synthesis route (Patent Document 6).
- Non-Patent Documents 17, 18, 24, 26, 28, 29 have been synthesized by the following synthesis route.
- Non-Patent Documents 32, 33, and 34 As a simpler coelenteramine synthesis method, a cross-coupling method (still coupling) via Pd has been established relatively recently.
- the synthesis route follows the following procedure (Non-Patent Documents 32, 33, and 34).
- the object of the present invention is to provide new knowledge about the optimal luminescent substrate for ALuc by examining the amino acid sequence of the artificial bioluminescent enzyme (ALuc) of the prior application and analyzing the structure of the three-dimensional structure.
- the super two-dimensional structure of ALuc was predicted while mobilizing the knowledge about the luminescent enzyme obtained so far.
- the chemical structure of the luminescent enzyme that can be suitably matched with the three-dimensional structure was studied.
- Non-patent Document 11 a method for specifying an enzyme active site by hydrophilicity search. Although this method estimates a rough enzyme active site, it did not give the knowledge of which amino acids are specifically important.
- Non-patent Document 3 the amino acid sequences of all bioluminescent enzymes derived from luminescent copepods obtained from NCBI were divided into three at arbitrary positions. After the front and back domains were overlapped and aligned, the amino acids corresponding to each other were compared in the amino acid sequence, and the amino acid sequence was determined in the direction of increasing similarity. In this manner, a large number of amino acid sequences that can become novel artificial luminescent enzymes were determined (Patent Document 7).
- the conformation of the main chain for each amino acid residue is determined for the X-ray crystal structure data (PDBID: 2hpsA, 2hq8A) of Coelenterazine-Binding Protein (CBP) using the method described in the prior document (Non-patent Document 35). Coding conversion was performed on three types of codes: helix type (h), ⁇ sheet type (s), and other type (o) ⁇ , and the super secondary structure was described.
- HyperProtein v1.0 http://www.hyper.com/Products/HyperProtein/tabid/504/Default.aspx
- Chem3D Ultra v8.0 http: //www.cambridgesoft.com/Ensemble_for_Chemistry/ChemBio3D/
- MM Molecular-Mechanics-
- the molecular model can be constructed with the three-dimensional structure of ALuc30, for example, maintaining the main super-secondary structure of CBP including the Loop structure.
- the hydroxyphenyl group of native coelenterazine and amino acid residues in the vicinity of 4 mm from the substrate reaction site for example, 17LYS, 20THR, 37ILE, 72ASN, 143VAL, 145LEU, 149CYS , 189LYS and 192GLY amino acid residues may have a particularly large effect on bioluminescence.
- FIG. 20-30 is located at the ⁇ -helix site that forms a rigid super-secondary structure, and the influence of the surrounding amino acid residues on the function of the original HIS tag Is also a concern.
- an artificial bioluminescent enzyme three-dimensional positional information (that is, a three-dimensional structure) of each amino acid constituting ALuc30 was analyzed. The model obtained by the analysis is shown in FIG. 1B.
- coelenterazines coelenterazine and its derivatives
- coelenterazines that react specifically with artificial bioluminescent enzymes were found (FIG. 1).
- coelenterazine is formed by an imidazole skeleton and three residue. Each residue interacts with an artificial luminescent enzyme with the following characteristics:
- Residue C (R-C)
- the amino acid sequence of the luminescent enzyme particularly interacts with the back amino acid.
- amino acids such as No. 165 Leu, No. 169 Cys, No. 209 Lys, No. 212 Gly.
- amino acids may be located at the end of an artificial bioluminescent enzyme and appear to be in a relatively hydrophilic environment in addition to a structurally soft structure.
- the optimal RC property for such an environment is that it has a certain hydrophilicity, it is ideal that the RC has a hydrophilic functional group (eg, a hydroxy group) in the skeleton of the benzene ring.
- a comparative experiment was conducted using a derivative (ie, coelenterazine 400A) having no functional group in the R-C benzene ring among the conventional coelenterazine derivatives (FIG. 3).
- the present invention was completed by clarifying the chemical structure of an ideal luminescent substrate from the scrutiny based on the three-dimensional structure of the artificial luminescent enzyme.
- the present invention specifically includes the following aspects.
- Item 1 A compound represented by the following general formula (1) for use as a luminescent substrate of a polypeptide comprising the amino acid sequence of (i) or (ii) below and having a copepod luciferase activity: use: (I) the amino acid sequence shown in SEQ ID NO: 1, or (Ii) An amino acid sequence in which one or more amino acids are deleted in at least one of positions 1-31 and 217-221 of the amino acid sequence shown in SEQ ID NO: 1.
- X a represents a phenyl group or a naphthyl group which may be substituted with a halogen atom.
- X b represents a phenyl group.
- M c represents a hydrogen atom, a hydroxyl group or a thiol group.
- Item 2 the compound represented by the general formula (1) is coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), or coelenterazine 400A (CTZ 400A)
- CTZ 400A coelenterazine 400A
- Item 3 A bioluminescence assay method comprising the steps of bringing the following compound (A) into contact with the following polypeptide (B) and measuring the luminescence intensity based on the contact:
- Compound (A) Compound represented by the following general formula (1);
- Item 5 The method according to Item 5, wherein two or more luminescent colors are measured in combination with another luminescent enzyme.
- Item 6 The method according to Item 3, which is a reporter gene assay method, a two-hybrid assay method, a luminescent capsule assay method, or a single-molecule bioluminescent probe measurement method.
- a bioluminescence resonance energy transfer (BRET) method comprising a step of measuring the luminescence brightness of the fluorescent protein having the luminescence energy transitioned:
- Compound (A) Compound represented by the following general formula (1);
- the present invention relates to the development of a luminescent substrate that is most suitable for the non-natural artificial bioluminescent enzyme (ALuc) of the inventor's already invented invention. And important amino acids), and the knowledge about the chemical structure of the substrate that fits in the three-dimensional structure was obtained. By investigating the luminescent properties of many coelenterazine derivatives that match this finding, we have clarified the optimal luminescent substrate for ALuc.
- ALuc non-natural artificial bioluminescent enzyme
- coelenterazine derivatives (CTZ n, CTZ i, CTZf, CTZ i, etc.) with controlled RA size selected in the examples of the present invention were prepared using Renilla luciferase 8.6-535 (RLuc -8.6-535; high selectivity (brightness standard) about 100 to 10,000 times that of ALuc compared to conventional representative).
- a multicolor imaging system (including a dual assay system) can be constructed by combining with RLuc8.6-535.
- three different luminescent enzymes (ALuc, RLuc8.6-535 and CLuc) of different colors are expressed in the same cultured cells, and then first luminescence is performed with a luminescent substrate dedicated to ALuc (eg, CTZ i) and then quenched.
- RLuc8.6-535 is caused to emit light with a specific substrate, and then the previous luminescent enzyme is inactivated (quenched) with SDS. Finally, light is emitted with a substrate dedicated to CLuc. Since CLuc is strong because it has many Cys, it is not inactivated by SDS, so this assay system can be constructed. Of course, at this time, by utilizing the difference in the color of each luminescent enzyme, the crosstalk between them can be prevented by attaching an appropriate filter to the photodetector.
- the bioassay method whose performance has been improved by the present invention can be further improved by using a combination of the optimal reaction solution for bioassay (Japanese Patent Application No. 2014-085311 (Japanese Patent Application No. 2012-236872)).
- the optimal reaction solution one-shot buffer
- background light is suppressed, and an increase in signal intensity and luminance are expected.
- the experimental procedure can be simplified and omitted, saving time and labor. As a result, the S / N ratio in the bioassay is improved, the reproducibility is improved, and the cost is reduced.
- ALuc Genetic phylogenetic characteristics and three-dimensional structure of artificial bioluminescent enzyme (ALuc).
- A Verification of genetic phylogenetic correlation between commercial copypod luminescent enzyme and ALuc. The dotted line indicates artificial bioluminescent enzymes developed by the present inventors.
- B Super two-dimensional structure of ALuc30. The chemical structure indicates the substrate (coelenterazine). R-A, R-B, and R-C each indicate the residue of the luminescent substrate. H1-H9 indicates the number of each helix. Chemical structure of coelenterazine derivatives used in this study. The top row shows derivatives with different R-A sites of coelenterazine. The lower row shows derivatives with different R-B sites of coelenterazine.
- Each arrow and box indicates a characteristic functional group of each derivative. Comparison of emission luminance and spectra of ALuc and RLuc8.6-535 with coelenterazine derivatives.
- A Comparison of emission luminance depending on coelenterazine derivatives. The blue bar and the blue Y axis (left) indicate RLuc8.6-535, and the red bar and the red Y axis (right) indicate ALuc34.
- Inset A shows an image of emission luminance depending on the coelenterazine derivative. Comparison of emission luminance and spectra of ALuc and RLuc8.6-535 with coelenterazine derivatives.
- B Comparison of emission spectra of ALuc and RLuc8.6-535 depending on coelenterazine derivatives.
- A The super two-dimensional structure of ALuc30 is shown. The center shows the skeleton of coelenterazine. Correlation between substrate selectivity and conformation of ALuc.
- B Internal profile of the three-dimensional structure of ALuc30. There is a cavity at the tip of R-A. A black and white reversal diagram (Monochromeoinversion image) is also shown. The results of the dual assay are shown. Since the luminescence activity of ALuc is almost deactivated by the SDS treatment, only the luminescence activity of CLuc remains. The lower table shows the quantitative values of the luminescence intensity. An example of the arrangement
- amino acid sequence shown in SEQ ID NO: 3 is shown.
- x means that any amino acid may be used.
- a lower case “y” means a hydrophobic amino acid.
- Z means a hydrophilic amino acid.
- ALucCM indicates the amino acid sequence shown in SEQ ID NO: 2.
- x means that any amino acid may be used (a blank (blank) may be used).
- “O” is a hydrophobic amino acid
- “j” is a hydrophilic amino acid
- “@” is a high molecular weight aliphatic amino acid
- “+” Means an amino acid having a positive charge
- “-” means an amino acid having a negative charge.
- ALucCM represents the amino acid sequence shown in SEQ ID NO: 1. In the figure, “x” means that any amino acid may be used (a blank (blank) may be used).
- “O” is a hydrophobic amino acid
- “j” is a hydrophilic amino acid
- “@” is a high molecular weight aliphatic amino acid
- “+” Means an amino acid having a positive charge
- “-” means an amino acid having a negative charge.
- the luminescent substrate of the present invention is a compound represented by the following general formula (1).
- X a represents a phenyl group which may be substituted with one halogen atom, or a naphthyl group (naphthalene group).
- the position of the substituent is not limited, but when the phenyl group has a substituent, the meta position or the para position, particularly the para position is preferable.
- halogen atom refers to chlorine, bromine, fluorine, and iodine.
- X b represents a phenyl group.
- M c represents one hydrogen atom, a hydroxyl group or a thiol group.
- the position of the hydroxyl group or thiol group on the benzene ring is not limited, but the meta position or para position, particularly the para position is preferred.
- Examples of the compound of the present invention typically include coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), coelenterazine 400A (CTZ 400A), and CTZ n.
- CTZ i, CTZ f, and CTZ h are particularly preferable, but not limited thereto.
- CTZ n coelenterazine n
- CTZ i coelenterazine i
- CTZ f coelenterazine f
- CTZ h coelenterazine h
- CTZ 400A coelenterazine 400A
- the luminescent substrate of the present invention can be synthesized, for example, according to the synthesis route indicated by the following document (Non-patent Documents 17, 18, 24, 26, 28, 29).
- the luminescent substrate is used as a luminescent substrate of the following artificial luminescent enzyme (sometimes referred to herein as “artificial luciferase” or “ALuc” (artificial luciferase)).
- Typical artificial luciferases (ALuc) in the present invention are ALuc10 (SEQ ID NO: 4), ALuc15 (SEQ ID NO: 5), ALuc16 (SEQ ID NO: 6), ALuc17 (SEQ ID NO: 7), ALuc18 (SEQ ID NO: 8), ALuc19 ( Sequence number 9), ALuc21 (sequence number 10), ALuc22 (sequence number 11), ALuc23 (sequence number 12), ALuc24 (sequence number 13), ALuc25 (sequence number 14), ALuc26 (sequence number 15), ALuc27 (sequence) 16, ALuc28 (SEQ ID NO: 17), ALuc29 (SEQ ID NO: 18), ALuc30 (SEQ ID NO: 19), ALuc31 (SEQ ID NO: 20), ALuc32 (SEQ ID NO: 21), ALuc33 (SEQ ID NO: 22) and ALuc34 (SEQ ID NO: 23) ).
- the artificial luciferase (ALuc) of the present invention can be expressed as a polypeptide having the amino acid sequence described in any of (i) to (iii) below and having chiacyl luciferase activity.
- everal means 1 to 20, preferably 1 to 10, and more preferably 1 to 5.
- amino acid sequence having 90% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 4 to 23,
- an amino acid sequence having 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity is more preferable.
- the amino acid sequences of the artificial luciferase (ALuc) of the present invention all have a common basic skeleton as shown in FIGS. As long as they have these basic skeletons, amino acids at other positions have the same high-performance chiacyl luciferase activity even if they are arbitrary amino acids. Therefore, the artificial luciferase (ALuc) of the present invention can also be expressed as a polypeptide having the amino acid sequence described in (iv) to (vii) below and having chiacyl luciferase activity.
- the amino acids up to the 1-20th position on the N-terminal side are a secretion signal (secretion peptide; SP), and the 21st- 21st position peptide on the C-terminal side Is a Glycine-rich linker peptide (commonly referred to as GS linker), and therefore part or all of the amino acids in any region may be deleted.
- secretion peptide secretion peptide
- GS linker Glycine-rich linker peptide
- amino acid sequence shown in SEQ ID NO: 2 positions 1-20 on the N-terminal side and positions 214-218 on the C-terminal side, and among the amino acid sequences shown in SEQ ID NO: 1, 1-position on the N-terminal side The same applies to the 20th position and the 217th to 221st positions on the C end.
- the secretion signal for example, it corresponds to amino acids 1-18 in the case of Metridia pacifica luciferase 1 (MpLuc1), which is a copepod luminescent enzyme, and corresponds to amino acids 1-19 in the case of pleuromamma luciferase, which may be deleted. I know that.
- amino acid sequences shown in SEQ ID NOs: 1 to 3 the amino acid represented by Xaa will be described in detail below.
- the amino acids at positions 145, 147, 156, 158, 185, 188, 199 and 203 may be any amino acid. Of these, positions 74-75 and 137-140 may be deleted.
- position 3 is E or G
- positions 20-27 are PTENKDDI sequence (SEQ ID NO: 26), ATINEEDI sequence (SEQ ID NO: 27), ATINENFE sequence (SEQ ID NO: 28), HHHHHHHH sequence (SEQ ID NO: 29), EKLISEE Sequence (SEQ ID NO: 30), MMYPYDVP sequence (SEQ ID NO: 31) or MMDYKDDD sequence (SEQ ID NO: 32)
- position 29 is I, L, Y or K
- position 30 is V, D or A
- Position is E, G or A
- position 35 is K
- position 62-64 is ANS sequence or DAN sequence
- position 67 is D or G
- position 75-76 is GG sequence Or K (1 residue deletion) or may be deleted
- positions 83-84 are LE, KA or KE sequences
- positions 87-88 are KE sequences, IE sequences, LE sequences or KI Sequence is 127, E, G or A, 137-145 is I
- amino acids at positions 13, 16, 36, 148, 171, and 215 are hydrophobic amino acids (for example, V, F, A, and L), and preferably, position 13 is V or F.
- the position is V or A
- position 36 is F or G
- position 148 is I or G
- position 168 is V or A
- position 215 is A or L.
- 5,65,73,99,117,211 is a hydrophilic amino acid (for example, Q, K, D, R, H, E, T.), preferably, the 5th position is Q or K, 65 is D or R, 73 is K, H, R or E, 99 is T or H, 117 is K, E or Q, 211 is K or T .
- a hydrophilic amino acid for example, Q, K, D, R, H, E, T.
- positions 4, 6, 7, 10, 11, 15, 31, 32, 37-39, 61, 66, 72, 76, 81, 136, 157, and 200 are aliphatic amino acids.
- positions 4, 6, 7, 10, 11, 15, 32, 61, 76, 81, 157 are high molecular weight aliphatic amino acids (eg, I, V, L, M.), more preferably 4th is I or V, 6th is V or L, 7th is L or I, 10th is L or V, 11th is I or L, 15th is L or V, position 32 is I or V, position 61 is L or V, position 76 is L or M, position 81 is L or M, and position 157 is L or M.
- positions 31, 34, 37-39, 66, 72, 136, 200 are low molecular weight aliphatic amino acids (eg, A, G, T, L), more preferably position 31 is G, L or A, position 34 is G or I, position 37 is G, A, S or F, position 38 is T or F, position 39 is T or A, position 66 is A Or G, position 72 may be G or may be deleted, position 136 is G or A, position 200 is T or G;
- Positions 70, 71, 95, and 108 are positively charged amino acids (basic amino acids such as K, R, and H).
- positions 70 and 71 are R or deleted.
- the 95th position is K or R
- the 108th position is H or K.
- Positions 60 and 208 are negatively charged amino acids (acidic amino acids, such as N, D, Q, E), preferably, position 60 is N or D, and position 208 is Q or E.
- the amino acids at positions 144, 148-151, 159, 161, 188, 191, 202, and 206 may be any amino acid.
- positions 22-23, 39-40, 76-77, 140, 148-151 may be deleted.
- positions 20-29 are PTENKDDI sequence (2 residue deletion, SEQ ID NO: 37), ATINEEDI sequence (2 residue deletion, SEQ ID NO: 38), ATINENFEDI sequence (SEQ ID NO: 39), HHHHHHHH sequence (2 residue deletion, SEQ ID NO: 40), EKLISEE sequence (2 residue deletion, SEQ ID NO: 41), MMYPYDVP sequence (2 residue deletion, SEQ ID NO: 42) or MMDYKDDD sequence (2 residues) Group deletion, SEQ ID NO: 43), position 31 is I, L, Y or K, position 32 is V or A, position 35 is E or G, position 37 is K or S , 64-66 is an ANS sequence or DAN sequence, 69 is D or G, 76-77 is GG sequence or K (1 residue deletion), or may be deleted , Positions 85-86 are LE, KA or KE sequences, positions 89-90 are KE sequences, IE sequences, LE sequences or KI sequences, positions 129
- amino acids at positions 13, 16, 174, and 218 are hydrophobic amino acids (for example, V, F, A, and L.), preferably, position 13 is V or F, and position 16 is V or A. Yes, position 174 is V or A, position 218 is A or L.
- positions 5, 67, 75, 101, 119, 214 are hydrophilic amino acids (for example, Q, K, D, R, H, E, T), and preferably, position 5 is Q or K.
- 67 is D or R
- 75 is K
- 101 is T or H
- 119 is K
- 211 is K or T .
- 4, 6, 7, 10, 11, 15, 33, 34, 39-41, 63, 68, 77, 78, 83, 138, 160, 203 are aliphatic amino acids. However, positions 39, 40 and 70 may be deleted.
- 4, 6, 7, 10, 11, 15, 34, 63, 78, 83, 160 are preferred high molecular weight aliphatic amino acids (eg, I, V, L, M), but less frequently May contain low molecular weight aliphatic amino acids.
- position 4 is I or V
- position 6 is V or L
- position 7 is L or I
- position 10 is L or V
- position 11 is I or L
- 15 Position is L or V
- position 34 is I or V
- position 63 is L or V
- position 78 is L or M
- position 83 is L or M
- position 160 is L or M It is.
- low molecular weight aliphatic amino acids eg, A, G, T
- are preferred at positions 33, 39-41, 68, 74, 137, and 203. is there.
- position 33 is G, L or A
- position 39 is G or A or may be deleted
- S or F position 40 is T or deletion 41 may be T or A
- 68 may be A or G
- 74 may be G or deleted
- 137 may be G or A
- 203 The place is T or G.
- Positions 72, 73, 97, and 110 are positively charged amino acids (basic amino acids. For example, K, R, and H.). However, positions 72 and 73 may be deleted. Preferably, positions 72 and 73 may be R or may be deleted, position 97 is K or R and position 110 is H or K.
- Positions 62 and 211 are negatively charged amino acids (acidic amino acids, such as N, D, Q, E), preferably 62 is N or D, and 211 is Q or E.
- the amino acids at positions 142, 155, 185, and 197 may be any amino acid, and the amino acids at positions 71 to 75 and 140 to 142 may be partially or completely deleted.
- hydrophilic amino acids are 3rd, 22nd, 27th, 33rd, 127th, 140th, 141st, 155th are E, 26th, 30th, 62nd, 67th, 185th is D, 35th, 87th is K, 37th is S, 38th, 39th, 138th, 142th, 197th is T, 63rd is N, Position 71 is R and position 73 is D or H.
- positions 3, 37, 67, 72, 74, 75, 138, and 197 are G, and positions 22, 27, and 141 are I.
- 30th is V
- 33rd, 39th, 62nd, 63rd, 127th, 140th, 155th, 185th is A
- 87th is L
- 26th, 38th F is 3rd, 22nd, 27th, 33rd, 127th, 140th, 141st, 155th are E, 26th, 30th, 62nd, 67th, 185th is
- amino acids at positions 4, 6, 7, 10, 11, 13, 15, 16, 20, 31, 34, 36, 61, 66, 81, and 168 are hydrophobic amino acids, preferably the 4th position.
- position 6 is V or L
- position 7 is I or L
- position 10 is V or L
- position 11 is I or L
- position 13 is V or F Yes
- position 15 is V or L
- position 16 is V or A
- position 20 is A or P
- position 31 is L or G
- position 34 is I or G
- position 36 is F
- position 61 is V or L
- position 66 is A or G
- position 81 is L or M
- position 168 is V or A.
- amino acids at positions 5, 24, 25, 60, 64, 65, 70, 95, 108, 153, 200, and 208 are hydrophilic amino acids, preferably, position 5 is Q or K, and position 24 is K or E, position 25 is D or N, position 60 is D or N, position 64 is N or S, position 65 is D or R, position 70 is K or R , Position 95 is K or R, position 108 is K or H, position 153 is E or D, position 200 is D or S, and position 208 is K, H or T.
- Typical examples of the amino acid sequence shown in SEQ ID NO: 3 include ALuc10, ALuc15, ALuc16, ALuc18, ALuc22, ALuc23 and ALuc25.
- the region at positions 1-71 in the amino acid sequence shown in SEQ ID NO: 1 (the region at positions 1-69 in the amino acid sequence shown in SEQ ID NO: 2, and This also corresponds to the region of positions 1 to 69 of the amino acid sequence shown in SEQ ID NO: 3.)
- Typical examples include ALuc15, ALuc16, ALuc17, ALuc18, ALuc24.
- the region at position 1-157 of the amino acid sequence shown in SEQ ID NO: 1 (the region at position 1-155 of the amino acid sequence shown in SEQ ID NO: 2, and This also corresponds to the region of positions 1-152 of the amino acid sequence shown in SEQ ID NO: 3.) has the amino acid sequence shown in SEQ ID NO: 25.
- Typical examples include ALuc22, ALuc25, ALuc26, ALuc27, ALuc28, ALuc29.
- an antibody recognition site is contained inside.
- An “antibody recognition site” or “epitope sequence” can be rephrased as an “antigen site”.
- ALuc30, ALuc31, ALuc32, and ALuc34 are applicable.
- the region at positions 20-29 in SEQ ID NO: 2 or 20-31 in SEQ ID NO: 1 contains the antibody recognition site (epitope sequence).
- Preferred examples of the antibody recognition site (epitope sequence) include His-tag (HHHHHH) (SEQ ID NO: 48), FLAG-tag (DYKDDDDK) (SEQ ID NO: 49), Myc-tag (EQKLISEEDL) (SEQ ID NO: 50), HA -tag (YPYDVPDYA) (SEQ ID NO: 51) can be mentioned, but is not limited thereto.
- all of the amino acids at positions 20-29 in SEQ ID NO: 2 or 20-31 in SEQ ID NO: 1 are H (His x 8 sequence).
- Typical examples include ALuc30 and ALuc31.
- the sequence at positions 20-29 in SEQ ID NO: 2 or 20-31 in SEQ ID NO: 1 is EQKLISEEDL (Myc-tag sequence, SEQ ID NO: 50). is there.
- a typical example is ALuc32.
- amino acid at positions 20-29 in SEQ ID NO: 2 or 20-31 in SEQ ID NO: 1 is YPYDVPDYA (HA-tag sequence, SEQ ID NO: 51).
- a typical example is ALuc33.
- amino acid at positions 20-29 in SEQ ID NO: 2 or 20-31 in SEQ ID NO: 1 is DYKDDDDK (FLAG-tag sequence, SEQ ID NO: 49).
- a typical example is ALuc34.
- the term “copepod luciferase” refers to a luminescent enzyme (luciferase) produced by a minute crustacean living as a luminescent plankton called a copepod. Specifically, MoLuc1, MoLuc2, PaLuc1, PaLuc2, LoLuc, HtLuc1, HtLuc2, HmLuc1, HmLuc2, Gaussia luciferase (GLuc), chiacyl luciferase (MLuc, MpLuc1, MpLuc2) and the like are included. “Copepod luciferase” specifically oxidizes “coelenterazine” as substrate specificity.
- the term “copepod luciferase” refers to a luciferase having an enzyme activity and a structural feature common to luciferases derived from known copepods. Specifically, it is an luciferase having an optimum pH of about 5 to 8, an optimum temperature of about 4 to 25 ° C., and having an enzyme activity that catalyzes a luminescence reaction using “coelenterazine” as a substrate.
- luciferase having one enzyme active domain, a secretory signal at the N-terminus, a molecular weight of about 20 kD (18 kD-28 kD) and the smallest molecular weight compared to other luminescent enzymes.
- Coelenterazine is not limited to natural coelenterazine (Native CTZ, n CTZ), but also includes various derivatives of natural coelenterazine. That is, “coelenterazine” can also be referred to as “coelenterazines”.
- coelenterazine examples include natural coelenterazine (Native CTZ), coelenterazine ip (CTZ ip), coelenterazine i (CTZ i), coelenterazine hcp (CTZ hcp), coelenterazine 400A (CTZ 400A), coelenterazine fcp (CTZ fcp), Examples include coelenterazine cp (CTZ cp), coelenterazine f (CTZ f), coelenterazine h (CTZ h), coelenterazine n (CTZ n), and the like.
- CTZ cp coelenterazine f
- CTZ h coelenterazine h
- CTZ n coelenterazine n
- an eukaryotic cell expression vector (eg, pcDNA3.1 (+)) encoding ALuc was introduced into COS-7 cells derived from African monkeys.
- An expression vector having a copepod luminescent enzyme is also introduced into cells by the same method. After a certain time (10 to 20 hours, for example, 16 hours) has passed since the introduction of the vector, each cell is lysed using a known cell lysis solution.
- ALuc An example of a base sequence encoding ALuc is shown as a sequence number.
- the cell lysate is mixed with a known buffer solution containing the optimum luminescent substrate of the present invention, and the color development intensity, luminescence stability over time, and the like are measured.
- the emission intensity can be measured by measuring the intensity at a specific wavelength using a conventional emission spectrophotometer after the addition of the optimum luminescent substrate of the present invention. Stability can be evaluated. To measure the shift to the long wavelength side, scan all wavelengths.
- the optimal luminescent substrate of the present invention is exhibited, such as 100 to 10,000 times selectivity for the present inventors' ALuc compared to conventional luciferases derived from marine organisms. By using it, it was confirmed that the luminescence specificity could be guaranteed in various bioassay systems using ALucs.
- the “reporter analysis method capable of using a luminescent substrate optimal for ALuc” of the present invention is classified into three categories “basic”, “inducible” and “activatable” shown in Non-Patent Document 16 by Niu et al. These will be described in three types.
- the “basic” method is the simplest reporter analysis system, and can be said to be a reporter analysis system in which various types of test proteins are simply linked with ALuc and labeled.
- a typical example is a bioluminescent enzyme fusion protein linked to an antibody (that is, a bioluminescent enzyme-labeled antibody).
- the “inducible” method differs from the “basic” method in that the expression of the reporter is controlled by a promoter.
- a so-called reporter gene assay and a two-hybrid assay (expressing a reporter depending on stimulation) can be mentioned.
- the “activatable” method is a reporter analysis method that utilizes the fact that the reporter itself reacts actively to ligand stimulation and emits light, and typically includes a single-molecule bioluminescent probe and a luminescent capsule. Besides, it can be applied to protein-complentation-assay (PCA) and protein-splicing-assay (PSA).
- PCA protein-complentation-assay
- PSA protein-splicing-assay
- fusion protein refers to (i) a protein that is integrally expressed from a gene encoding a fusion protein comprising a reporter protein that is ALuc and a target protein or a peptide that recognizes the target protein, (ii) ) A reporter protein that is ALuc and a target protein or a peptide that recognizes the target protein are separately expressed and linked by a chemical reaction.
- means for linking separately expressed proteins and the like by chemical reaction include, for example, linking using a crosslinker, linking using avidin-biotin binding ability, linking using the chemical reactivity of amino acid residues, etc.
- Analysis systems included in the ⁇ inducible method '' include reporter gene assay, Yeast Two-hybrid assay, Mammalian Two-hybrid assay, protein splicing assay (PSA), protein complementation assay (PCA), circular permutation assay, Bioluminescence resonance energy transfer assay (BRET) is available, but the measurement performance of the assay can be dramatically improved by using ALuc as a reporter gene essential for these analysis systems.
- PSA protein splicing assay
- PCA protein complementation assay
- BRET Bioluminescence resonance energy transfer assay
- reporter gene assay and a two-hybrid assay, which are typical “inducible method” analysis systems, will be described in detail.
- the reporter gene assay is widely used as an analysis tool for transcription factor activation and gene expression regulation by external stimuli, but typically disrupts signal transduction via nuclear receptors. It is used to detect endocrine disruptors (environmental hormones).
- the expression of a target gene eg, hormone responsive gene
- the expression of a target gene related to signal transduction via a nuclear receptor is determined by the cis region (hormone response element; hormone response element) in which the complex of ligand and receptor regulates transcription of the gene. ).
- This is a method for detecting the amount of hormone molecules or endocrine disrupting substances that can serve as ligands based on the amount of bioluminescence, etc., by introducing a plasmid incorporating a reporter gene such as luciferase downstream of the cis region of these various hormone-responsive genes. .
- COS cells COS cells, CHO-K1 cells, HeLa cells, HEK293 cells, NIH3T3 cells, etc. of mammalian cells used for general gene recombination are preferably used.
- Bacteria such as yeast cells and Escherichia coli Although it may be a cell, an insect cell, etc., it is often used in a living body of a mammal including human beings or in a mammalian cell in vitro.
- ALuc is used as a reporter protein together with the luminescent substrate of the present invention, there is an advantage that measurement can be performed in a very short time after stimulation because the luminescence intensity of the reporter is extremely high. Therefore, the measurement time can be significantly shortened compared with the conventional reporter protein, and the stability of luminescence over time is high, so that luminescence can be measured even in cell lines with poor gene transfer efficiency. Further, since the shift to the long wavelength side increases the permeability through the cell membrane and the skin, the background value decreases and the measurement accuracy is high.
- the luminescent enzyme is linked to a known eukaryotic expression vector carrying a special promoter upstream, and eukaryotic. After introduction into a cell and after a certain period of time, it may be used for measurement under the condition that there is no signal (stimulation) (Non-patent Document 20).
- a known pTransLucent vector can be used and can be easily mounted using a known method.
- the two-hybrid method is one of the methods for investigating the interaction between proteins.
- yeast two-hybrid Y2H
- yeast Sacharomyces cerevisiae
- the system was first built.
- GAL4 DBD and any protein A bait
- GAL4 DBD separability of the DNA-binding domain
- transcriptional activation domain of the transcriptional activator GAL4 protein. It can be determined whether or not the transcription activation domain (TA) expressed in the cell interacts with the protein B (prey) as a fusion protein.
- the DNA binding domain (DBD) binds to the “UASG” nucleotide sequence because DBD and TA are close to each other, so that expression of a reporter gene linked downstream thereof is promoted. If the reporter gene is luciferase, the affinity of both A and B proteins can be measured by monitoring bioluminescence in the presence of the specific substrate, and screening for proteins and peptides that interact with protein A (bait) Can do. Protein B (prey) at that time can also be provided by an expression library.
- Host cells are not limited to yeast cells, and bacteria such as E. coli, mammalian cells, and insect cells are also used.
- “LexA” of a repressor protein derived from Escherichia coli can be used in addition to GAL4DDBD which is a transcriptional activator derived from yeast.
- a DNA encoding these and a DNA encoding a bait protein (that is, the above-mentioned arbitrary protein A) such as a ligand-binding region of a ligand-responsive transcription regulatory factor are ligated and downstream of a promoter that can function in a host cell. Link.
- transcription activation region of transcription activator for example, the transcription activation region of GAL4, the B42 acidic transcription activation region derived from E. coli, the transcription activation region of herpes simplex virus VP16, etc. can be used. .
- the DNA encoding these transcriptional activation regions and the DNA encoding the prey protein are linked and downstream of a promoter that can function in the host cell.
- the plasmid pGBT9 (manufactured by Clontech) can be cited as a vector having DNA encoding the DNA binding region of the transcriptional regulatory factor GAL4 and capable of using budding yeast in host cells.
- plasmid pGAD424 (manufactured by Clontech) and the like can be mentioned.
- vectors that have a DNA encoding the DNA binding region of GAL4 and can be used in mammalian cells include pM (Clontech), pBIND (Promega), etc., and herpes simplex virus VP16
- examples of vectors having a DNA encoding a transcription activation region and usable in mammalian cells include pVP16 (manufactured by Clontech), pACT (manufactured by Promega), and the like.
- examples of vectors that have DNA encoding the DNA binding region of LexA and can be used in mammalian cells include pLexA (manufactured by Clontech) and the like.
- vectors that can be used in the above include pB42AD (manufactured by Clontech).
- a vector in which the ALuc gene is inserted as a reporter gene downstream of the region to which GAL4 binds may be constructed.
- a commercially available pG5Luc vector Promega
- pFR-Luc vector (Stratagene)
- it can be used by simply mounting ALuc together with the substrate of the present invention by a known method instead of firefly luciferase mounted on the vector.
- It can also be used instead of Chloramphenicol acetyltransferase (CAT) of a commercially available pG5CAT vector (Clontech).
- CAT Chloramphenicol acetyltransferase
- the luminescent fusion protein having this structure As a typical example, when a DEVD sequence or IETD sequence that responds to cell death is inserted at the joint of each protein, it actively responds with caspase-3 or caspase-8 activity as a signal during cell death. And work as a visualization system.
- This luminescent capsule has the advantage of exhibiting extremely high brightness and stable luminescence characteristics compared to conventional luminescent probes and responding to specimens that cannot penetrate the cell membrane.
- This luminescent capsule has a basic skeleton with a “membrane localization signal (MLS)” attached to the “C-terminus of the luminescent enzyme body”. Even if ALuc is connected in tandem to increase the amount of luminescence, the effects of compounds that cause cell surface morphological changes, such as compounds that induce cell death, can be visualized as morphological changes on the cell membrane surface, making observation easy. become.
- MLS membrane localization signal
- a polypeptide that causes a morphological change on the surface of the cell membrane or a partial recognition sequence thereof specifically, for example, G-protein coupled receptor (GPCR) or c-
- GPCR G-protein coupled receptor
- c- c-
- cell death can be visualized by inserting a polypeptide that induces cell death or a recognition sequence thereof as a cargo between the C-terminus of the luminescent enzyme body and MLS.
- caspases and proteases serine protease, cysteine protease, etc.
- peptide sequences recognized by digestive enzymes trypsin, amylase, etc.
- trypsin, amylase, etc. usually 20 amino acids or less, preferably 10 amino acids or less
- cell death can be visualized by Caspase-3 activity.
- a fluorescent protein or other luminescent enzyme as a cargo between the luminescent enzyme body and MLS, the amount of light generated on the cell membrane surface is the same as when the luminescent enzyme is connected in tandem with the luminescent substrate shown in the present invention. Therefore, it becomes easier to observe the morphology of the cell membrane and responds to ligands that cannot penetrate the cell membrane, so that a wide range of stimulants can be screened.
- the luminescent capsule is a luminescent fusion protein in which an arbitrary protein or polypeptide to be expressed on the cell membrane surface is inserted between the C-terminal side of ALuc and a membrane localization signal (MLS), and is typically ,
- MLS membrane localization signal
- A Luminescent fusion protein in which a fluorescent protein or luciferase is inserted between the C-terminal side of ALuc and a membrane localization signal (MLS) (note that other lucucases may be other ALuc.
- a polypeptide that changes the morphology of the cell membrane a polypeptide that induces cell death is preferable, and a polypeptide of 20 amino acids or less including Caspase and its recognition sequence “DEVD” or “IETD” is particularly preferable. .
- ALuc is a single molecule type luminescent probe according to the invention for which the present inventors have already applied for a patent (Non-patent Documents 4, 6, 9, 10, and Patent Documents 1-4)
- a bimolecular luminescent probe Non-patent Documents 7 and 8
- the presence or absence of the ligand and the activity intensity of the ligand can be observed with high luminance.
- a high-performance luminescent probe can be constructed in a form in which a recognition protein for recognizing that a ligand is bound to is linked.
- the recognition protein recognizes that a ligand is bound to the ligand binding protein
- the enzyme fragment divided into two can complement and change the enzyme activity.
- the detection limit can be improved and the measurement can be performed with high reliability.
- the term “single molecule luminescent probe” is one of the known bioluminescent probes characterized in that all components used for visual imaging are integrated in a single fusion molecule ( Patent Document 1-2).
- it is a fusion protein comprising N- and C-terminal fragments obtained by dividing ALuc in two as a basic component, a ligand-binding protein and a ligand-binding protein recognition protein.
- the term “bimolecular luminescent probe” refers to a type of organism in which the N-terminal fragment and C-terminal fragment of ALuc are present in a fusion protein containing a ligand binding protein and a fusion protein containing a recognition protein, respectively. Refers to a luminescent probe.
- a specific method for use as a single-molecule light-emitting probe using ALuc follows the method described in detail in (Patent Documents 1-4). Specifically, a chimeric DNA encoding a luminescent probe in which ALuc is divided into two and linearly bound to a ligand-binding protein and a peptide sequence that recognizes a three-dimensional structural change when a ligand is bound to the protein. design.
- the clone is subcloned into a vector suitable for the cell in which the chimera DNA is to be expressed, and the vector is introduced into the cell and expressed in the cell. It can also be introduced directly into.
- the cells of interest are preferably cells derived from mammals including humans, and may be cells that exist in vivo or cultured cells that maintain their original functions. .
- prokaryotic cells such as Escherichia coli may be used.
- the specific type of vector is not particularly limited, and a vector that can be expressed in the host used for expression can be appropriately selected.
- a method for introduction into cells a known transfection method such as a microinjection method or an electroporation method can be used.
- intracellular introduction methods using lipids BioPORTER (Gene Therapies Systems), Chariot (Active Motif), etc.
- BioPORTER Gene Therapies Systems
- Chariot Active Motif
- the bioluminescent probe using the high-intensity luminescent enzyme together with the optimal luminescent substrate referred to in the present invention is expressed as a fusion protein in the cell after being introduced into the cell as a chimeric DNA. Then, by measuring the change in the amount of luminescence from the cells, the nature of the ligand, the degree of activity, etc. can be evaluated.
- the “ligand binding protein” that can be mounted with the ALuc is intended to be a protein that binds a ligand to the ligand binding site.
- the ligand-binding protein can be, for example, one that changes the three-dimensional structure by binding a ligand, causes phosphorylation, or promotes protein-protein interaction.
- a ligand-binding protein for example, a nuclear receptor (NR), a cytokine receptor, or various protein kinases having a hormone, chemical substance or signal transduction protein as a ligand is used.
- the ligand binding protein is appropriately selected depending on the target ligand.
- the ligand that binds to the ligand-binding protein is not particularly limited as long as it binds to the ligand-binding protein, and may be an extracellular ligand that is taken into the cell from outside the cell. It may be an intracellular ligand produced in For example, it can be an agonist or antagonist for a receptor protein (eg, nuclear receptor, G protein-coupled receptor, etc.).
- signaling proteins such as cytokines, chemokines, and insulin that specifically bind to proteins involved in intracellular signal transduction, intracellular second messengers, lipid second messengers, phosphorylated amino acid residues, G protein-coupled receptor ligands Etc.
- the binding domain of each second messenger can be used as a ligand binding protein.
- the second messenger is intended to be another type of intracellular signaling substance that is newly generated in the cell by binding extracellular signaling substances such as hormones and neurotransmitters to receptors present on the cell membrane.
- Examples of the second messenger include cGMP, AMP, PIP, PIP 2 , PIP 3 , inositol triphosphate (IP 3 : inositol triphosphate), IP 4 , Ca 2+ , diacylglycerol, and arachidonic achid.
- calmodulin CaM
- CaM calmodulin
- the gene encoding ALuc can be stably introduced into various cell lines.
- the ALuc can be stably introduced into undifferentiated cells in embryos, ES cells, or new universal cells (iPS). Since the cells themselves do not shine, it is very difficult to search for molecular phenomena and tissue specificities that occur inside.
- a molecular probe containing the ALuc is introduced into a somatic cell, and then an embryo is created and differentiated into various organ tissues. Then, a specific molecular phenomenon that occurs in each organ can be measured with high sensitivity.
- Non-patent Document 36 Following the method of Yamanaka et al. (Non-patent Document 36).
- a suitable signal peptide to the ALuc, it can be used for high-intensity imaging of each organelle.
- a suitable signal peptide for example, by attaching a “MLCCMRRTKQV sequence” (SEQ ID NO: 52) derived from GAP-43 to the N or C terminus of ALuc, it can be localized to the cell membrane.
- “GRKKRRQRRR sequence” (SEQ ID NO: 53) is attached for cytoplasmic localization.
- localization in the endoplasmic reticulum (ER) and cell nucleus can be achieved by connecting “KDEL” (SEQ ID NO: 54) and “DPKKKRKV sequence” (SEQ ID NO: 55), respectively.
- HIS-tag (HHHHHH) (SEQ ID NO: 48), FLAG-tag (DYKDDDDK) (SEQ ID NO: 49), Myc-tag (EQKLISEEDL) (SEQ ID NO: 50), HA-tag (YPYDVPDYA) (SEQ ID NO: 51)
- antigen sites such as V5-tag (GKPIPNPLLGLDST) (SEQ ID NO: 556) and T7-tag (MASMTGGQQMG) (SEQ ID NO: 57
- a known technique such as immunostaining or immunocytochemistry can be applied.
- reaction solution for bioassay having luminescent substrate of the present invention Reaction solution for bioassay (1-1) Lysis buffer (cell lysate) and assay buffer (reaction solution) Conventionally, bioassays have been performed by dividing into lysis buffer (cell lysate) and assay buffer (reaction solution). In order to rapidly lyse cells, high lysis power and low inhibitory activity against luminescent enzymes are considered essential, while in order to reduce stable assay conditions and background during bioassay reactions, This is because it was thought that the removal and examination of components that induce self-luminescence were essential.
- Promega also sold the lysis buffer and assay buffer under the product name Luciferase Assay Buffer (catalog number: E290A) and product name Luciferase Lysis Buffer (catalog number: E291A), respectively. It is sold under the product names Luciferase Assay ⁇ ⁇ ⁇ ⁇ Buffer (catalog number: E3300S) and Luciferase Lysis Buffer (catalog number: B3321).
- Luciferase Assay ⁇ ⁇ ⁇ ⁇ Buffer catalog number: E3300S
- Luciferase Lysis Buffer catalog number: B3321
- reaction solution components that can be used together with the optimal luminescent substrate for ALuc referred to in the present invention is being investigated.
- A Surfactant: Polyoxyethylene octylphenyl ether (TritonX-100; TX100), Nonidet P-40 (NP40), polyoxyethylene sorbitan monolaurate (Tween20; TW20), polyoxyethylene sorbitan monooleate (TW80), polyoxyethylene cetyl ether (Brij58), sodium dodecyl sulfate (SDS) etc.
- the degree of hydrophilicity is TW20>Brij58>TW80>TX100> NP40, and the degree of surface activity is NP40>TX100>Brij58>TW20> TW80.
- B Salts: NaCl, KCl, (NH 4 ) 2 SO 4 etc.
- SH reagent mercaptoethanol, DTT etc.
- Polyol glycerol, glucose, sucrose etc.
- Glycols Polyethylene glycol (PEG) Polypropylene glycol (PPG)
- chelating reagents EGTA, EDTA, etc.
- Protease Inhibitor aprotinin (molecular weight 6.5 kD), leupeptin (molecular weight: 427), pepstatin A (pepstatin, molecular weight: 686), phenylmetylsulfonyl fluoride (PMSF , Molecular weight: 174), antipain (Antipain, molecular weight: 605), chymostatin (chymostatin, molecular weight: 608), pefabloc SC (AEBSF, 240 Da), DFP (184 Da), p-APMSF (216 Da), STI ( 20,100 Da), Leupeptin (460 Da), N-Tosyl-L-phenylalaninechloromethylketone, 3,4-dichloroisocoumarin (215 Da), EDTA-Na 2 (372 Da), EGTA (380 Da), 1,10-phenanthroline (198 Da), phosphoramidon (580 Da), Dithiobis (2-amino-4-methylpent
- Buffer p-Toluenesulphonic acid, tartaric acid, citric acid, phthalate, glycine, trans-aconitic acid, formal aicd, 3,3-dimethylglutaric acid , Phenylacetic acid, sodium acetate, succinic acid, sodium cacodylate, sodium hydrogen maleate, maleic acid, sodium phosphate, KH 2 PO 4 , imidazole, 2,4,6-trimethylpyridine, Triethanolamine hydrochloride, sodium 5,5-diethylbarbiturate, N- ethylmorpholine, sodium pyrophosphate, Tris (hydroxymethyl) aminomethane, bicine, 2-amino-2-methylpropane-1,3-diol, diethanolamine, potassium p-phenolsulphonate, boric acid, sodium borate, ammonia, glycine, Na 2 CO 3 / NaHCO 3 , sodium borate, or combinations thereof (i) Other: Sodium molybdate (
- Basic reaction solution component 1 which can add an optimal luminescent substrate to the ALuc of the present invention 1:
- HBSS buffer Hanks' Balanced Salt Solution
- the preparation method is in accordance with a known protocol (for example, the one described in the website of http://cellbank.nibio.go.jp/legacy/sheet/att00011.htm of the National Institute of Biomedical Innovation) as follows. It was prepared as follows.
- -Solution 1 1.4% NaHCO3 solution-Solution 2: NaCl 80.0 g, KCl 4.0 g, MgSO4 ⁇ 7H2O 2.0 g, Na2HPO4 ⁇ 2H2O 0.6 g, glucose 10.0 g, KH2PO4 0.6 g in 800 ml water.
- -Solution 3 A solution of 1.4 g CaCl2 in 100 ml water.
- -Solution 4 Weigh 0.4 g of Phenol red, paste it with a small amount of water, then add water to 150 ml.
- Basic reaction solution component 2 that can add an optimal luminescent substrate to ALuc Tris buffer itself is a buffer component that has been widely used (Tris here is an abbreviation for trishydroxymethylaminomethane, and its general composition is a tris salt adjusted to pH with 10 mM HCl. EDTA 1mM may be included), and it is used in various biological research because of its high biocompatibility. However, studies on the effect of Tris buffer on the bioluminescence reaction have not been sufficiently conducted so far.
- Tris buffer can be suitably used for bioluminescence, and is a basic buffer component commonly used for cell lysis (Lysis) and assay.
- Buffer composition in the present invention The basic buffer components HBSS buffer and Tris-buffer are used in combination, and in this case, both are used in a volume (v / v)% of 20-50: 50-20, preferably Is blended at 40-60: 60-40, most preferably 60:40.
- NP-40 and TW80 and further SDS are used in combination.
- NP-40: TW80: SDS is used in a volume (v / v)% of 1: 0.1 to 1: 0 to 0.5, preferably Is blended at a ratio of 1-2: 0.5-2: 0.1-1 and most preferably 1: 1: 0.1.
- Surfactant TW80 is contained in combination with other surfactants, but the concentration at that time is 1 to 10 (v / v)%, preferably 5 to 10 (v / v)%.
- polyethylene glycol PEG
- sugar component sucrose, glucose
- PEG400 is in the range of 0.01 to 10 (v / v)%
- sugar component sucrose, glucose
- sugar component is in the range of 0 to 20 mg / mL.
- PEG400 is added in the range of 0.1 to 10 (v / v)%
- sugar components are added in the range of 2 to 10 mg / mL.
- the heavy metal (Fe (III), Cu (II), Mo (VI) or Zn (II)) is contained alone or in combination, but the concentration at that time is in the range of 0.01 to 1 PPM, preferably 1 PPM.
- Halogen ions (Br - or I) is alone or in combination is contained, the concentration at that time 1 ⁇ 100 mM, preferably added in amounts of 50 ⁇ 100 mM.
- the buffer composition preferable as a one-shot reaction solution containing a luminescent substrate specific for ALuc was narrowed down as follows.
- the basic composition of the “one-shot reaction solution” in the bioluminescent enzyme utilization technology is based on the C3 buffer.
- Tris-HCl buffer combined with HBSS buffer, surfactant NP-40 or SDS, salts Al (III), Ca (II), Cu (II), Fe (III), or Mg (II), PEG or PPG, halogen ions (I -, Br -), may be constructed by combining a D (+) glucose or Glycine.
- Ligand activity may be measured according to a normal bioluminescence assay, and a conventional protocol can be applied without particular limitation.
- a luminometer eg, MiniLumat LB 9506 (Berthold); GloMax 20 / 20n (Promega)
- a cell lysate is prepared by applying a lysate buffer to the cells cultured on the plate, and the luminescence value immediately after mixing with the ALuc optimal luminescent substrate of the present invention is measured.
- a ready-made bioluminescence plate reader eg, Mithras LB 940 (Berthold); SH-9000 (Corona)
- a ready-made bioluminescence plate reader eg, Mithras LB 940 (Berthold); SH-9000 (Corona)
- bioluminescence by the expressed probe can be instantaneously introduced into the substrate and measured for luminescence by using an automatic substrate solution injector attached to the plate reader.
- test substances that are targets of these screening methods include organic or inorganic compounds (particularly low molecular weight compounds), biologically active proteins, peptides, and the like. These substances may be known or unknown in function and structure. Further, the “combinatorial chemical library” is an effective means as a test substance group for efficiently specifying a target substance.
- the preparation and screening of combinatorial chemical libraries is well known in the art (see, eg, US Pat. Nos. 6,004,617; 5,985,365).
- libraries for example, libraries such as those manufactured by ComGenex in the US, manufactured by Russian Asinex, manufactured by US Tripos, Inc., manufactured by Russian ChemStar, Ltd, manufactured by 3D Pharmaceuticals, manufactured by Martek Biosciences, etc.
- high-throughput screening can be performed by applying a combinatorial chemical library to a population of cells expressing the probe.
- Kit The present invention also provides a bioassay kit containing an ALuc-specific luminescent substrate.
- the kit of the present invention can contain various components for carrying out a bioassay as necessary. Examples of such components include luminescent enzymes, vectors containing genes encoding luminescent enzymes, cells expressing luminescent enzymes, ALuc-specific luminescent substrates of the present invention, various instruments (96-well plates or tubes, etc.), and controls. Although a sample etc. are illustrated, it is not limited to this. In addition, a protocol describing the method for carrying out the bioassay method of the present invention may be included.
- luminescent enzyme examples include bioluminescent enzymes derived from insects and marine animals, typically fireflyizicferase, click beetle luciferase, Renilla luciferase, copepod luciferase (Metridia longa luciferase), etc.
- the artificial luciferase (ALuc) of the present invention is a suitable example.
- Artificial luciferase (ALuc) is one of the particularly preferred embodiments.
- the vector containing the gene encoding the luminescent enzyme is the intended bioassay (reporter-gene assay, two-hybrid assay, protein complementation assay, protein via intein).
- bioassay reporter-gene assay, two-hybrid assay, protein complementation assay, protein via intein.
- intein-mediated protein-splicing assay, single-chain probe-based assay, etc. it can be produced by a known method.
- control sample examples include a positive control containing a predetermined amount of luminescent enzyme and a negative control containing no luminescent enzyme.
- the kit of the present invention can be produced by combining the above components by a known method.
- the kit of the present invention can be used for carrying out the bioassay method of the present invention described above.
- Example 1 Consideration of genetic phylogenetic characteristics and three-dimensional structure of artificial bioluminescent enzyme A series of artificial bioluminescent enzymes (ALuc) were synthesized according to the prior patent of the present inventors. The information about was not yet clear. Therefore, we first elucidated the genetic correlation between ALuc and other luminescent enzymes from a genetic phylogenetic perspective.
- Metridia longa is a typical marine organism-derived luminescent enzyme using coelenterazine as a substrate using CLUSTALW2.1, a multiple alignment analysis program for amino acid sequences provided by the National Center for Biological Information (NCBI).
- NCBI National Center for Biological Information
- the sequences of luciferase (MLuc), Metridia pacifica luciferase 2 (MpLuc2), Gaussia ucluciferase (GLuc) and Lucia were compared with ALuc.
- MLuc Metridia pacifica luciferase 2
- Lucia were compared with ALuc.
- FIG. 1 (A) it was found that ALuc was located at a system position greatly different from the conventional arrangement. This characteristic on the genetic line strongly suggested the possibility that ALuc may exhibit a luminescent substrate specificity different from the conventional one.
- we clarified the three-dimensional structure of ALuc and came up with the idea of finding the substrate structure that matches
- Non-patent Document 35 X-ray crystal structure data (PDBID: 2hpsA, 2hq8A) of Coelenterazine-Binding Protein (CBP), the conformation of the main chain for each amino acid residue is determined.
- the coding was converted into three types of codes, ⁇ -helix type (h), ⁇ -sheet type (s), and other type (o), and the super secondary structure was described.
- HyperProtein v1.0 http://www.hyper.com/Products/HyperProtein/tabid/504/Default.aspx
- Chem3D Ultra v8.0 http: //www.cambridgesoft.com/Ensemble_for_Chemistry/ChemBio3D/
- MM Molecular Mechanics
- ALuc30 is formed by nine ⁇ -helices, and it can be seen that each helix is arranged so as to surround the luminescent substrate (the red compound at the center).
- the substrate is formed of an imidazole skeleton and three residues (ie, R-A, R-B, and R-C), and is inserted into ALuc in a manner that penetrates from R-A to the back side.
- R-A has the structure of a broken arrowhead, and it can be seen that there is a cavity around it.
- R-B is pierced upward in a manner opposite to R-A and is surrounded by hydrophobic amino acids.
- R-C is located near the entrance of the pocket of the luminescent enzyme and interacts with the C-terminal amino acid of ALuc30 (for example, the 9th helix).
- FIG. 2 The upper left of FIG. 2 is the chemical structure of native coelenterazine.
- the top row is a list of chemical structures considering the functionality of Residue A (RA).
- the bottom row enumerates the chemical structure mainly focusing on the functional group of Residue B (RB).
- a surrounding line and an arrow show the difference between each residue and functional group.
- Coelenterazine is composed of an imidazole skeleton and three residue (shown here as R-A, R-B, and R-C) linked to it. It is thought that each luciferase specificity comes out by the structure and functional group of each residue.
- Native coelenterazine has a phenol structure at the R-A and R-C positions, and a benzene structure at the R-B position.
- a relatively large functional group can be allowed in R-A. Focusing on this point, among the coelenterazine derivatives, a series of luminescent substrates were selected centering on the size of the functional group that binds to R-A (the arrow in the upper part of FIG. 2).
- residue capable of ⁇ - ⁇ stacking is an essential condition for ALuc-specific luminescent substrates.
- Example 3 Comparison of bioluminescence brightness of ALuc with coelenterazine derivatives Based on the above example, the following experiment was conducted to clarify a luminescent substrate specific to ALuc.
- COS-7 a cultured cell derived from African monkey kidney, was cultured in a 96-well plate and cultured until the culture area covered 90% of the area under the plate. At this point, the cells in the plate well were divided into two, and pcDNA3.1 (+) vectors expressing ALuc34 and RLuc8.6-535 were introduced in the lipofection method (TransIT-LT1) and cultured for about 16 hours. . After incubation, the cells are lysed and 10 ⁇ L each is dispensed into another 96-well plate, each luminescent substrate is added simultaneously using a multichannel pipette, and the relative luminescence intensity is measured using the luminescence imaging device LAS-4000 (FujiFilm). Immediately measured using.
- luminescent substrates with a residue other than the benzene ring at the RB position generally showed weak emission luminance and selectivity (eg, CTZ ip, CTZ cp, CTZ fcp, CTZ hcp compared) ).
- the RC position is preferably a phenol (hydroxybenzene) group
- the emission luminance of CTZ400A and CTZ h was compared. Compared with the strong emission in the case of having (CTZ ⁇ h), almost no emission luminance was observed in the case without (CTZ400A).
- the optimal luminescent substrate for ALuc is preferably a benzene ring skeleton in RA that allows a functional group of a certain size in the skeleton, more preferably a halogen ion as a functional group, Particularly preferred is a luminescent substrate containing iodine, fluorine and chlorine.
- R—B is preferably a benzene ring skeleton.
- R-C is preferably a benzene ring residue having a hydrophilic functional group (for example, a hydroxyl group or a thiol group), more preferably a phenol (hydroxybenzene) structure.
- the bioluminescence spectrum was examined from a cell lysate (lysate) prepared in the same manner as in Example 3A. First, 5 ⁇ L of the cell lysate was mixed with 30 ⁇ L of the luminescent substrate shown in FIG. 3B, and then immediately measured using a high-precision luminescence spectrometer (AB-1850, ATTO) capable of simultaneous measurement of all wavelengths.
- a high-precision luminescence spectrometer AB-1850, ATTO
- RLuc8.6-535 emits light having a wavelength longer by about 30 nm than ALuc34 even for the same luminescent substrate.
- CTZ i had a slightly longer wavelength shift than CTZ f (Table 2).
- Coelenterazine is composed of an imidazole skeleton and three residences connected to it (indicated here as R-A, R-B, and R-C). It is thought that each luciferase specificity comes out by the structure and functional group of each residue.
- Native coelenterazine has a phenol structure at the R-A and R-C positions and a benzene structure at the R-B position (FIG. 4A).
- FIG. 4A the internal structure of ALuc30 was revealed (FIG. 4B).
- FIG. 4B the internal structure of ALuc30 was revealed (FIG. 4B).
- a certain cavity is seen, which explains from the three-dimensional structure that even a relatively large functional group attached to R-A is acceptable.
- iodine is generally an infrequent element in biological proteins and is also an electron donor.
- the surprising luminescent enzyme selectivity exhibited by CTZ i is not only due to the “size effect” but also as a result of favorable interactions with amino acids in ALuc as electron donors. Strongly suggest.
- Example 5 Luminescence detection by dual assay in the presence of ALuc and Cypridina Luciferase (CLuc).
- the ability to perform a dual assay in the presence of two or more luminescent enzymes is advantageous for simultaneous measurement of a large number of biomarkers. Focusing on this point, luminescence measurement was performed under conditions where two luminescent enzymes (ALuc and CLuc) coexist (FIG. 5).
- ALuc and CLuc two luminescent enzymes
- COS-7 cells were cultured in a 96-well plate, and a plasmid expressing ALuc or CLuc was introduced into each cell compartment by Lipofection. Under this condition, CLuc is secreted, but ALuc remains in the cell due to the endoplasmic reticulum localization signal (KDEL) attached to the end. After 16 hours, 10 ⁇ L of CLuc culture solution and 2 ⁇ L of ALuc rice set were mixed. Furthermore, the emission luminance was compared under the condition of 0.1% SDS. At this time, the brightness was compared by adding each specific luminescent substrate.
- KDEL endoplasmic reticulum localization signal
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Abstract
Description
本出願は、2013年10月18日に出願された、日本国特許出願第2013-217560号明細書(その開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
人工生物発光酵素の一例としてALuc30を構成する各アミノ酸の3次元的位置情報(即ち、立体構造)を解析した。解析により得られたモデルを図1Bに示す。
(i)配列番号1に示されたアミノ酸配列、又は、
(ii)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水素原子、水酸基又はチオール基を示す。]
項2、前記一般式(1)で表される化合物が、セレンテラジンn(CTZ n)、セレンテラジンi(CTZ i)、セレンテラジンf(CTZ f)、セレンテラジンh(CTZ h)またはセレンテラジン400A(CTZ 400A)である、項1に記載の使用。
上記接触に基づく発光輝度を測定する工程を含む、生物発光アッセイ方法:
化合物(A):下記の一般式(1)で表される化合物;
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水酸基又はチオール基を示す。]
ポリペプチド(B):(i)又は(ii)に記載のアミノ酸配列を含み、かつカイアシ類ルシフェラーゼ活性を有するポリペプチド
(i)配列番号1に示されたアミノ酸配列、又は、
(ii)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水素原子、水酸基又はチオール基を示す。]
項5、項3に記載の方法において、他の発光酵素と組み合わせて2つ以上の発光色を測定することを特徴とするマルチカラーイメージング法。
上記接触に基づく発光エネルギーが他の蛍光蛋白質に遷移する工程、
上記発光エネルギーが遷移した蛍光蛋白質の発光輝度を測定する工程を含む、生物発光共鳴エネルギー移動(BRET)法:
化合物(A):下記の一般式(1)で表される化合物;
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水素原子、水酸基又はチオール基を示す。]
ポリペプチド(B):(i)又は(ii)に記載のアミノ酸配列を含み、かつカイアシ類ルシフェラーゼ活性を有するポリペプチド
(i)配列番号1に示されたアミノ酸配列、又は、
(ii)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
1.人工発光酵素(ALuc)群に最適な発光基質について
(発光基質)
本願発明の発光基質は、下記の一般式(1)で表される化合物である。
本発明において、上記発光基質は、下記の人工発光酵素(本明細書において、「人工ルシフェラーゼ」もしくは「ALuc」(artificial luciferase)と言う場合がある。)の発光基質として用いる。
(i)配列番号4~23のいずれかに示されたアミノ酸配列、
(ii)配列番号4~23のいずれかに示されたアミノ酸配列において、1もしくは数個のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列、
なお、ここで数個とは、1~20個、好ましくは1~10個、より好ましくは1~5個を表す。
(iii)配列番号4~23のいずれかに示されたアミノ酸配列と90%以上の同一性を有するアミノ酸配列、
ここで、例えば、95%以上、96%以上、97%以上、98%以上、99%以上、99.5%以上の同一性を有するアミノ酸配列であればさらに好ましい。
(iv)配列番号2に示されたアミノ酸配列、又は、
(v)配列番号2に示されたアミノ酸配列のうち、1-29位、又は214-218位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
(iv)配列番号1に示されたアミノ酸配列、又は、
(v)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
(vi)配列番号3に示されたアミノ酸配列、又は、
(vii)配列番号3に示されたアミノ酸配列のうち、1-29位、又は211-215位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
最適発光基質によるALucの酵素活性は、例えば、以下の方法で検証することができる。
本発明のALucをレポーター蛋白質として「basic法」に適用する場合、ALucを単純に標的蛋白質に繋げた融合蛋白質を作ればよい。この際に非制御型プロモーターで発現する点が他のレポーター分析法とは異なる。
生物発光酵素をレポーター蛋白質として「inducible法」に適用することは、組換えDNA技術によって組換え蛋白質が作製される際の遺伝子発現の時期及び発現量の解析のために従来から用いられており、特に外部刺激に応答した発現時期及び発現量変化を示す指標として広く用いられている。「inducible法」に含まれる分析システムとしては、レポータージーンアッセイ(reporter gene assay)、Yeast Two-hybrid assay、Mammalian Two-hybrid assay、protein splicing assay(PSA)、protein complementation assay(PCA)、circular permutation assay、bioluminescence resonance energy transfer assay(BRET)等があるが、これらの分析システムに必須のレポーター遺伝子としてALucを用いることで、アッセイの計測性能を飛躍的に向上できる。
レポータージーンアッセイ法は、外部刺激による転写因子の活性化及び遺伝子の発現調節の解析手段として繁用されているが、典型的には核内受容体を介したシグナル伝達を攪乱する内分泌攪乱物質(環境ホルモン)の検出に用いられている。核内受容体を介したシグナル伝達に関連した標的遺伝子(例えば、ホルモン応答性遺伝子)の発現は、リガンドと受容体の複合体が当該遺伝子の転写調節するシス領域(ホルモン応答配列;hormone response element)に結合することで引き起こされる。この各種ホルモン応答性遺伝子のシス領域の下流にルシフェラーゼなどのレポーター遺伝子を組み込んだプラスミドを細胞内に導入し、リガンドとなり得るホルモン分子又は内分泌攪乱物質量を生物発光量などで検出するアッセイ法である。
ツー・ハイブリッド法(Two-hybrid法)は蛋白質間の相互作用を調べる手法の1つであり、1989年に酵母(Saccharomyces cerevisiae)を用いたyeast two-hybrid(Y2H)システムがまず構築された。転写活性化因子であるGAL4蛋白質のDNA結合ドメイン(GAL4 DBD)と転写活性化ドメインが分離可能であることを利用して、GAL4 DBDと任意の蛋白質A(bait)を融合蛋白質として発現させ、同時に細胞内で発現させた転写活性化ドメイン(TA)と融合蛋白質とした蛋白質B(prey)と相互作用をするかどうかを判定できる。前記蛋白質AとBが結合する場合にはDBDとTAが近接してDNA結合ドメイン(DBD)が、「UASG」塩基配列に結合するのでその下流に連結したレポーター遺伝子発現を促すことになる。レポーター遺伝子がルシフェラーゼであれば、その特異的な基質存在下で生物発光をモニターすれば、A,B両蛋白質の親和性が測定でき、蛋白質A(bait)と相互作用をする蛋白質、ペプチドのスクリーニングができる。その際の蛋白質B(prey)は発現ライブラリーによって提供させることもできる。
生物発光酵素を“activatable”法のレポーター蛋白質として搭載する分析システムも、従来から本発明者らが「生物酵素発光プローブ」技術として研究開発し、発展させてきた。以下、典型的な“activatable”法の例として、ALucの「生物酵素発光プローブ」への適用例、及びそれを用いた「細胞内イメージング法」について述べるが、それに先立ち、まず、以前開発した「発光性融合蛋白質(発光カプセル)」について述べる。その他、ALucは、“activatable”法に含まれるprotein complentation assay(PCA),protein splicing assay (PSA)におけるレポーター蛋白質として好適に適用できる。このような方法を、本明細書では、発光カプセル法もしくは発光カプセルアッセイ法という場合がある。
ALucのC末端側に膜局在シグナル(MLS)を結合することで、ALuc本体を細胞膜に局在させることができるが、このように細胞膜に局在する分子設計を行うことによって、基質と酸素の供給が円滑になるため、極めて高輝度で安定的な生物発光可視化が可能になる。その際、ALuc本体とシグナルペプチドをコードする核酸の間に任意のポリペプチドや蛋白質の遺伝子をカーゴ(貨物という)として挿入することが可能である。こうすることによってカーゴとなる蛋白質を細胞膜表面に効率的に運ぶことができ、しかも運ばれた場所が光るように仕掛けたことになる。典型的な例としては、各蛋白質の繋ぎ目に、細胞死に応答するDEVD配列やIETD配列を入れ込んだ場合では、細胞死の際caspase-3やcaspase-8の活性を信号として能動的に応答し、可視化するシステムとして働く。本発明者はこの構造の発光性融合蛋白質を「発光カプセル」と名付けた。
(a)ALucのC末端側と膜局在シグナル(MLS)との間に、蛍光蛋白質又はルシフェラーゼが挿入された発光性融合蛋白質、(なお、ルシフェラーゼとしては他のALucであってもよい。
(b)ALucのC末端側と膜局在シグナル(MLS)との間に、細胞膜の形態を変化させるポリペプチド又は当該ポリペプチドが認識する20アミノ酸以下の、好ましくは10アミノ酸以下のポリペプチドが挿入された発光性融合蛋白質。ここで、特に、細胞膜の形態を変化させるポリペプチドとして、細胞死を誘発するポリペプチドが好ましく、Caspase及びその認識配列である「DEVD」又は「IETD」を含む20アミノ酸以下のポリペプチドが特に好ましい。
また、ALucを、本発明者らが既に特許出願している発明に係る一分子型発光プローブ(非特許文献4,6,9,10、特許文献1-4)、又は二分子型発光プローブ(非特許文献7,8)に組み込むことによって、リガンドの有無、リガンドの活性強度を高輝度で観測できる。当該プローブの構成要素として、[1]2つに分割された当該発光酵素(NとC末側断片)の近傍に、[2]標的リガンドに応答するリガンド結合蛋白質と、[3]リガンド結合蛋白質にリガンドが結合したことを認識する認識蛋白質を繋げた形態で高性能の発光プローブを構成できる。この発光プローブ中、前記リガンド結合蛋白質にリガンドが結合したことを前記認識蛋白質が認識した場合に、2つに分割された前記酵素断片が相補して酵素活性を変化させることができる。この時、当該分割酵素の高輝度と安定性のため、検出限界の向上と信頼性の高い計測が可能となる。
また、当該ALucをコードする遺伝子を用いることで、様々な細胞株に安定的に導入できる。一例として、胚内未分化細胞、ES細胞や新型万能細胞(iPS)に当該ALucを安定的に導入できる。前記細胞そのものは、光らないため内部で起こる分子現象、組織特異性を探索するのは大変困難であった。この困難を克服するために、まず、体細胞に当該ALucを含む分子プローブを導入してから胚を作成し、様々な臓器組織に分化させる。すると、臓器ごとに起こる特異的な分子現象を高感度に計測できる。
1.バイオアッセイ用反応溶液
(1-1)ライシスバッファー(細胞溶解液)とアッセイバッファー(反応液)
従来、バイオアッセイはライシスバッファー(細胞溶解液)とアッセイバッファー(反応液)に分けてアッセイを行ってきた。細胞を速やかに溶解するためには、高い溶解力、発光酵素への低い阻害性が必須であると考えられ、一方、バイオアッセイ反応の際には安定したアッセイ条件及びバックグラウンドを下げるために、自己発光を誘発する成分の除去や検討が必須であると考えられていたことによる。
(a)界面活性剤:polyoxyethylene octylphenyl ether(TritonX-100;TX100),Nonidet P-40(NP40),polyoxyethylene sorbitan monolaurate(Tween20;TW20),polyoxyethylene sorbitan monooleate(TW80),polyoxyethylene cetyl ether(Brij58),sodium dodecyl sulfate(SDS)等。
親水度の度合いとしては、TW20>Brij58>TW80>TX100>NP40、界面活性度の度合いとしてはNP40>TX100>Brij58>TW20>TW80の順であるとされている。(b)塩類:NaCl,KCl,(NH4)2SO4など
(c)SH試薬:メルカプトエタノール、DTT等
(d)ポリオール:グリセロール、グルコース、スクロース等
(e)グリコール類:ポリエチレングリコール(PEG)、ポリプロピレングリコール(PPG)(f)キレート試薬:EGTA、EDTA等
(g)Protease Inhibitor:アプロチニン(分子量6.5kD)、ロイペプチン(分子量:427)、ペプスタチンA(pepstatin,分子量:686)、phenylmetylsulfonyl Fluoride(PMSF、分子量:174)、アンチパイン(Antipain,分子量:605)、キモスタチン(chymostatin,分子量:608)、pefabloc SC(AEBSF,240 Da),DFP(184 Da),p-APMSF(216 Da),STI(20,100 Da),Leupeptin(460 Da),N-Tosyl-L-phenylalaninechloromethylketone,3,4-dichloroisocoumarin(215 Da),EDTA-Na2(372 Da),EGTA(380 Da),1,10-phenanthroline(198 Da),phosphoramidon(580 Da),Dithiobis(2-amino-4-methylpentane),E-64(357 Da),cystatin,bestatin,Epibestatin hydrochloride,aprotinin,minocycline,ALLN(384 Da)等
(h)バッファー剤:p-Toluenesulphonic acid,tartaric acid,citric acid,phthalate,glycine,trans-aconitic acid,formic aicd,3,3-dimethylglutaric acid,phenylacetic acid,sodium acetate,succinic acid,sodium cacodylate,sodium hydrogen maleate,maleic acid,sodium phosphate,KH2PO4,imidazole,2,4,6-trimethylpyridine,Triethanolamine hydrochloride,sodium 5,5-diethylbarbiturate,N-ethylmorpholine,sodium pyrophosphate,Tris(hydroxymethyl)aminomethane,bicine,2-amino-2-methylpropane-1,3-diol,diethanolamine,potassium p-phenolsulphonate,boric acid,sodium borate,ammonia,グリシン(glycine),Na2CO3/NaHCO3,sodium borate,またはこれらの組み合わせ
(i)その他:Sodium molybdate(受容体の安定化)、ジチオトレイトール(dithiothreitol,DTT)(還元剤)。
-溶液1:1.4% NaHCO3 溶液
-溶液2:NaCl 80.0g,KCl 4.0g,MgSO4・7H2O 2.0 g,Na2HPO4・2H2O 0.6g,glucose 10.0g,KH2PO4 0.6gを水800mlに溶かした溶液。
-溶液3:CaCl2 1.4 gを水100mlに溶解した溶液。
-溶液4:Phenol red 0.4gを秤量し,少量の水でペースト状にし,ついで水を加えて150mlとする。
トリスバッファーそのものは従来から広く使われているバッファー成分(ここでいうトリスはトリスヒドロキシメチルアミノメタンの略称であり、一般的な組成はトリス塩10mMにHClでpHを調整したもので、添加剤としてEDTA 1mMを入れる場合もある)であり、生体適合性が高い理由で、さまざまなバイオ研究で用いられている。しかし、トリスバッファーが生物発光反応に与える影響に関する研究は今まで十分されてなかった。
上記基本的なバッファー成分のHBSSバッファー及びTris-バッファーを組み合わせて用いるが、その際両者を容量(v/v)%で20~50:50~20、好ましくは40~60:60~40、最も好ましくは、60:40で配合する。
リガンド活性の測定は、通常の生物発光アッセイに準じて実施すればよく、特に制限なく従来のプロトコールが適用できる。
これらのスクリーニング方法の対象となる被検物質には、例えば、有機または無機の化合物(特に低分子量の化合物)、生物活性を持つ蛋白質、ペプチド等が含まれる。これらの物質は、機能や構造が既知のものであっても未知のものであってもよい。また、「コンビナトリアルケミカルライブラリー」は、目的物質を効率的に特定するための被検物質群として有効な手段である。コンビナトリアルケミカルライブラリーの調製およびスクリーニングは、当該技術分野において周知である(例えば、米国特許第6,004,617号;5,985,365号を参照)。さらには、市販のライブラリー(例えば、米国ComGenex社製、ロシアAsinex社製、米国Tripos,Inc.社製、ロシアChemStar,Ltd社製、米国3D Pharmaceuticals社製、Martek Biosciences社製などのライブラリー)を使用することもできる。また、コンビナトリアルケミカルライブラリーを、本プローブを発現する細胞の集団に適用することによって、いわゆる「ハイスループットスクリーニング」を実施することもできる。
本発明はまた、ALuc特異的な発光基質を含むバイオアッセイ用キットをも提供する。本発明のキットは、バイオアッセイを実施するための各種成分を必要に応じて含めることができる。このような成分としては、発光酵素、発光酵素をコードする遺伝子を含むベクター、発光酵素を発現する細胞、本発明のALuc特異的な発光基質、各種器具(96穴プレート若しくはチューブなど。)、対照試料などが例示されるが、これに限定されない。また、本発明のバイオアッセイ法を実施するための方法を記載した手順書を含めてもよい。
本発明におけるその他の用語や概念は、発明の実施形態の説明や実施例において詳しく規定する。なお、用語は基本的にはIUPAC-IUB Commission on Biochemical Nomenclatureによるものであり、あるいは当該分野において慣用的に使用される用語の意味に基づくものである。また発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。例えば、遺伝子工学および分子生物学的技術はJ.Sambrook,E.F.Fritsch & T.Maniatis,"Molecular Cloning: A Laboratory Manual (2nd edition)",Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York(1989);D.M.Glover et al.ed.,"DNA Cloning",2nd ed.,Vol.1 to 4,(The Practical Approach Series),IRL Press,Oxford University Press(1995);Ausubel,F.M.et al.,Current Protocols in Molecular Biology,John Wiley & Sons,New York,N.Y,1995;日本生化学会編、「続生化学実験講座1、遺伝子研究法II」、東京化学同人(1986);日本生化学会編、「新生化学実験講座2、核酸III(組換えDNA技術)」、東京化学同人(1992);R.Wu ed.,"Methods in Enzymology",Vol.68(Recombinant DNA),Academic Press,New York(1980);R.Wu et al.ed.,"Methods in Enzymology",Vol.100(Recombinant DNA,Part B) & 101(Recombinant DNA,Part C),Academic Press,New York(1983);R.Wu et al.ed.,"Methods in Enzymology",Vol.153(Recombinant DNA,Part D),154(Recombinant DNA,Part E) & 155(Recombinant DNA,Part F),Academic Press,New York(1987)などに記載の方法あるいはそこで引用された文献記載の方法またはそれらと実質的に同様な方法や改変法により行うことができる。また、本発明で使用する各種蛋白質やペプチド、あるいはそれらをコードするDNAについては、既存のデータベース(URL:http://www.ncbi.nlm.nih.gov/等)から入手することができる。
本発明者らの先願特許により一連の人工生物発光酵素(ALuc)が合成されたが、その特異的な発光基質に関する情報は未だに明らかになっていなかった。そこで、まず、遺伝系統学的な観点からALucと他の発光酵素間の遺伝的相関性を明らかにした。
前記実施例により明らかになったALucの立体構造に基づき、この立体構造に合致するセレンテラジン誘導体の化学構造を考察した(図2)。図2の左上はネイティブセレンテラジンの化学構造である。上段は、レジデューA(R-A)の官能基を考慮した化学構造の羅列である。一方、下段は、主にレジデューB(R-B)の官能基に焦点を合わせて化学構造を羅列した。囲み線及び矢印は、各レジデューや官能基の違いを示す。
前記実施例を根拠に、ALucに特異的な発光基質を明らかにするために、以下の実験を行った。
一般的に生物発光はルシフェリンとルシフェラーゼ間の酸化触媒反応により産生される。多様なルシフェラーゼの種類が知られているが、ルシフェリンにおいては化学構造の多様性がない。海洋生物由来の発光酵素に用いられるルシフェリンも同様に数少なく、セレンテラジン、ウミホタルルシフェリンが代表的である。特にセレンテラジンは多数の海洋生物由来の発光酵素に用いられるため、以前から約50種類以上の誘導体が合成されてきた(非特許文献30、31)。
Claims (7)
- 前記一般式(1)で表される化合物が、セレンテラジンn(CTZ n)、セレンテラジンi(CTZ i)、セレンテラジンf(CTZ f)、セレンテラジンh(CTZ h)またはセレンテラジン400A(CTZ 400A)である、請求項1に記載の使用。
- 下記の化合物(A)と、下記のポリペプチド(B)とを接触させる工程、及び
上記接触に基づく発光輝度を測定する工程を含む、生物発光アッセイ方法:
化合物(A):下記の一般式(1)で表される化合物;
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水素原子、水酸基又はチオール基を示す。]
ポリペプチド(B):(i)又は(ii)に記載のアミノ酸配列を含み、かつカイアシ類ルシフェラーゼ活性を有するポリペプチド
(i)配列番号1に示されたアミノ酸配列、又は、
(ii)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。 - 請求項3に記載の方法において、他の発光酵素と組み合わせて2つ以上の発光色を測定することを特徴とするマルチカラーイメージング法。
- レポータージーンアッセイ法、ツーハイブリットアッセイ法、発光カプセルアッセイ法又は一分子型生物発光プローブ測定法である、請求項3に記載の方法。
- 下記の化合物(A)と、下記のポリペプチド(B)とを接触させる工程、及び
上記接触に基づく発光エネルギーが他の蛍光蛋白質に遷移する工程、
上記発光エネルギーが遷移した蛍光蛋白質の発光輝度を測定する工程を含む、生物発光共鳴エネルギー移動(BRET)法:
化合物(A):下記の一般式(1)で表される化合物;
Xaは、ハロゲン原子により置換されていてもよいフェニル基またはナフチル基を示す。
Xbは、フェニル基を示す。
Mcは、水素原子、水酸基又はチオール基を示す。]
ポリペプチド(B):(i)又は(ii)に記載のアミノ酸配列を含み、かつカイアシ類ルシフェラーゼ活性を有するポリペプチド
(i)配列番号1に示されたアミノ酸配列、又は、
(ii)配列番号1に示されたアミノ酸配列のうち、1-31位、又は217-221位の少なくともいずれかの領域において、1個以上のアミノ酸が欠損しているアミノ酸配列。
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US11136361B2 (en) | 2016-06-03 | 2021-10-05 | Kyushu University, National University Corporation | Fusion protein for improving protein expression from target mRNA |
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JP6153140B2 (ja) | 2017-06-28 |
JPWO2015056762A1 (ja) | 2017-03-09 |
US20160281129A1 (en) | 2016-09-29 |
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