WO2014020933A1 - Nouvelle substance fluorescente - Google Patents

Nouvelle substance fluorescente Download PDF

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WO2014020933A1
WO2014020933A1 PCT/JP2013/057206 JP2013057206W WO2014020933A1 WO 2014020933 A1 WO2014020933 A1 WO 2014020933A1 JP 2013057206 W JP2013057206 W JP 2013057206W WO 2014020933 A1 WO2014020933 A1 WO 2014020933A1
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protein
fluorescent
modified
amino acid
fluorescence
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Japanese (ja)
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精二 宮谷
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アルバイオ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag

Definitions

  • the present invention relates to a novel fluorescent material obtained by modifying a gene originally possessed by organisms including humans.
  • fluorescent genes have been isolated and identified from various lower organisms.
  • lower organisms whose fluorescent genes have been isolated and identified include, for example, Owan jellyfish (Non-patent Document 1), flower worms (Patent Document 1), tropical Renilla (Patent Document 2), thrip coral (Patent Document 3), Examples include Okawari sea anemone (Patent Document 4), non-Owan jellyfish hydroworm (Patent Document 5), co-poda (Patent Document 6), Suboikuikumeishi (Patent Document 7) and the like.
  • fluorescent genes label target genes, compounds, cells, organisms, etc. as tracer genes and reporter genes that can be easily detected in vivo or in vitro as biochemistry, molecular biology, cell It is frequently used in fields such as biology and pharmaceutical research.
  • Specific applications used in known fluorescent genes include, for example, cell identification, selection and purification applications described in Patent Document 8, gene expression analysis described in Patent Document 9, and target substances (cells) Labeling of proteins, compounds, etc.), traces of target substance behavior (pharmacokinetics, etc.), use as reporter or tracer protein for in vivo imaging, use as molecular weight markers on protein gels and Western blots, calibration of FACS equipment Can also be used as a marker for microinjection into cells and tissues, and can be used for intracellular traces of target genes described in Patent Document 10, transplanted cells described in Patent Document 11 As a biosensor that applies fluorescence resonance energy transfer (FRET) described in Patent Document 12
  • FRET fluorescence resonance energy transfer
  • biopharmaceuticals such as cell therapy using stem cells such as ES cells, iPS cells, and somatic stem cells, nucleic acid drugs such as siRNA, and protein drugs such as antibodies have made remarkable progress.
  • stem cells such as ES cells, iPS cells, and somatic stem cells
  • nucleic acid drugs such as siRNA
  • protein drugs such as antibodies
  • the cells, tissues, nucleic acids, and proteins used in these biopharmaceuticals are engrafted and functioning in the recipient's body, and the in vivo kinetics such as transport and metabolic pathways.
  • a fluorescent gene as a highly safe reporter and tracer that can be confirmed in vivo such as how it is and whether it has side effects.
  • non-biopharmaceuticals that is, low-molecular-weight pharmaceuticals
  • fluorescent genes are highly functional reporters and tracers.
  • stem cells such as ES cells and iPS cells
  • these stem cells are usually not directly used for treatment, but are differentiated into target cells in vitro or ex vivo. Differentiated cells are used for treatment.
  • the problem of canceration risk caused by the mixture of cells that cannot be differentiated in this process that is, undifferentiated cells, hinders practical application.
  • it is essential to prevent contamination of undifferentiated stem cells in target cells used for treatment.
  • it is also important for the practical application of cell therapy to remove unnecessary cells other than the target cells and obtain high-purity target cells. Needless to say.
  • Non-Patent Document 22 As a useful means for purifying such target cells, currently, cell sorting methods using flow cytometry such as FACS, dielectric cytometry, fully automatic single cell isolation analyzer and the like are used (Patent Document 22). 23, 24, Non-Patent Document 2).
  • a general cell sorting method by FACS is a labeled binding molecule in which a binding molecule such as an antibody against a cell surface marker is labeled with a fluorescent substance such as a low molecular weight organic compound (for example, FITC) or a fluorescent gene product (for example, GFP derived from Aequorea jellyfish). Is a method in which only cells that emit fluorescence are selected and purified by associating them with cells (Non-patent Documents 3 and 4).
  • Non-Patent Documents 5 and 6 describe the practical application of cell therapy using stem cells that are not labeled outside the cell, but express the fluorescent substance inside the cell and select only the target cells by FACS. Studies have been described.
  • the fluorescent protein which is the gene product of the fluorescent gene, is recognized as a foreign substance by a biological immune reaction. Therefore, it is considered that an adverse event such as a rejection reaction is caused (see, for example, Patent Document 25, Paragraph [0004], Non-Patent Documents 7, 8, 9, and 10). Furthermore, since it has been reported that an antibody against a fluorescent gene is produced (for example, see Non-Patent Document 11), the fluorescent gene described in the above document has a risk of anaphylactic shock, and When used for the second time and thereafter, the fluorescence intensity is significantly reduced and it cannot be used repeatedly. Therefore, the fluorescent gene described in the above document is not preferable as a substance to be administered in vivo.
  • the fluorescent genes described in the above documents were administered to subjects because not all substances, including those considered to have low toxicity, are derived from subjects who undergo cell therapy (including human patients). There is a problem of causing an adverse event.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a nucleic acid-derived fluorescent substance that is highly safe even when administered to a living body.
  • the present inventor has found through experiments that bacterial-derived DHODH® (Dihydroorotate® dehydrogenase) emits stable detectable fluorescence in cells derived from multicellular organisms.
  • DHODH® Dihydroorotate® dehydrogenase
  • fluorescence could not be detected stably even when introduced into cells derived from multicellular organisms. Therefore, in order to obtain a highly safe fluorescent protein even when administered to a living body, further breakthrough is necessary.
  • the present inventor decided to further modify the amino acid sequence of the human homolog.
  • the result was that when a human homologue cofactor or substrate with a modified interaction region was introduced into the cell, the cell fluoresced. This fluorescence is considered to be generated as a result of the stable formation of a complex with the cofactor or substrate by the modified human homolog.
  • the present inventors have found that similar results can be obtained for other proteins capable of interacting with a cofactor or substrate by modifying the interaction region with the cofactor or substrate.
  • the inventors have found that similar results can be obtained with proteins derived from other animals.
  • the present invention includes the following as one aspect thereof. (1) (a) a modified protein or a fusion protein thereof, wherein the amino acid in the interaction region for the cofactor or substrate is modified, (b) a nucleic acid encoding the modified protein of the above (a) or a fusion protein thereof, (c) a fluorescent protein complex that is a complex of the modified protein of (a) or a fusion protein thereof and a cofactor or substrate, and (d) the modified protein of (a) or a fusion protein thereof, A cell having at least one substance selected from the group consisting of the nucleic acid of (b) above, the fluorescent protein complex of (c) above, A fluorescence test material comprising at least one biological substance selected from the group consisting of:
  • the modified protein (a) can emit stable detectable fluorescence in the presence of a cofactor or substrate. Therefore, the modified protein (a) can be used as a material for fluorescence examination as described above (for example, for cell identification) in animal or plant cells or in vivo.
  • the nucleic acid (b) can express the modified protein (a) in cells of animals and plants. Therefore, the nucleic acid of (b) can be used as a material for fluorescence examination as described above in animal or plant cells or in vivo for the same reason as the modified protein of (a).
  • the fluorescent protein complex (c) can emit fluorescence in the cells of animals and plants.
  • the fluorescent protein complex of the above (c) can be used as a material for fluorescence examination as described above in animal or plant cells or in vivo.
  • the cell (d) has at least one of the substances (a) to (c). Therefore, the cells of (d) above can be used as a material for fluorescence examination as described above (for example, confirmation of cell pharmacokinetics) in the living body of animals and plants.
  • a method for detecting fluorescence comprising: (a) a modified protein or a fusion protein thereof, wherein the amino acid in the interaction region for the cofactor or substrate is modified, (b) a nucleic acid encoding the modified protein of the above (a) or a fusion protein thereof, (c) a fluorescent protein complex that is a complex of the modified protein of (a) or a fusion protein thereof and a cofactor or substrate, and (d) the modified protein of (a) or a fusion protein thereof, A cell having at least one substance selected from the group consisting of the nucleic acid of (b) and the fluorescent protein complex of (c), A method comprising the step of applying excitation light to at least one biological substance selected from the group consisting of:
  • the modified protein (a) can form a stable complex with a cofactor or a substrate in animal and plant cells.
  • the complex can emit fluorescence by applying excitation light. Therefore, the method (a) for detecting the fluorescence of soot can be applied to a technique (for example, cell identification) using the fluorescence test as described above.
  • the nucleic acid (b) can express the modified protein (a).
  • the fluorescent protein complex of (c) can emit fluorescence when irradiated with excitation light.
  • the cell (d) has at least one of the substances (a) to (c). Therefore, even when at least one of the substances (b) to (d) is used, a technique using the fluorescence test as described above for the same reason as when the modified protein (a) is used. It can be applied to.
  • a method for detecting fluorescence in a living body (a) a modified protein in which an amino acid in an interaction region for a cofactor or a substrate is modified, (b) a nucleic acid encoding the modified protein of (a) above, (c) a fluorescent protein complex that is a complex of the modified protein of (a) and a cofactor or substrate, and (d) the modified protein of (a), the nucleic acid of (b), and A cell having at least one substance selected from the group consisting of the fluorescent protein complex of (d) above, Introducing at least one biological substance selected from the group consisting of: Including the method.
  • an animal or plant-derived material can be used as the biological substance.
  • the biological material derived from animals and plants is administered into animals and plants, adverse events are unlikely to occur. From the viewpoint of further suppressing adverse events, it is preferable that the animal or plant from which the biological substance is derived and the organism to be administered are of the same species.
  • the modified protein described in (4) above can emit fluorescence that can be stably detected in the presence of a cofactor or a substrate. For this reason, the modified protein described in (4) above can be used as a material for fluorescent examination as described above (for example, for cell identification) in cells or in vivo.
  • the nucleic acid described in (4) above can express the modified protein described in (4) above in a cell. Therefore, the nucleic acid described in (4) above can be used as a material for fluorescence examination as described above in cells or in vivo.
  • a modified protein comprising the nucleic acid translation product according to (4) above.
  • a nucleic acid that encodes a protein that forms a fluorescent protein complex with a cofactor or substrate, or a fluorescent protein (9) A method of fluorescentizing a protein, comprising a step of modifying an amino acid in an interaction region of a protein with a cofactor or a substrate. (10) A method for producing a modified protein that forms a fluorescent protein complex together with a cofactor or substrate, the method comprising a step of modifying an amino acid in an interaction region of the protein with respect to the cofactor or substrate .
  • nucleic acid-derived fluorescent substance that is highly safe even when administered to a living body can be obtained.
  • the base sequence and amino acid sequence of isolated Bacillus DHODH are shown.
  • the upper row shows the base sequence, and the lower row shows the deduced amino acid sequence encoded by the base sequence.
  • An asterisk (*) indicates a start codon.
  • Amino acid sequence comparison of isolated bDHODH, Bacillus methanolicus MGA3, and Bacillus cereus G9241 is shown. When these three amino acid sequences were compared, isolated bDHODH showed a high homology of 81% with Bacillus derived DHODH.
  • the expression of isolated bDHODH (bDHODH) in DH5 ⁇ cells of E. coli cell line and HEK293 cells of human cell line is shown.
  • the upper panel shows expression with DH5 ⁇
  • the lower panel shows expression with HEK293.
  • a control in DH5 ⁇ only the pGEX4T-1 vector was used, and as a control in HEK293, only the pCS2 + vector was used.
  • Expression in DH5 ⁇ was photographed with a fluorescence microscope (macro zoom microscope MVX10 for research, Olympus) using a GFP filter unit, and expression in HEK293 was fluorescence microscope (inverted microscope for research ECLIPSE TE2000-U standard phase difference set, Nikon Corporation) using a GFP filter unit.
  • the base sequence Genbank Accession No. NP_001352
  • amino acid sequence Genbank Accession No.
  • FIG. 4B is a continuation of FIG. 4A.
  • the upper panel shows expression with DH5 ⁇
  • the lower panel shows expression with HEK293.
  • As a control in DH5 ⁇ only the pGEX4T-1 vector was used, and as a control in HEK293, only the pCS2 + vector was used.
  • DH5 ⁇ was photographed with a fluorescence microscope (macro zoom microscope MVX10 for research, Olympus) using a GFP filter unit, and expression in HEK293 was fluorescence microscope (inverted microscope for research ECLIPSE TE2000-U standard phase difference set, Nikon Corporation) using a GFP filter unit.
  • a homology comparison between the amino acid sequence of wild-type hDHODH and the amino acid sequence of isolated bDHODH is shown.
  • An asterisk (*) indicates the same amino acid
  • a colon (:) indicates an amino acid that is very similar
  • a dot (.) Indicates an amino acid that is somewhat similar in nature.
  • region which interacts with FMN in wild type hDHODH by molecular simulation software is shown.
  • the center compound indicates FMN, the circle indicates the amino acid of wild-type hDHODH and its position, and the circle directly connected to FMN with an arrow indicates the amino acid of wild-type hDHODH that interacts.
  • region which interacts with DHO in wild type hDHODH by molecular simulation software is shown.
  • the center compound indicates DHO, the circle indicates the amino acid of wild-type hDHODH and its position, and the circle directly connected with DHO by an arrow indicates the amino acid of wild-type hDHODH that interacts.
  • bDHODH indicates isolated bDHODH
  • EGFP Enhanced Green Fluorescent Protein
  • ⁇ 1-74hDHODH indicates ⁇ 1-74 wild-type hDHODH
  • Table 4 One of the 2 + 5 + 6 + 8 fluorescent hDHODH in the region No./combination is shown, and five modifications-1 indicates one of the 2 + 5 + 6 + 8 + 11 fluorescent hDHODH in the interaction region No./combination described in Table 4, and five modifications -2 represents one of the 2 + 5 + 6 + 8 + 12 fluorescent hDHODH in the interaction region No./combination described in Table 4.
  • pCS2 + vector was used as a control.
  • Expression in HEK293 was photographed using a GFP filter unit with a fluorescence microscope (inverted microscope for research ECLIPSE TE2000-U standard phase difference set, manufactured by Nikon Corporation). 2 shows expression of N-terminal deletion type fluorescent hDHODH and granular expression of full length fluorescent hDHODH including mitochondrial signal in HEK293 cells.
  • WT full length wild type hDHODH, ⁇ 1-28WT: ⁇ 1-28 wild type hDHODH, ⁇ 1-41WT: ⁇ 1-41 wild type hDHODH, ⁇ 1-50WT: ⁇ 1-50 wild type hDHODH, ⁇ 1-74WT: ⁇ 1-74 wild type hDHODH, Full Fluor-D: full length fluorescent hDHODH, ⁇ 1-28 Fluor-D: ⁇ 1-28 fluorescent hDHODH, ⁇ 1-41 Fluor-D: ⁇ 1-41 fluorescent hDHODH, ⁇ 1-50 Fluor-D: ⁇ 1- 50 fluorescent hDHODH, ⁇ 1-741-Fluor-D: ⁇ 1-74 fluorescent hDHODH.
  • the vector used was pCS2 +.
  • Expression in HEK293 was photographed using a GFP filter unit with a fluorescence microscope (inverted microscope for research ECLIPSE TE2000-U standard phase difference set, manufactured by Nikon Corporation). The flow cytometry analysis result of the fluorescence hDHODH introduction cell is shown.
  • HEK293 cells were used as cells, and pCS2 + was used as a vector.
  • the flow cytometer uses a FACS Calibur (Becton, Dickinson and Company), the excitation wavelength is 488 nm with an argon laser, and the fluorescence wavelength is 530 ⁇ 15 nm with the detector FL-1.
  • A shows the flow cytometry analysis result of the mixed cell of the cell into which only the pCS2 + vector is introduced and the non-introduced cell
  • B shows the flow cytometry analysis result of the mixed cell of the wild type hDHODH-introduced cell and the non-introduced cell
  • C shows the results of flow cytometry analysis of mixed cells of non-introduced cells and cells introduced with fluorinated hDHODH modified at 4 locations of 2 + 5 + 6 + 8 in the interaction region No./combination shown in Table 4.
  • region No./ combination of description is shown.
  • the horizontal axis of the graph indicates relative fluorescence, and the vertical axis indicates cell count.
  • a.u. represents an arbitrary unit.
  • C and D it shows that the fluorescence peak of the fluorescent human DHOHD-introduced cells (right side of the peak) and the non-fluorescent peak of the non-introduced cells (left side of the peak) can be clearly distinguished.
  • Fluor-D fluorescent hDHODH
  • mycmyFluor-D myc-His-labeled fused fluorescent hDHODH in which a myc-His label is fused to the C-terminus of fluorescent hDHODH.
  • PcDNA3.1pcmyc-HiscA was used as the vector.
  • Expression in HEK293 was photographed using a GFP filter unit with a fluorescence microscope (inverted microscope for research ECLIPSE TE2000-U standard phase difference set, manufactured by Nikon Corporation).
  • the present inventor has developed a fluorescence that can be stably detected by modifying a protein that is originally possessed by higher organisms such as humans and mice and that cannot stably detect fluorescence.
  • We succeeded in creating a protein modified to emit When this protein (or a nucleic acid encoding this protein, etc.) is administered to an organism, it is considered that adverse events such as rejection are particularly unlikely to occur if the origin of the protein itself is the subject of administration.
  • adverse events such as rejection are particularly unlikely to occur if the origin of the protein itself is the subject of administration.
  • it is thought that the problem that repeated administration cannot be performed due to a decrease in fluorescence intensity due to antibody production can be prevented.
  • the conventional method aims to improve the fluorescence intensity of the fluorescent protein.
  • the present inventor succeeded in changing the non-fluorescent protein to a state in which fluorescence can be stably emitted. That is, if the conventional GFP modification technology is a technology that produces 1 to 1 ′, it can be said that the present inventors have succeeded in producing 0 to 1.
  • the “modified protein” includes a modified protein in which an amino acid in an interaction region for a cofactor or a substrate is modified.
  • the modified protein can stably form a complex with the cofactor or substrate.
  • the complex emits fluorescence that can be stably detected. Therefore, the modified protein can be suitably used as a fluorescent examination material. More specific examples of fluorescence test materials include reporter or tracer for cell identification, isolation, selection, purification, gene expression analysis, target substance labeling, or target substance behavior tracking (such as pharmacokinetics). Materials for use can be mentioned.
  • the target cells can be purified by the following procedure. First, an antibody against an antigen specifically expressed on the cell surface of the target cell is fused with the modified protein. The obtained fusion protein is brought into contact with a cell population containing target cells and non-target cells. Then, if cells that emit fluorescence are selected using a flow cytometer, the target cells can be purified.
  • the target cell can be identified by the following procedure. First, a vector encoding both a target gene to be introduced into a cell and a nucleic acid encoding the modified protein is prepared. Next, if necessary, the cell population and the vector are mixed and cultured in the presence of a transformation reagent. When excitation light is applied to the cultured cell population, only cells in which the vector has been introduced / maintained fluoresce, so that the cells in which the target gene has been introduced / maintained can be identified using this fluorescence as an index. Thereafter, the cells can be purified using a flow cytometer.
  • the modified protein may be, for example, a protein derived from animals or plants. This modified protein is preferably derived from an animal from the viewpoint of suppressing adverse events after in vivo administration of the animal.
  • the modification may be made by introducing a mutation into a gene encoding a wild type protein.
  • the modification may be performed by introducing a mutation into a gene encoding a protein that cannot form a stable complex with a cofactor or a substrate.
  • the protein before modification may contain mutations and deletions.
  • the modified protein also includes an interaction region in a pre-modification protein that interacts with a cofactor, an interaction region in a pre-modification protein that interacts with a substrate, and an interaction in a pre-modification protein that interacts with both the cofactor and the substrate.
  • the modified protein is derived from a protein having an interaction region for a cofactor or a substrate, and the amino acid sequence in the interaction region of the modified protein is different from the amino acid sequence of the interaction region of the wild-type protein. It may be modified to have an amino acid sequence.
  • the modified protein may be a protein that forms a fluorescent protein complex with a cofactor or substrate.
  • a complex that is a complex of a protein and a cofactor or a substrate and that can stably emit fluorescence in a cell is referred to as a fluorescent protein complex.
  • the fluorescent protein complex may contain other components other than the protein, cofactor, and substrate.
  • the cofactor or substrate contained in the fluorescent protein complex may be capable of emitting fluorescence alone, or may not be stable alone but complexed. It may have the ability to emit fluorescence for the first time.
  • An example of the former is FMN.
  • An example of the latter is BV.
  • the modification may be one or more modifications of amino acids in the interaction region.
  • “one or more” is preferably two or more, more preferably four or more, and even more preferably ten or more. is there. This number is not particularly limited, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 or more, or within the range of any two of them There may be.
  • examples of the “protein” include a protein containing an interaction region for a cofactor or a substrate.
  • a protein can be appropriately selected from, for example, NCBI GenBank.
  • examples of such proteins include DHODH, BVR-A, dihydrothymine dehydrogenase (Q12882), methionine synthase reductase (Q9UBK8), nitric oxide synthase (P29474, P35228, or P29475), phosphopantothenoylcysteine decarboxylase (Q96CD2), FAD synthase (Q8NFF5), hydroxy acid oxidase (Q9UJM8, or Q9NYQ3), iodotyrosine dehalogenase (Q6PHW0), cytochrome P450 reductase (P16435), NADPH-dependent diflavin oxidoreductase (Q9UHB4), NADH dehydrogena
  • the alphabets and numbers in parentheses described after each protein name are examples of UniProt accession numbers.
  • the above protein is preferably a monomer, and preferably has a low molecular weight.
  • the modified protein contained in the fluorescent protein complex is a compound having an FMN, DHO, BV, NAD (P) H, FAD, porphyrin ring from the viewpoint that the fluorescent protein complex emits fluorescence more stably. It is preferably derived from a protein having an interaction region for vitamin A or cobalamin.
  • the “cofactor” includes a coenzyme, a prosthetic group, and a metal ion.
  • coenzymes are loosely bound to enzymes and prosthetic groups are strongly bound.
  • the coenzyme is reversibly bound to the enzyme and the prosthetic group is bound irreversibly.
  • the “substrate” includes a substance that interacts with a protein to a certain degree.
  • Cofactors or substrates include, for example, FMN, DHO, BV, NAD (P) H, cofactors or substrates listed in Tables 11A and B, pyrroloquinoline quinone, topaquinone, tryptophan-tryptophyll quinone, lysine tyrosylquinone , Cysteinyl-tryptophan quinone, thiamine diphosphate, PALP, PLP, NAD, coenzyme A, biotin, folic acid, vitamin B12, adenosine triphosphate, pyridoxal phosphate, uridine diphosphate glucose, copper, manganese, molybdenum, nickel, And one or more selected from the group consisting of selenium.
  • the “interaction region” includes an amino acid in a protein that directly or indirectly interacts with a cofactor or substrate.
  • the interaction region includes amino acids before and after the amino acid that directly or indirectly interacts with the cofactor or substrate.
  • This interaction region can be identified using an existing method, and there is no particular limitation on the method. For example, molecular simulation software (MOE; Integrated Computational Chemistry System) Analysis using software such as Discovery Studio (Accelrys Inc.), LIGPLOT (University College London).
  • the interaction region may be identified based on publicly known literature describing the results of an experiment analyzing the binding with a substrate such as a substrate that interacts with a protein or a ligand such as a coenzyme or the results of an experiment performed by itself. Identification of the interaction region using molecular simulation software is performed by inputting the information of the protein such as the amino acid sequence of the target protein and the ID of the protein structure data bank (PDB), etc., and the substrate or coenzyme that interacts with the protein. By inputting the ligand information such as the ligand structure and CAS registration number, it is possible to automatically identify the interaction region between the protein and the ligand.
  • a substrate such as a substrate that interacts with a protein or a ligand such as a coenzyme or the results of an experiment performed by itself.
  • Identification of the interaction region using molecular simulation software is performed by inputting the information of the protein such as the amino acid sequence of the target protein and the ID of the protein structure data bank (PDB), etc.,
  • the region specified by using any of the existing methods such as the above-described molecular simulation software may not have an interaction ability under an in vitro experiment. Even such a region is included in the interaction region of one embodiment of the present invention as long as it is a region specified by using any existing method such as the molecular simulation software. That is, a region corresponding to the interaction region is included in the interaction region of one embodiment of the present invention. Even if a region identified using any of the existing methods such as the above molecular simulation software does not actually have the ability to interact, it can be stabilized together with the cofactor or substrate by modifying the region. It is possible to produce modified proteins that form complex complexes.
  • front and back amino acids include, for example, a plurality of amino acids that are continuously adjacent on the N-terminal side or C-terminal side of amino acids that directly or indirectly interact with a cofactor or substrate.
  • the number of amino acids is preferably 10 or less, more preferably 7 or less, from the viewpoint of increasing the fluorescence intensity. This number may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the amino acids before and after this may interact indirectly with the cofactor or substrate.
  • interaction includes a phenomenon in which force acts between two or more substances.
  • the interaction includes, for example, an action in which association or binding occurs between two or more substances.
  • the interaction may be, for example, ionic bond, hydrogen bond, hydrophobic interaction, hydrophilic interaction, intermolecular force, chelate bond, coordination bond, van der Waals bond, electrostatic bond, covalent bond, or non-covalent bond including.
  • the interaction may be direct or indirect. In the following, an embodiment of the interaction will be described, with the cofactors, substrates, and amino acids in the protein being alphabetized for convenience.
  • the amino acid having the effect of assisting or stabilizing the direct interaction is indirectly interacting with the cofactor A or the substrate B.
  • the amino acid D and the amino acid C may interact directly so as to assist or stabilize the direct interaction.
  • the above-mentioned cofactor A or substrate B and the amino acid D in the protein interact indirectly.
  • amino acid F that interacts indirectly with cofactor A or substrate B may be located.
  • modification of amino acid F may cause a structural change of the protein, and amino acid F may directly interact with cofactor A or substrate B.
  • the amino acid F directly interacts with the cofactor A or the substrate B the complex of the protein and the cofactor A or the substrate B is further stabilized.
  • amino acid H that indirectly interacts with cofactor A or substrate B may be located.
  • modification of amino acid C may cause a structural change of the protein, and amino acid H may directly interact with cofactor A or substrate B.
  • the amino acid H directly interacts with the cofactor A or the substrate B since the amino acid H directly interacts with the cofactor A or the substrate B, the complex of the protein and the cofactor A or the substrate B is further stabilized.
  • amino acid G that does not indirectly interact with cofactor A or substrate B may be located.
  • modification of amino acid G may cause a structural change in the protein, and amino acid G may interact directly or indirectly with cofactor A or substrate B.
  • the amino acid G directly or indirectly interacts with the cofactor A or the substrate B, the complex of the protein and the cofactor A or the substrate B is further stabilized.
  • amino acid I that does not indirectly interact with cofactor A or substrate B may be located.
  • modification of amino acid C may cause a structural change of the protein, and amino acid I may interact directly or indirectly with cofactor A or substrate B.
  • the amino acid I interacts directly or indirectly with the cofactor A or the substrate B, so that the complex of the protein and the cofactor A or the substrate B is further stabilized.
  • near amino acid C includes a state where the amino acid C is located near the protein structure or space.
  • the number of amino acids directly or indirectly interacting with the cofactor or substrate is 1 or more, preferably 3 or more, more preferably 7 or more, from the viewpoint of increasing the fluorescence intensity. It is.
  • the number of amino acids is not particularly limited, and may be, for example, 1, 2, 3, 4, 5, 6, 10, or 15 or more, or any two of them. Further, the number of interaction regions to be modified is 1 or more, preferably 3 or more, more preferably 4 or more, from the viewpoint of increasing the fluorescence intensity. The number of the amino acids is not particularly limited, and may be, for example, 1, 2, 3, 4, 5, 6, 10, 12, or 15 or more, or within the range of any two values thereof.
  • the “fluorescent protein” includes a protein capable of stably detecting fluorescence.
  • This fluorescent protein can be obtained, for example, by modifying a gene that encodes a protein that cannot stably detect fluorescence (sometimes referred to herein as “non-fluorescent protein”). This modification may be caused by an interaction region in a protein that interacts with a cofactor, an interaction region in a protein that interacts with a substrate, or one or more amino acids in an interaction region in a protein that interacts with both a cofactor and a substrate. It may be modified.
  • the “fluorescent gene” or “fluorescent gene” includes a nucleic acid encoding a fluorescent protein.
  • fluorescent protein or “fluorescent protein” refer to the formation of a stable complex with a cofactor or substrate due to modification of a non-fluorescent protein, resulting in a stable overall complex. In some cases, it is used for proteins when they become fluorescent. In other words, a fluorescent protein does not necessarily have to emit fluorescence, but only when a complex with a cofactor or a substrate is formed, a protein having such a characteristic that the whole complex emits fluorescence. Including. On the other hand, “non-fluorescent protein” includes a protein having such a characteristic that a complex cannot be stably formed with a cofactor or a substrate, and therefore the fluorescence of the entire complex cannot be stably emitted. .
  • a non-fluorescent protein can stably form a complex with a cofactor or substrate, but has a characteristic that it cannot stably emit fluorescence as a whole complex due to the structural problem of the complex. It may be a protein.
  • the non-fluorescent protein may be a wild type protein or a modified protein as long as the protein cannot stably detect fluorescence.
  • the non-fluorescent protein may be a polypeptide that cannot stably detect fluorescence (non-fluorescent polypeptide). Whether or not the fluorescence emitted from the test substance can be stably detected may be determined by whether or not the fluorescence derived from the test substance can be observed for a certain period using a fluorescence microscope.
  • fluorescence can be observed for a certain period using a fluorescence microscope. Therefore, they may be judged to emit fluorescence that can be stably detected.
  • fluorescence cannot be observed for a certain period with a fluorescence microscope. Therefore, ⁇ 1-74 hDHODH may be determined not to emit stable detectable fluorescence.
  • the fluorescence may be detected using, for example, a green fluorescence detector or a near infrared fluorescence detector as appropriate.
  • animals and plants are organisms classified in the animal kingdom (sometimes referred to as “animals” in this specification), or organisms classified in the plant kingdom (referred to as “plants” in this specification). Is also included.
  • Animals include, for example, organisms classified as mammals or mammals. Examples of animals include humans, mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, chimpanzees, and frogs.
  • Plants include, for example, organisms classified as red plant gates, gray plant gates, green algae plant gates, or streptophores. Plants include organisms classified as, for example, fern planta, gymnospermia or angiosperm. Examples of the plant include Arabidopsis thaliana, rice, corn, and sesame.
  • the “nucleic acid” includes those in which a plurality of nucleotides or equivalents thereof are combined.
  • the nucleic acid, DNA strand or RNA strand is included.
  • the nucleic acid is a cell uptake promoting substance (for example, PEG or a derivative thereof), a labeling tag (for example, a fluorescent labeling tag), a linker (for example, a nucleotide linker), or a chemotherapeutic agent (for example, an antineoplastic substance).
  • Etc. may be combined.
  • Nucleic acids can be synthesized using a nucleic acid synthesizer. In addition, it can also be purchased from a trust company (for example, Invitrogen).
  • nucleic acids may form salts or solvates.
  • in vivo nucleic acids may be chemically modified.
  • the term nucleic acid includes, for example, nucleic acids that form salts or solvates, or nucleic acids that have undergone chemical modification.
  • the nucleic acid may be an analog of nucleic acid.
  • the “salt” is not particularly limited. For example, an anion salt formed with any acidic (eg, carboxyl) group, or a cationic salt formed with any basic (eg, amino) group.
  • Salts include inorganic salts or organic salts, for example, salts described in Berge et al., J J Pharm. Sci., 1977, 66, 1-19.
  • a “solvate” is a compound formed by a solute and a solvent.
  • the solvent for example, J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953) can be referred to.
  • the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
  • “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.
  • Nucleic acids also include cDNA, genomic DNA, or chemically synthesized DNA or RNA strands and can be single-stranded or double-stranded.
  • the above “protein” includes those in which a plurality of amino acids or their equivalents are combined.
  • the chain length of the protein is not particularly limited, and may be, for example, 20, 50, 100, 300, 500, 1000, 5000, or 10000 amino acids, or any of those values or a range of any two values. It may be within.
  • the protein includes, for example, an enzyme, a receptor, or a structural protein. Protein is a concept including a polypeptide, and is not limited by the presence or absence of functionality or the chain length.
  • the protein includes, for example, a polypeptide having homology to a protein registered in the NCBI protein database and having an interaction region for a cofactor or a substrate. Whether there is homology can be confirmed by homology search using BLAST or the like.
  • the homology at this time may be, for example, 80, 90, 95, 98, 99, or 100%, or a range of any two of them.
  • the term protein includes, for example, a protein that forms a salt or a solvate, or a protein that has undergone chemical modification.
  • a synthesizer for example, PSSM-8 (SHIMADZU CORPORATION)
  • PSSM-8 SHIMADZU CORPORATION
  • the protein includes a wild type protein or a mutant protein. In this specification, when “in the range of two values” is specified, the range includes the two values themselves.
  • the modified protein may be a protein fragment of the modified protein that forms a fluorescent protein complex with a cofactor or substrate.
  • This protein fragment can fluoresce by forming a fluorescent protein complex with a cofactor or substrate.
  • the length of this protein fragment is not limited as long as it can form a fluorescent protein complex with a cofactor or substrate.
  • This protein fragment can be bound or fused with a compound other than the protein fragment.
  • amino acid is a general term for organic compounds having an amino group and a carboxyl group.
  • any amino acid in the amino acid sequence may be chemically modified. Any amino acid in the amino acid sequence may form a salt or a solvate. Further, any amino acid in the amino acid sequence may be L-type or D-type. Even in such a case, it can be said that the protein according to the embodiment of the present invention includes the above-mentioned “specific amino acid sequence”. That is, for example, R in the amino acid sequence may be arginine, or a chemically modified product, salt, or solvate thereof.
  • Examples of chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (for example, acetylation, myristoylation, etc.), C-terminal modification (for example, amidation, glycosylphosphatidylinositol addition, etc.), or side chain Modifications (for example, phosphorylation, sugar chain addition, etc.) are known.
  • N-terminal modification for example, acetylation, myristoylation, etc.
  • C-terminal modification for example, amidation, glycosylphosphatidylinositol addition, etc.
  • side chain Modifications for example, phosphorylation, sugar chain addition, etc.
  • the modified protein stably emits fluorescence with a cofactor or substrate, (i) an amino acid sequence encoding one or more proteins selected from the group consisting of DHODH and the like, (ii) ⁇ described in (i) above An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added, (iii) 80% or more homology to the amino acid sequence described in (i) above And (iv) a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid consisting of a base sequence complementary to the base sequence encoding the amino acid sequence described in (i) above.
  • At least one amino acid modification may be included. This modification may be at least one of the modifications shown in Table 2, Table 3, Table 4, Table 5, Table 6, or Table 7. Further, the modified protein is (ix) amino acid sequence encoding BVR-A, (x) ⁇ ⁇ ⁇ ⁇ one or more amino acids are deleted, substituted, inserted from the amino acid sequence described in (ix), Or (xi) amino acid sequence having 80% or more homology to the amino acid sequence described in (ix) above, and (xii) amino acid sequence described in (ix) above.
  • One or more amino acid sequences selected from the group consisting of an amino acid sequence encoded by a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid consisting of a base sequence complementary to the base sequence, and At least one region corresponding to the interaction region shown in FIG. 14 may have at least one amino acid modification.
  • This modification may be at least one of the modifications shown in Table 8, Table 9, or Table 10.
  • the modification shown in the table or figure may be modification of any one or more amino acids, and when there are two or more modifications, all possible combinations can be adopted. .
  • the “plurality” may be, for example, 20, 15, 10, 8, 6, 4, 3, or 2, or may be less than any of these values.
  • the “80% or more” is preferably 90% or more, more preferably 95% or more from the viewpoint of reducing antigenicity. This value is not particularly limited, and may be, for example, 80, 85, 90, 95, 97, 98, 99, or 100% or more, and may be in the range of any two of them.
  • the above-mentioned “homology” may be calculated according to a method known in the art, based on the ratio of the number of amino acids homologous in two or more amino acid sequences.
  • the amino acid sequences of the group of amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence when necessary to maximize the ratio of the same amino acids.
  • Methods for alignment, percentage calculation, comparison methods, and related computer programs are well known in the art (eg, BLAST, GENETYX, etc.).
  • “homology” can be expressed by a value measured by NCBI BLAST unless otherwise specified.
  • Blastp can be used as the default algorithm for comparing amino acid sequences with BLAST. Although the measurement result is quantified as Positives or Identities, this embodiment preferably employs Positives.
  • the following conditions can be adopted as the “stringent conditions”.
  • Use low ionic strength and high temperature for washing eg, 50 ° C., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate
  • a denaturing agent such as formamide (eg, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, And 750 mM sodium chloride, 75 mM sodium citrate) or (3) 20% formamide, 5 ⁇ SSC, 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denaturation Incubate overnight at 37 ° C.
  • the N-terminal or C-terminal amino acid of the modified protein or the protein before being modified may be deleted.
  • the number of deleted amino acids is preferably 74 or less, more preferably 20 or less, from the viewpoint of reducing the molecular weight. This value is not particularly limited, and may be, for example, 1, 5, 10, 20, 50, 74, 90, or 100 or less, or may be within the range of any two of them.
  • the deletion may be a deletion of a portion that inhibits the main activity inherent in the protein. Even when the protein chain length is shortened as a result of deletion of the N-terminal or C-terminal amino acid, it is included in the concept of the protein of one embodiment of the present invention.
  • the amino acids before being modified are, for example, Met, Ara, Val, Leu, Ile, Pro, Phe, Trp, Cys, Gly, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, It may be Lys, Arg, or His.
  • the amino acid after the modification may be substituted with an amino acid other than the amino acid before the modification.
  • This modification may be performed by modifying the nucleic acid encoding the interaction region of the target protein with the cofactor or substrate. Further, before the step of modifying, there may be a step of determining the interaction region in the target protein. Further, after the modifying step, there may be a step of selecting one having an increased fluorescence intensity.
  • the modification may be replaced with an amino acid having an isoelectric point larger or smaller than the amino acid before modification.
  • the amino acid before modification is a hydrophobic amino acid, a hydrophilic amino acid, an amino acid having an aliphatic side chain, an amino acid having a hydroxyl group-containing side chain, an amino acid having a sulfur atom-containing side chain, a carboxylic acid and an amide-containing side chain.
  • the amino acids may be classified into any of amino acids having a base, amino acids having a base-containing side chain, and aromatic-containing side chains. At this time, the amino acid after modification may be a different class of amino acid from that before modification.
  • the type of “fluorescence” may be green, near infrared, ultraviolet, purple, blue, yellow, orange, red, infrared, or far infrared.
  • the modified protein emits green fluorescence for example, it may have a maximum fluorescence wavelength between 450 nm and 600 nm. This wavelength is not particularly limited, and may be, for example, 450, 500, 550, or 600 nm, and may be in the range of any two values thereof.
  • the modified protein emits green fluorescence for example, it may have a maximum excitation wavelength between 300 nm and 550 nm.
  • This wavelength is not particularly limited, and may be, for example, 300, 350, 400, 450, 500, or 550 nm, and may be in the range of any two values thereof.
  • the modified protein may have a maximum fluorescence wavelength between 650 nm and 2500 nm.
  • This wavelength is not particularly limited and may be, for example, 650, 700, 750, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, or 2500 nm, and within the range of any two of them. There may be.
  • the modified protein emits near-infrared fluorescence for example, it may have a maximum excitation wavelength between 600 nm and 1100 nm.
  • This wavelength is not particularly limited, and may be, for example, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1100 nm, and is within the range of any two of them. Also good.
  • the fluorescence wavelength or the excitation wavelength may be modified so as to shift compared to the protein before modification.
  • the fluorescence is preferably near-infrared fluorescence or fluorescence having a maximum fluorescence wavelength between 650 nm and 2500 nm. Note that the fluorescent color detected by a fluorescence microscope or the like may change depending on the filter. Therefore, the modified protein may have a plurality of detectable fluorescent colors.
  • the fluorescence intensity of the modified protein can be determined, for example, by using a commercially available fluorescence microscope (for example, an inverted microscope for research ECLIPSE TE2000-U standard phase difference set, manufactured by Nikon Corporation), or a fluorescence spectrophotometer (for example, Hitachi High-Tech product). It is preferable that it can be used and stably detected.
  • the fluorescence intensity of the modified protein is substantially or significantly increased with respect to the protein before modification when the protein before modification has such a small fluorescence intensity that it cannot be stably detected. Also good. Note that “significantly” may include a case where, for example, statistical significance is evaluated using Student's t test (one-sided or two-sided) and p ⁇ 0.05.
  • the fluorescence intensity of the modified protein is preferably 50 times or more, more preferably 500 times or more compared to the protein before modification, from the viewpoint of increasing the accuracy or efficiency of the fluorescence test.
  • This number is not particularly limited, for example, 1.5, 2, 3, 4, 5, 10, 50, 100, 500, 1000, 2000, or may be increased more than 10,000 times, any of those two values It may rise to within the range.
  • Whether the modified protein is stably emitting fluorescence or whether the fluorescence intensity of the modified protein can be detected stably is determined by using the software of BD CellQuest Pro in FACS Calibur (Becton, Dickinson and Company). May be used (see, eg, FIG. 12).
  • ODDYSEY® manufactured by LI-COR
  • the “complex” includes a substance in which two or more substances are collected as a group.
  • the complex is, for example, an assembly of a protein and a cofactor or substrate.
  • the protein and the cofactor or substrate may interact.
  • the “biological substance” includes, for example, a protein, a nucleic acid, a cell, a sugar chain, or a lipid.
  • modification includes a phenomenon in which an amino acid is substituted, deleted, inserted, or added.
  • “at least one” may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more, or any two values thereof. .
  • “dynamics” includes a state in which a specific object is moving.
  • the pharmacokinetics includes, for example, a mode of movement or change in a living body of a component taken into the body.
  • “excitation light” includes light that causes excitation in a substance such as a fluorescent substance.
  • “modified protein in which an amino acid in an interaction region for a cofactor or a substrate is modified” refers to, for example, modification to an amino acid in an interaction region for a cofactor or substrate of a protein before modification. Including the state after applying.
  • the modified protein may be a modified protein having a modified amino acid in an interaction region for a cofactor or a substrate. Further, the modified protein may be a modified protein having an amino acid sequence in an interaction region with a cofactor or substrate that is different from the amino acid sequence before the modification.
  • the “expression control sequence” includes a nucleic acid sequence that enables transcription control or translation control, such as a promoter, an enhancer, a silencer, a terminator, an operator, and an inducer.
  • “operable” is in the correct position and orientation with respect to the nucleic acid encoding the modified protein, and includes controlling RNA polymerase initiation and gene expression.
  • “operably linked” functions in concert for the purpose, eg, transcription initiation in a promoter, and continues through a DNA sequence encoding the modified protein of the invention. , Including that the segments are arranged.
  • a “flow cytometer” is an apparatus using an analysis technique called flow cytometry.
  • flow cytometry an analysis technique
  • a device having a cell sorting function has only a cell analysis function. It may be a device (cell analyzer).
  • flow cytometers include, but are not limited to, the FACS series (Becton, Dickinson and Company).
  • the method for producing the modified protein is not particularly limited, but a known technique such as a gene recombination technique or chemical synthesis may be used.
  • the modified protein can be bound or fused with a compound other than the protein.
  • the modified protein can be labeled with the above compound.
  • the compound may be, for example, a biopolymer, a low molecular compound, or a high molecular compound.
  • Biopolymers include, for example, proteins, peptides, nucleic acids, sugars, lipids and the like.
  • the low molecular weight compound or the high molecular weight compound includes, for example, a therapeutically effective compound and a radioactive substance.
  • the peptide may be an oligopeptide or a polypeptide.
  • an antibody eg, a tumor-specific antibody, stem cell-specific antibody, nerve-specific antibody, etc.
  • enzyme eg, ligand, receptor, peptide tag (eg, myc tag, HA Tag, GST tag, etc.) and signal peptides (for example, secretory signal peptide, organelle signal peptide, etc.), but are not limited thereto.
  • the peptide may be localized in the cell, on the cell surface, or outside the cell.
  • the compound to be fused with the modified protein can be fused on the N-terminal side or the C-terminal side of the modified protein, and can be appropriately designed based on the purpose of use. Protein fusion or labeling can be performed using known techniques such as chemical synthesis or genetic recombination techniques.
  • nucleic acid encoding modified protein One embodiment of the present invention provides a nucleic acid encoding the modified protein.
  • the nucleic acid according to the embodiment of the present invention can be produced by a known method such as a phosphoramidite method or a polymerase chain reaction (PCR) using a specific primer, but is not limited thereto.
  • One embodiment of the present invention is a protein obtained by a method for producing the modified protein or a fusion protein thereof, a nucleic acid obtained by a method for producing a nucleic acid encoding the modified protein, a recombinant vector containing the nucleic acid, or these Recombinant hosts having at least one are provided. Also provided are compositions comprising at least one of these nucleic acids, recombinant vectors, proteins, or recombinant hosts.
  • the method of fluorinating a wild-type protein or a protein that does not stably emit fluorescence by amino acid modification includes, for example, a nucleic acid sequence encoding an interaction region for a cofactor or a substrate. And a method for introducing a mutation.
  • a method for introducing a mutation for example, a known technique such as a site directed random mutagenesis method for introducing a random mutation or a PCR method using a degenerate oligonucleotide for introducing a desired mutation is appropriately used. However, it is not limited to these.
  • the amino acid sequence to be modified is a modification of one or more amino acids in the interaction region for the cofactor or substrate.
  • This amino acid modification is a modification of at least one of the above interaction regions.
  • the 12 interaction regions confirmed by the simulation are preserved across species as shown in FIG.
  • the above 12 interaction regions are the interaction region between 6 FMN and DHODH, the interaction region between 4 DHO and DHODH, and the interaction region between 2 FMN and DHODH and DHODH. A region having 3 to 7 consecutive amino acids.
  • One embodiment of the present invention includes a nucleic acid encoding a modified protein obtained by the above method, a recombinant vector containing the nucleic acid, a modified protein that is a translation product of the nucleic acid, a fusion protein of the modified protein, or these
  • a recombinant host having at least one of the following is provided: Also provided are compositions comprising at least one of these nucleic acids, recombinant vectors, proteins, and recombinant hosts.
  • the nucleic acid can be used by inserting it into an appropriate vector.
  • the type of “vector” is not particularly limited, and a wide variety of recombinant vectors can be used.
  • This vector is engineered, for example, to express a modified protein, and thereby used to deliver the modified protein to cells.
  • the method for introducing the recombinant vector into the cell is not particularly limited, and may be a known method, and examples thereof include transfection, electroporation, lipofection, and a viral vector.
  • the vector is an expression vector.
  • an “expression vector” comprises a nucleic acid sequence encoding a modified protein, and any gene construct that can be transcribed in animal cells, plant cells, or microorganisms including human cells. including. This expression vector is directed to translation into a modified protein.
  • the expression vector may contain a sequence generally used in the vector such as a restriction enzyme cleavage site used in the vector.
  • the expression vector may also contain an origin of replication and a promoter located in front of the gene to be expressed, such as a polyadenylation sequence (eg, from SV40 or adenovirus 5E1b region), a terminator (eg, human growth hormone terminator).
  • the vector may contain a sequence encoding a cofactor or substrate synthase that can synthesize a cofactor or substrate of the protein.
  • the vector may further contain a selection marker.
  • Selectable markers include, for example, genes lacking complement in host cells such as dihydrofolate reductase and Schizosaccharomyces pombe TPI gene, ⁇ -lactamase and geneticin that give resistance to ampicillin. Examples include, but are not limited to, drug resistance genes such as neomycin resistance gene that give resistance.
  • the vector useful in one embodiment of the present invention is capable of autonomous replication, for example, plasmids and Sendai virus exist outside the chromosome, and the replication is not necessarily directly linked to the replication of the host cell genome.
  • replication of the vector may be replication on the host chromosome, for example, the vector may be integrated into the host cell chromosome, as achieved by a retroviral vector.
  • Vectors useful in one embodiment of the present invention include, but are not limited to, plasmid vectors, viral vectors, cosmid vectors, lambda phage vectors, and artificial chromosome vectors.
  • plasmid vector for example, for bacteria such as pGEX (GE ⁇ helthcare), pQE (Quiagen), pBK-CMV, pESC (Agilent ⁇ Technologies), pCS2, pCMV-SC, pSG (Agilent Technologies), pSVL (GE helthcare), Examples include eukaryotic organisms such as pCDNA3.1 (Life Technologies), but are not limited thereto.
  • viral vectors include, but are not limited to, paramyxovirus vectors (such as Sendai virus vectors), retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, alphaviruses, and the like. Not.
  • cosmid vectors include, but are not limited to, pKS334, pAT2, and pAT3 (Toyobo, JP-A-8-214881).
  • ⁇ phage vectors include, but are not limited to, ⁇ -based vectors such as Lambda ZapII or Lambda-Zap vector (Agilent Technologies) capable of inducibly expressing the polypeptide encoded by the insert.
  • ⁇ -based vectors such as Lambda ZapII or Lambda-Zap vector (Agilent Technologies) capable of inducibly expressing the polypeptide encoded by the insert.
  • Artificial chromosome vectors include human, mouse, and bacterial artificial chromosome (HAC) vectors (for example, Patent No. 4022835, Patent No. 4165532, Patent No. 4293990, Patent No. 3030092, JP 2007-295860, WO2011) / 083870, WO2010 / 038904, WO2008 / 013067, WO2004 / 031385, Special Table 2003-530113), but are not limited thereto.
  • HAC bacterial artificial chromosome
  • a “promoter” includes a DNA sequence necessary to initiate transcription of a particular gene, recognized by the cell's synthetic machinery or by a synthetic machinery introduced into the cell.
  • the promoter used in one embodiment of the present invention may be operable in animal cells including human cells, plant cells, or microorganisms.
  • promoters useful in one embodiment of the present invention include, but are not limited to, constitutive promoters, cell / tissue specific promoters, developmental stage specific promoters, or inducible promoters. Although the following are illustrated as a constitutive promoter, It is not limited to these.
  • Promoters that can operate in prokaryotes
  • promoters operable in yeast host cells include GAL1, GAL4, other glycolytic gene promoters, alcohol dehydrogenase gene promoters, TPI1 promoters, ADH2-4c, etc. .
  • promoters examples include polyhedrin promoter, P10 promoter, autographa calihornica polyhedrossis basic protein promoter, baculovirus immediate early gene promoter, or baculovirus 39K delayed early gene promoter. Can be mentioned.
  • promoters operable in mammalian cells include, for example, actin promoter, EF-1 ⁇ promoter, metallothionein gene promoter, SV40 promoter, cytomegalovirus (CMV) promoter, retrovirus LTR promoter, adenovirus 2 major late promoter, and others Virus promoters, and the like.
  • cell / tissue-specific promoters or developmental stage-specific promoters include, but are not limited to, those described in Tables 1A and 1B below.
  • Inducible promoters include promoters that are controlled in response to chemical (eg, alcohol, steroids, antibiotics, hormones, metal ions, etc.) or physical (eg, heat, light, ionizing radiation, etc.) signals. Is mentioned. Specific examples include, but are not limited to:
  • Tetracycline regulated promoter Tetracycline regulated promoter, rapamycin regulated promoter, glucocorticoid steroid regulated promoter, sex hormone steroid regulated promoter, ecdysone regulated promoter, lipopolysaccharide (LPS) regulated promoter, isopropylthiogalactoside (IPTG) regulated promoter
  • Cytochrome P450 promoter activated by a range of toxic compounds, heat shock protein (hsp) promoter, fos promoter or jun promoter inducible by ionizing radiation, etc.
  • One embodiment of the present invention includes at least one biological substance selected from the group consisting of the above-mentioned modified protein, fusion protein, nucleic acid, fluorescent protein complex, and cell having at least one substance thereof.
  • a material for fluorescence inspection Since this fluorescence inspection material can emit fluorescence by being irradiated with excitation light, it can be used for various applications described above or below.
  • This fluorescent examination material can be used, for example, to measure the dynamics of the biological substance.
  • This fluorescent test material can also be used to identify, isolate, sort or purify cells.
  • this fluorescent test material can be produced by modifying non-fluorescent proteins of animals and plants, it can be suitably used for in vivo use of animals and plants. At this time, if this fluorescent examination material is used for in vivo use of organisms of the same species as the derived organism such as the modified protein, adverse events are particularly unlikely to occur.
  • the term “material” when described with a particular application conceptually includes “material for use in a particular application”. The material at this time may have both the specific application and an application other than the specific application.
  • the term “material” includes, for example, a substance used as a raw material or component of a product or intermediate. This material may contain one or more substances. The shape and material of this material are not particularly limited. This material may be in the form of, for example, a protein, a nucleic acid, or a cell.
  • One embodiment of the present invention provides at least one biological substance selected from the group consisting of the above-mentioned modified protein, fusion protein, nucleic acid, fluorescent protein complex, and cell having at least one substance thereof, or
  • This method can be applied to various uses described above or below by using the detected fluorescence as an index.
  • This method can be applied to reporters or tracers such as cell identification, isolation, selection, purification, gene expression analysis, or behavior tracking (pharmacokinetics, etc.) of a target substance.
  • This method can also be applied to measure the dynamics of the biological material.
  • This method can also be applied to measure the dynamics of the biological material.
  • This method can also be applied to prevent, treat or diagnose a disease.
  • the modified protein can be produced by modifying a non-fluorescent protein of animals and plants, the above method can be suitably used for detecting fluorescence in animals and plants in vivo. At this time, if it is used for detecting fluorescence in the living organism of the same species as the derived organism such as the modified protein, adverse events are particularly unlikely to occur.
  • the method may further include a step of introducing at least one substance selected from the group consisting of the modified protein, the fusion protein, the nucleic acid, and the fluorescent protein complex into the cell.
  • the excitation light may be applied to the biological material or may be applied to the periphery of the biological material.
  • the modified protein when the modified protein is irradiated with excitation light, the modified protein emits fluorescence, thereby enabling a fluorescence test. At this time, even if the excitation light is irradiated to the modified protein and its surroundings, the modified protein emits fluorescence, so that a fluorescence test can be performed.
  • the modified protein existing in the periphery of the nucleic acid is irradiated with excitation light, whereby the modified protein emits fluorescence, thereby enabling a fluorescence test.
  • the periphery of the nucleic acid includes, for example, the inside of the cell where the nucleic acid exists.
  • a method for producing an identified, isolated, sorted, or purified cell comprising the step of performing the above method. For example, identifying, isolating, selecting, or purifying therapeutically effective cells from a cell population containing therapeutically effective cells and non-therapeutic cells using the fluorescence of the fluorescent protein complex as an index Can do.
  • This therapeutically effective cell can be used, for example, for prevention, treatment, diagnosis, or examination of a disease.
  • therapeutically effective cells include cells for cell therapy.
  • cells for cell therapy include stem cells and immune cells.
  • the therapeutically effective cells may be used in the form of a prophylactic composition, pharmaceutical composition, or diagnostic composition after mixing with a carrier.
  • a method for fluorescentizing a complex of a target protein derived from animals and plants, and a cofactor or substrate, wherein the target protein has an interaction region with the cofactor or substrate comprising the step of modifying an amino acid.
  • a method for increasing the fluorescence intensity of a complex of a target protein and a cofactor or substrate comprising a step of modifying an amino acid in an interaction region of the target protein derived from animals or plants with respect to the cofactor or substrate.
  • a method for stably forming a complex of a target protein and a cofactor or substrate comprising a step of modifying an amino acid in an interaction region for the cofactor or substrate of a target protein derived from an animal or plant.
  • a method for trapping a cofactor or a substrate in a target protein which comprises a step of modifying an amino acid in an interaction region for the cofactor or substrate of a target protein derived from an animal or plant.
  • (C-1) a method for screening a target protein modified to emit fluorescence, comprising a step of modifying an amino acid in a region interacting with a cofactor or a substrate of a target protein derived from animals and plants .
  • This method further includes (C-2) a step of bringing the modified target protein into contact with a cofactor or a substrate, and (C-3) a step of applying excitation light to the modified target protein or the target protein and its surroundings. , (C-4) a step of measuring the intensity of fluorescence resulting from the above (C-3), or (C-5) a step of selecting a modified target protein when the fluorescence is stably observed , May be included.
  • the case where fluorescence is observed stably includes, for example, a state in which fluorescence can be visually observed with a fluorescence microscope. Or, when the fluorescence is observed stably, when the protein before modification has a small fluorescence intensity that cannot be stably detected, the protein before modification is substantially or Includes significantly elevated conditions.
  • the “recombinant host” includes a cell into which the nucleic acid or recombinant vector has been introduced, or a tissue or organism containing the cell.
  • the cell may be any exogenous DNA fragment containing the nucleic acid sequence of the modified protein, or any cell that can express a gene. Examples of such cells include, but are not limited to, bacteria, fungi such as yeast, or higher eukaryotic cells such as insect cells and mammalian cells.
  • the cell may express a modified protein.
  • bacterial cells include gram-positive bacteria such as Bacillus bacteria, and Gram-negative bacteria such as Escherichia coli or Streptomyces.
  • the introduction of the nucleic acid or the recombinant vector into bacteria is not particularly limited, and may be a known introduction method, for example, introduction method using competent cells or protoplasts (COHEN, SN et al, Proc Natl Acad Sci U S A, 1972, Vol.69, p.2110-4, DUBNAU, D. et al, J Mol Biol, 1971, Vol.56, p.209-21, CHANG, S. et al, Mol Gen Genet, 1979, Vol. 168, p.111-5).
  • yeast cells include cells belonging to Saccharomyces such as Saccharomyces cerevisiae or Saccharomyces reteyveri, cells belonging to Schizosaccharomyces, cells belonging to the genus Pichia, cells belonging to the genus Krivellomyces, and the like.
  • the introduction of the nucleic acid or the recombinant vector into yeast is not particularly limited, but may be a known introduction method, for example, electroporation method, spheroblast method, lithium acetate method (HINNEN, A. et al, Proc Natl Acad Sci U S A, 1978, Vol.75, p.1929-33, ITO, H. et al, J Bacteriol, 1983, Vol.153, p.163-8).
  • Examples of other fungal cells include cells belonging to the genus Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • a recombinant host cell can usually be obtained by integrating a nucleic acid or a recombinant vector into a host chromosome. Integration of a nucleic acid or a recombinant vector into a host chromosome can be performed by homologous recombination or heterologous recombination according to a known method.
  • a recombinant vector or baculovirus is co-introduced into the insect cells to obtain the recombinant virus in the culture supernatant of the insect cells, and then the recombinant virus is further infected with the insect cells, Protein can be expressed.
  • insect cells include Sf9, Sf21, HiFive (Life Technologies) and the like.
  • baculoviruses include outgrapha, californica, nuclea, polyhedronosis, and virus.
  • the method for co-introducing the recombinant gene introduction vector into the insect cell and the baculovirus for preparing the recombinant virus is not particularly limited, and may be a known introduction method, such as the calcium phosphate method or the lipofection method. It is done.
  • mammalian cells examples include HEK293 cells, VERO cells, HepG2 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells.
  • cells obtained from animal subjects including humans can also be used as cell preparations that are reintroduced into animals including humans after introduction of nucleic acids or recombinant vectors.
  • Examples of such cells include skin cells, bone marrow cells, nerve cells, hepatocytes, pancreatic cells, somatic cells such as pancreatic cells, retinal cells, ES cells, iPS cells, other stem cells, cells derived from these stem cells, and the like.
  • These mammalian cells include cases where they have a therapeutic effect and cases where they have no therapeutic effect.
  • the targeted in vivo cells can contain a modified protein.
  • transgenic mice All of the above cells, and tissues and organisms (for example, transgenic mice) containing the cells fall within the scope of the description of “recombinant host” used in the present embodiment.
  • All diseased, defective or healthy cells are included in one embodiment of the invention in this manner.
  • ⁇ Recombinant expression> The above recombinant host cells are cultured in an appropriate nutrient medium under conditions that allow expression of exogenous DNA fragments or genes containing the nucleic acid sequence of the introduced modified protein.
  • an appropriate nutrient medium under conditions that allow expression of exogenous DNA fragments or genes containing the nucleic acid sequence of the introduced modified protein.
  • isolating and purifying a modified protein or a fusion protein containing the same from the culture those skilled in the art can use protein isolation and purification methods commonly used.
  • the fluorescent protein complex can emit fluorescence. Therefore, a modified protein or a fusion protein thereof according to an embodiment of the present invention, a nucleic acid encoding the modified protein, a fluorescent protein complex, a fluorescent test material, or a fluorescence-related substance derived from at least one of them ( Hereinafter, it may be referred to as “modified protein or the like”), and can be used for any application utilizing the fluorescence characteristics used in fluorescent substances such as existing fluorescent proteins and fluorescent dyes.
  • reporter or tracer protein for cell identification, cell sorting, cell purification, gene expression analysis, labeling of target substances (cells, proteins, compounds, etc.), behavior tracking of target substances (pharmacokinetics, etc.)
  • target substances cells, proteins, compounds, etc.
  • behavior tracking of target substances pharmacokinetics, etc.
  • Applications can be mentioned, but the invention is not limited to these.
  • This use includes the use described in the literature described in the above-mentioned background art column, for example.
  • modified proteins were created based on proteins originally possessed by organisms including humans, there is a probability that even when administered to organisms including humans, humoral immunity or cellular immunity is not induced. It can be said that it is more useful than known fluorescent genes because of its advantages such as high safety and high safety.
  • the above-mentioned modified protein having high safety can be administered into the body of living organisms including humans, and fluorescent reagents (in vivo tracers, in vivo reporters) in in vivo fluorescent bioimaging and humans
  • the present invention can be used for all purposes such as purification of cells prepared for the purpose of administering to living organisms, such as purification of cells and advantages in utilizing fluorescence properties.
  • Nucleic acid encoding the above modified protein that is highly safe and applicable to the uses described above and below a recombinant vector having the nucleic acid, a modified protein, a fusion protein of the modified protein, and a fluorescent protein complex A body, a fluorescent test material, or a recombinant host containing at least one of these can be applied to a subject including a human and used in a disease prevention / treatment method, a diagnostic method, and a test method.
  • a nucleic acid encoding a modified protein, a recombinant vector having the nucleic acid, a modified protein, for use in disease prevention, treatment, diagnosis, or testing A composition is provided comprising a fusion protein of the modified protein, a fluorescent protein complex, a fluorescent test material, or a recombinant host comprising at least one of these.
  • the use of the above modified protein includes purification of cells. That is, it is possible to purify target cells by selecting target cells and unnecessary cells using the fluorescence derived from the modified protein as an index (ie, purifying target cells by positive selection or negative selection). In particular, it is expected to be useful for isolating and purifying only target cells obtained by inducing differentiation of stem cells such as ES cells and iPS cells. Examples of methods for selecting, sorting, isolating and purifying target cells include flow cytometry including fluorescence activated cell sorting (FACS) and fully automated single cell isolation analyzer (As One cell picking system, manufactured by ASONE) ), Can be performed by a fluorescence microscope or the like, but is not limited thereto.
  • FACS fluorescence activated cell sorting
  • ASONE fully automated single cell isolation analyzer
  • an antibody against an antigen specifically expressed on the cell surface of a target cell or an unnecessary cell is fused with the modified protein, or cells as shown in Tables 1A and 1B above. ⁇ Identify or select target cells or unwanted cells that emit fluorescence by introducing a recombinant vector in which a nucleic acid encoding the modified protein is linked to a tissue-specific promoter or a developmental stage-specific promoter into the cell. It is possible to purify the cells.
  • the target cells purified in this way include cells having therapeutic efficacy, but are not limited thereto.
  • One embodiment of the present invention also provides a method for treating, diagnosing or examining a disease using cells having therapeutic efficacy thus purified, or a pharmaceutical composition for treating, diagnosing or examining a disease. It also includes things.
  • a tracer that tracks the pharmacokinetics of therapeutic agents (including compounds and cells). For example, it is possible to fuse or bind a therapeutic agent to a modified protein and to follow fluorescence as an indicator.
  • fluorescent reagents in in-vitro or in-vivo fluorescent bioimaging for diagnosis.
  • diagnostic applications such as the identification of the presence or absence of tumor cells or pathogenic cells, etc. using fluorescence as an indicator by fusing cells / tissue-specific antibodies such as tumors and pathogenic cells, ligands, receptors, etc. to modified proteins can be used.
  • biosensors in prokaryotic or eukaryotic cells include as biosensors in prokaryotic or eukaryotic cells, eg, Ca 2+ ion indicators, pH indicators, phosphorylation indicators, or other ions (eg, magnesium, sodium, potassium, chloride or halogen).
  • a fluorescent indicator For example, when detecting Ca 2+ ions, a confocal microscope shows the movement from the cytosol to the plasma membrane for a fusion of a protein containing an EF motif such as calmodulin, valve albumin, recoverin, or calcineurin with a modified protein. It is monitored by observation, and the EF hand-containing protein at that time can be used as a fluorescent indicator of intracellular Ca 2+ .
  • the modified protein is used as a marker for whole cells to detect changes in multicellular reorganization and migration, such as changes in cell migration, wound healing, or neurite outgrowth through angiogenesis by endothelial cells. It can be used.
  • the modified protein according to the above embodiment can be used as, for example, a fluorescent indicator of cell activity in signal transduction. It is also used for screening used as a marker fused to a peptide (for example, target sequence) or a protein (for example, antibody, ligand, receptor, etc.) that detects a positional change (transfer of transcription factor, etc.) in a cell. It is possible. High content for detecting co-localization of other fluorescent fusion proteins using the modified protein as a localization marker (for example, as a fluorescent indicator of intracellular fluorescent protein or peptide movement) or as a marker alone It can also be used in screening (HCS).
  • it can be used as a transcription reporter for drug discovery, in which case it is possible to detect promoters (eg, NF ⁇ B, STAT, Smad, ER, etc.) in signal transduction pathways .
  • promoters eg, NF ⁇ B, STAT, Smad, ER, etc.
  • it can be used as a secondary information transmitter by fusing the modified protein to a specific domain such as SH2 domain or SH3 domain.
  • it can be used as an in vivo fluorescent label for organelles and cells and a fluorescent label for tracking their transport, that is, a tracer protein.
  • the modified protein can be used as an in vivo marker in genetically modified organisms.
  • the method can be used in research and development for gene therapy such as examining the expression efficiency of a transgene.
  • FRET fluorescence resonance energy transfer
  • One embodiment of the present invention includes a nucleic acid encoding the modified protein, a recombinant vector containing the nucleic acid, a modified protein that is a translation product of the nucleic acid, a fusion protein of the modified protein, a fluorescent protein complex, a fluorescence Compositions comprising one or more of a test material or a recombinant host having at least one of these are provided. Also provided by one embodiment of the present invention is a composition for use in one or more of the applications described above.
  • the composition according to an embodiment of the present invention can be provided as a reagent composition or a pharmaceutical composition, and in the case of a pharmaceutical composition, a pharmaceutically acceptable carrier, excipient, or dilution.
  • This pharmaceutical composition can be used for humans or animals in human or veterinary medicine.
  • Pharmaceutically acceptable carriers, excipients, or diluents are well known to those skilled in the art, and an appropriate substance can be arbitrarily selected according to the administration route and intended use.
  • the pharmaceutical composition may also include any suitable binder, lubricant, suspending agent, coating agent, solubilizer, etc. as or in addition to the carrier, excipient or diluent.
  • preservatives, stabilizers, dyes, or flavoring agents can be included in the pharmaceutical composition.
  • a composition comprising a pharmaceutical composition can be administered by various modes of administration including systemic administration, local administration, or localized administration, depending on the intended use.
  • Any suitable mode of administration can be employed.
  • subcutaneous injection intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, intracardiac injection, intratumoral injection, intravaginal injection, intrapulmonary injection, intranasal injection, intratracheal injection, blood vessel Administration by injection, such as internal injection, intraarterial injection, intracoronary injection, intraventricular injection, percutaneous (local) injection, or direct injection into lymph nodes.
  • compositions comprising the above pharmaceutical compositions can also be administered via mucosal routes such as oral / dietary routes, nasal routes, intratracheal routes, intravaginal routes, or rectal routes.
  • a composition containing the above pharmaceutical composition for example, diagnosis of a disease or pharmacokinetics of a test substance can be confirmed.
  • the disease can be treated with the cells, for example, by administering a pharmaceutical composition comprising therapeutically effective cells purified with a flow cytometer.
  • a composition comprising a therapeutically effective amount of the above pharmaceutical composition may be administered to a patient in need of treatment for a disease.
  • the reagent composition may be, for example, a reagent used for a fluorescent examination for research, or may be used for an in vitro test, a cell test, or a tissue test.
  • kits for use in one or more of the applications described above are also provided by one embodiment of the present invention.
  • the kit typically includes a construct constituting a vector containing an element for expressing the modified protein, for example, a nucleic acid sequence encoding the modified protein.
  • the components of the kit are typically present in a suitable storage medium (eg, a buffer solution, etc.) in a suitable container.
  • the kit also includes a number of different vectors that each encode the modified protein.
  • the vector is designed for expression under different circumstances or under different conditions, for example the vector is designed for constitutive expression comprising a strong promoter for expression in mammalian cells, or And a promoter-free vector having a large number of cloning sites for insertion of a promoter and whose expression is uniquely regulated.
  • the vector includes instructions for practicing any embodiment of the invention.
  • Dihydroorotate dehydrogenase is a widely preserved enzyme from Escherichia coli to multicellular organisms, and it converts dihydroorotic acid (DHO) to orotic acid, a pyrimidine biosynthetic pathway (uracil synthesis pathway).
  • DHO dihydroorotic acid
  • FMN flavin mononucleotide
  • LIU flavin of vitamin B2 derivative as a coenzyme
  • Non-patent document 4 describes an in vitro binding assay between FMN and DHODH that utilizes the property of FMN emitting green fluorescence. In vivo, it has not been known that endogenous DHODH and exogenous DHODH emit green fluorescence.
  • the “fluorescent DHODH” is a green fluorescence that can be detected stably by modifying the amino acids of DHODH inherent in all living organisms including humans (including animals, plants, and microorganisms) themselves. Including those that emit. Fluorescent DHODH does not necessarily fluoresce itself, but only when a complex with a cofactor or substrate is formed, a protein having such a characteristic that the complex as a whole fluoresces. Including.
  • the amino acid modification is, for example, modification of one or more amino acids in the interaction region of DHODH that interacts with FMN or DHO.
  • DHODH that interacts with FMN or DHO.
  • the 12 interaction regions confirmed by the simulation are preserved across species as shown in FIG.
  • the above 12 interaction regions are the interaction region between 6 FMN and DHODH, the interaction region between 4 DHO and DHODH, and the interaction region between both FMN and DHO and DHODH.
  • the amino acid modification is, for example, modification of at least one of the interaction regions.
  • the amino acid modification may be, for example, an amino acid modification corresponding to glycine at position 305 of hDHODH.
  • the amino acid corresponding to the 305th glycine can be confirmed by comparing the sequences of hDHODH and other organism-derived DHODH using BLAST or the like.
  • the amino acid corresponding to the glycine at position 305 includes the DHODH amino acid derived from other organisms corresponding to the glycine at position 305 of hDHODH in the comparison with BLAST and the like.
  • “corresponding amino acid” or “corresponding region” may be determined based on the position of the amino acid after sequence comparison, as in the above example.
  • the fluorescent DHODH emits green fluorescence that can be stably detected by the above modification, and preferably has a modified amino acid sequence in each interaction region described in Table 5. More preferably, the modified amino acid sequences of these interaction regions are combined with the modified amino acid sequences of two or more interaction regions, and more preferably the combinations of modified amino acid sequences described in Table 4. It is.
  • the amino acid sequence of DHODH other than the above interaction region may be deleted, substituted, inserted, or added from one to several amino acids as long as it emits green fluorescence.
  • the signal sequence may be deleted.
  • the range of “1 to several” in “deletion, substitution, insertion or addition of 1 to several amino acids” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, More preferably, it means 1 to 7, more preferably 1 to 5, particularly preferably about 1 to 3.
  • Example 3 In Example 3 etc. described later, experimental data when the DHODH gene is modified are described.
  • the modified form of this modified DHODH is a modification of 1 to 3 of 3 to 7 consecutive amino acids in the interaction region with FMN or DHO, and the interaction region is scattered, Antibody production and induction of cytotoxic T cells are thought to be particularly difficult to cause.
  • the antigen is decomposed into peptides and bound to HLA class I and HLA class II molecules and presented to the T cells.
  • This peptide functions as an antigen to produce antibodies and induce cytotoxic T cells. Must meet two conditions.
  • the peptide must be derived from a different species and not an autoantigen.
  • autoantigens all self-proteins, sugars, lipids, etc.
  • immune tolerance does not occur because immune tolerance is established through education within the thymus.
  • the peptide needs to be embedded and bound in the peptide-containing groove within the HLA class I and HLA class II antigens.
  • the modified DHODH described in the examples below when only one to three consecutive amino acids in the interaction region of DHODH, which is a self protein, are mutated, the antigenicity is remarkably increased. Low. Furthermore, the modified DHODH described in the Examples below includes a peptide structure that binds to HLA class I molecules and HLA class II molecules in the nearby 9 to 30 amino acid sequences including the mutated amino acids.
  • ⁇ fluorinated DHODH '' and ⁇ fluorinated DHODH '' are an interaction region in DHODH that interacts with FMN, an interaction region in DHODH that interacts with DHO, or both FMN and DHO.
  • Fluorescent DHODH was able to create a fluorescent gene by modifying mDHODH not only in humans, but also in mice, using the same principle, other animals, It is possible to create a fluorescent gene from DHODH possessed by organisms such as plants and microorganisms.
  • the wild-type DHODH derived from fluorescent DHODH may be derived from any species.
  • the amino acid sequence and base sequence of DHODH derived from such a biological species for example, in the case of animal origin, human (Homo sapiens, Genbank Accession No. NP_001352, NM_001361), monkey (Macaca mulatta, Genbank Accession No. XP_001104448, XM_001104448), chimpanzees (Pan troglodytes, Genbank Accession No. XP_001171601, XM_00117160), cattle (Bos taurus, Genbank Accession No.
  • NP_001015650, NM_001015650 rabbits (Oryctolagus cuniculus, Genbank_Accession Matt Genbank Accession No. NP_001008553, NM_001008553), mice (Mus musculus, Genbank Accession No. XP_064430, XM_020046), frogs (Xenopus laevis, Genbank Accession No. NP_001085026, NM_001091557), etc. thaliana, Genbank Accession No. NP_568428, NM_122236), rice (Oryza sativa) Japonica Group, Genbank Accession No.
  • NP_001054255, NM_001060790 Corn (Zea maymay, Genbank Accession No. NP_001152058, NM_001158586), Sesame (Ricinus communis, Genbank Accession No.
  • cloning may be performed by a method known to those skilled in the art based on the known DHODH base sequence or amino acid sequence.
  • a fluorescent gene having a detectable fluorescence intensity can be created by modifying the amino acid in the interaction region of the test protein.
  • Candidate genes include, but are not limited to, the proteins listed in Table 11A or B.
  • flavoproteins that interact with FMN for example, Dihydrolipoamide dehydrogenase, Acyl CoA dehydrogenase, etc. can be mentioned, but are not limited thereto.
  • A-2) The nucleic acid according to (A-1), wherein at least one region of the interaction regions is modified.
  • A-3) The nucleic acid according to any one of (A-1) to (A-2), wherein there are twelve interaction regions.
  • A-4) The above 12 interaction regions are the interaction region between 6 FMNs and DHODH, the interaction region between 4 DHOs and DHODH, and the two interaction regions between FMN and DHO and DHODH.
  • the above 12 interaction regions are any of the above (A-3) to (A-4), which is a region having 12 to 3 consecutive amino acids as shown in FIG.
  • the amino acid modification is any one or more of the modified amino acid sequences in each interaction region described in Table 5, according to any one of (A-1) to (A-5) above Nucleic acid.
  • the amino acid modification is a combination of the modified amino acids in each interaction region shown in Table 5 with the modified amino acid sequences in two or more interaction regions.
  • the nucleic acid according to any one of (A-8) The nucleic acid according to any one of (A-6) to (A-7) above, wherein the amino acid sequence is a combination of modified amino acid sequences described in Table 4.
  • the wild type DHODH is one or more wild type DHODH selected from animal origin, plant origin, and microorganism origin, any one of (A-1) to (A-8) above Nucleic acids.
  • the animal-derived wild-type DHODH is one or more wild-type DHODH selected from human-derived, monkey-derived, bovine-derived, rabbit-derived, dog-derived, rat-derived, mouse-derived, and frog-derived,
  • A-12 The nucleic acid according to (A-11) above, wherein the peptide is one or more peptides selected from those localized in the cell, on the cell surface, and outside the cell.
  • A-13 The nucleic acid according to any one of (A-11) to (A-12) above, wherein the peptide is one or more selected from an antibody, an enzyme, a ligand, a receptor, and a peptide tag .
  • a recombinant vector comprising the nucleic acid according to any one of (A-1) to (A-13) above.
  • A-15 A recombinant vector comprising an expression control sequence operably linked to the nucleic acid described in (A-1) to (A-13) above.
  • A-18 The recombinant vector according to (A-17) above, wherein the constitutive promoter is one or more promoters selected from viral promoters and gene promoters derived from animals and plants.
  • the inducible promoter is one or more promoters selected from chemical promoters such as alcohol, steroids, and tetracycline, and physical promoters such as heat, light, and ionizing radiation,
  • A-17 A recombinant vector according to 1.
  • A-20 The recombinant vector according to (A-17) above, wherein the cell / tissue-specific promoter and the developmental stage-specific promoter are one or more promoters selected from the promoters described in Table 1A or 1B.
  • (A-22) Translation product of nucleic acid according to any one of (A-1) to (A-13) above and translation produced by the recombinant vector according to any one of claims 1 to 21 Fluorescent DHODH or fusion protein containing one or more translation products selected from the products.
  • (A-23) The fluorescent DHODH protein or fusion protein thereof according to (A-19), which is bound to one or more substances selected from nucleic acids other than the fluorescent DHODH, compounds, and other substances .
  • A-24 The fluorescent DHODH or fusion protein thereof according to (A-23) above, wherein the nucleic acid, compound, or other substance labels the protein according to (A-23).
  • A-25 the nucleic acid according to any one of (A-1) to (A-13) above, the recombinant vector according to any one of (A-14) to (A-21) above, And a recombinant host comprising one or more proteins selected from the proteins described in any one of (A-22) to (A-24) above.
  • A-26 The recombinant host according to (A-25) above, wherein the recombinant host is one or more hosts selected from cells, tissues, organisms, and their progeny.
  • A-27 The recombinant host according to (A-26) above, wherein the tissue or its progeny contains the cell.
  • A-28 The recombinant host according to (A-27) above, wherein the organism comprises one or more selected from the cells and the tissue.
  • A-29 The above cell or its progeny is one or more cells selected from ES cells, iPS cells, somatic stem cells, other stem cells, and cells derived therefrom or progeny thereof (A- 26) A recombinant host according to any one of (A-28).
  • the method comprising the step of introducing into the cell one or more substances.
  • A-31) The method according to (A-30) above, further comprising a step of identifying, sorting, or purifying cells by a flow cytometer including fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • A-32) The above cells (A-30) to (A-), wherein the cells are one or more cells selected from ES cells, iPS cells, somatic stem cells, other stem cells, and cells derived therefrom.
  • A-33) The method according to any one of (A-30) to (A-32), wherein the cell is one or more cells selected from plants and microorganisms.
  • the animal-derived cell is one or more cells selected from human origin, monkey origin, bovine origin, rabbit origin, dog origin, rat origin, mouse origin, and frog origin, and plant origin is derived from Arabidopsis thaliana
  • (A-36) The nucleic acid according to any one of (A-1) to (A-13) above, for identifying, sorting, or purifying cells using the fluorescence of green fluorescent DHODH as an index, 1 selected from the recombinant vector according to any one of (A-14) to (A-21) above and the protein according to any one of (A-22) to (A-24) above A composition comprising the above substances.
  • (A-37) The cell is the nucleic acid according to any one of (A-1) to (A-13) above, or the nucleic acid according to any one of (A-14) to (A-21) above.
  • composition Any one of (A-36) to (A-37) above, wherein the cells are identified, sorted, or purified by a flow cytometer including fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the animal-derived cell is one or more cells selected from human origin, monkey origin, cow origin, rabbit origin, dog origin, rat origin, mouse origin, and frog origin, and plant origin is derived from Arabidopsis thaliana
  • the composition according to (A-40) above which is one or more cells selected from rice, corn, and castor bean.
  • A-42 A composition comprising cells identified, sorted, or purified by the method according to any one of (A-30) to (A-34) above.
  • A-43 A prophylactic / therapeutic method using cells identified, selected, or purified by the method according to any one of (A-30) to (A-34) above.
  • A-44) A diagnostic method using cells identified, selected, or purified by the method according to any one of (A-30) to (A-34) above.
  • A-45 A test method using cells identified, sorted, or purified by the method according to any one of (A-30) to (A-34) above.
  • A-46) The composition according to any one of (A-35) to (A-42), wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
  • A-47 The composition according to any one of (A-35) to (A-42) and (A-46) above for preventing / treating a disease.
  • A-48 The composition according to any one of (A-35) to (A-42) and (A-46) above for diagnosing a disease.
  • A-49 The composition according to any one of the above (A-35) to (A-42) and (A-46) for examining a disease.
  • A-50 A method for producing the composition according to any one of (A-35) to (A-42) and (A-46) to (A-49).
  • A-51) For producing the composition according to any one of (A-35) to (A-42) and (A-46) to (A-49) above, (A-1 ) To (A-13). Use of the nucleic acid according to any one of (A-13).
  • A-52 For producing the composition according to any one of (A-35) to (A-42) and (A-46) to (A-49) above, (A-14 Use of the recombinant vector according to any one of (A) to (A-21).
  • A-53 For producing the composition according to any one of (A-35) to (A-42) and (A-46) to (A-49) above, (A-22 Use of the protein according to any one of (A-24).
  • (A-54) (A-25) to (A-29) for producing the composition according to any one of (A-35) and (A-46) to (A-49) ) Use of a recombinant host according to any one of (A-55) The above (A-30) to (A-34) for producing the composition according to any one of (A-35), (A-46) to (A-49) ) Use of cells identified, sorted, or purified by the method according to any one of (1).
  • (A-56) A method for fluorescentizing DHODH by modifying one or more amino acids present in the interaction region of wild-type DHODH with FMN or DHO.
  • A-58 The method according to any one of (A-56) to (A-57) above, wherein the amino acid modification is modification of at least one of the interaction regions.
  • A-59 The method according to any one of (A-56) to (A-58), wherein the interaction region is present at 12 sites.
  • the above 12 interaction regions are the interaction region between 6 FMNs and DHODH, the interaction region between 4 DHOs and DHODH, and the two interaction regions between FMN and DHO and DHODH.
  • (A-61) Any of the above (A-59) to (A-60), wherein the 12 interaction regions are regions having 12 to 3 consecutive amino acids shown in FIG.
  • the method according to one.
  • (A-62) The amino acid modification described in any one of (A-56) to (A-61) above, which is any one or more of the modified amino acid sequences in each interaction region described in Table 5.
  • Method. (A-63) The amino acid modification described above is a combination of the modified amino acids in each interaction region described in Table 5 with the modified amino acid sequences in two or more interaction regions, (A-56) to (A-62) ).
  • (A-64) The method according to any one of (A-62) to (A-63) above, wherein the amino acid sequence is a combination of modified amino acid sequences described in Table 4.
  • wild-type DHODH is one or more wild-type DHODH selected from animal origin, plant origin, and microorganism origin the method of.
  • the animal-derived wild-type DHODH is one or more wild-type DHODH selected from human-derived, monkey-derived, bovine-derived, rabbit-derived, dog-derived, rat-derived, mouse-derived, and frog-derived, The method according to (A-64) above, wherein the plant is one or more wild-type DHODH selected from Arabidopsis thaliana, rice, corn, and castor bean.
  • A-68 A nucleic acid encoding fluorescent DHODH produced by the method according to any one of (A-57) to (A-66) above.
  • A-69 Any one of (A-67) to (A-68) above, wherein the nucleic acid is operably linked to a nucleic acid encoding an oligopeptide or polypeptide other than fluorescent DHODH. The nucleic acid described in 1.
  • A-70 The nucleic acid according to (A-69) above, wherein the peptide is one or more peptides selected from those localized in the cell, on the cell surface, and outside the cell.
  • A-71 The nucleic acid according to any one of (A-69) to (A-70), wherein the peptide is one or more selected from an antibody, an enzyme, a ligand, a receptor, and a peptide tag. .
  • A-72 A recombinant vector comprising the nucleic acid according to any one of (A-67) to (A-71) above.
  • A-73 A recombinant vector comprising an expression control sequence operably linked to the nucleic acid described in (A-67) to (A-71) above.
  • A-76 The recombinant vector according to (A-75) above, wherein the constitutive promoter is one or more promoters selected from viral promoters and gene promoters derived from animals and plants.
  • the inducible promoter is one or more promoters selected from chemical promoters such as alcohol, steroids, and tetracycline, and physical promoters such as heat, light, and ionizing radiation.
  • a recombinant vector according to 1. (A-78) The recombinant vector according to (A-75) above, wherein the cell / tissue-specific promoter and the developmental stage-specific promoter are one or more promoters selected from the promoters described in Table 1A or 1B .
  • A-81 The fluorescent DHODH or a fusion protein thereof according to (A-80), which is bound to one or more substances selected from nucleic acids, compounds, and other substances other than the fluorescent DHODH.
  • A-82) The fluorescent DHODH or fusion protein thereof according to (A-81) above, wherein the nucleic acid, compound, or other substance labels the protein according to (A-80).
  • A-83) the nucleic acid according to any one of (A-67) to (A-71) above, the recombinant vector according to any one of (A-72) to (A-79) above, And a recombinant host comprising one or more proteins selected from the proteins according to any one of (A-80) to (A-82) above.
  • A-84 The recombinant host according to (A-83) above, wherein the recombinant host is one or more hosts selected from cells, tissues, organisms, and their progeny.
  • A-85 The recombinant host according to (A-84) above, wherein the tissue or its progeny contains the cell.
  • A-86 The recombinant host according to (A-84) above, wherein the organism comprises one or more selected from the cells and the tissue.
  • A-87 The cell or its progeny is one or more cells selected from ES cells, iPS cells, somatic stem cells, other stem cells, and cells derived therefrom or progeny thereof (A-87).
  • the fluorescent protein is not limited to DHODH, and may be, for example, one or more proteins selected from the group consisting of the above-mentioned DHODH and the like.
  • the FMN described in (A-1) to (A-87) may be a cofactor other than FMN, and DHO may be a substrate other than DHO.
  • the interaction regions described in (A-3) to (A-8) can be specified for each protein using any of the existing methods such as the above-described molecular simulation software.
  • Biliverdin reductase is an enzyme present in animals, and generally reduces biliverdin (BV), a heme degradation product decomposed by heme oxygenase, using NAD (P) H as a coenzyme. It is an enzyme that converts to bilirubin (BR). BVR is known to have BVR-A, which is expressed and functioning in adults, and BVR-B, which is expressed and functioning in fetuses. Both convert heme into BR and excrete it from the body. It is thought to function in the process (Cunningham, O. et al, J Biol Chem, 2000, Vol. 275, No. 25, p. 19009-17). It has not been conventionally known that endogenous BVR and exogenous BVR emit near-infrared fluorescence in vivo.
  • fluorescent BVR is a near-reduce that can be stably detected by modifying the amino acids of BVR inherent in all living organisms including humans (including animals, plants, and microorganisms) themselves. Including those that emit external fluorescence. Fluorescent BVR does not necessarily fluoresce itself, but only when a complex with a cofactor or substrate is formed, a protein having such a characteristic that the complex as a whole emits fluorescence. Including.
  • the amino acid modification is, for example, modification of one or more amino acids in the interaction region of BVR that interacts with NAD (P) H or BV.
  • NAD NAD
  • BV NAD
  • the amino acid modification is, for example, modification of one or more amino acids in the interaction region of BVR that interacts with NAD (P) H or BV.
  • the 10 interaction regions confirmed in the prior art document are conserved across species as shown in FIG.
  • the above 10 interaction regions are 6 amino acid regions interacting with NAD (P) H, 3 amino acid regions interacting with BV, and 1 NAD (P) H and BV.
  • the amino acid modification is, for example, modification of at least one of the interaction regions. When modifying a protein that interacts with BV, it is prefer
  • the fluorescent BVR emits near-infrared fluorescence that can be stably detected by the above modification.
  • the modified amino acid sequence is within each interaction region shown in FIG. More preferably, two or more interaction regions are modified, more preferably a combination of modified amino acid sequences described in Table 9 or 10.
  • Example 9 describes experimental data when the BVR-A gene was modified.
  • the amino acid sequence other than the interaction region may be deleted, substituted, inserted, or added as long as it emits near-infrared fluorescence.
  • a signal sequence such as a nuclear translocation signal sequence or a sequence that does not participate in fluorescence generation such as a DNA binding region is included, these sequences may be appropriately deleted or substituted.
  • BVR-A fluorescent in the near-infrared in Example 9 is located at a position where each interaction region is distant, and only a mutation is introduced into 1 to 4 amino acids. The antigenicity is considered to be extremely low.
  • fluorescent BVR and “fluorescent BVR” are an interaction region in BVR that interacts with NAD (P) H, an interaction region in BVR that interacts with BV, or NAD.
  • P NAD
  • P Fluorescence that can stably detect wild-type BVR that cannot stably detect fluorescence by modifying one or more amino acids in the interaction region in BVR that interacts with both H and BVR BVR modified to emit
  • the wild-type BVR derived from the fluorescent BVR may be derived from any biological species.
  • the amino acid sequence and base sequence of BVR derived from such biological species include human (Homo sapiens, Genbank Accession No. NP_000703, NM_000712), monkey (Macaca mulatta, Genbank Accession No. NP_001245073, NM_001258144), chimpanzee (Pan troglodytes, Genbank Accession No. JAA33413, GABE01011326), cattle (Bos taurus, Genbank Accession No.
  • NP_001091040, NM_001097571 rabbits (Oryctolagus cuniculus, Genbank 27 Accession ⁇ ⁇ ⁇ No. XP. NM_053850), mice (Mus musculus, Genbank Accession No. NP_080954, NM_026678), frogs (Xenopus laevis, Genbank Accession No. NP_001108283, NM_001114811), and the like.
  • cloning may be performed by a technique known to those skilled in the art based on the known BVR base sequence or amino acid sequence.
  • One embodiment of the present invention is a modified BVR.
  • This modified BVR preferably has a mutation in, for example, E96A and Y97F.
  • E96A and Y97F the reaction in which BVR reacts with BV to generate BR (red fluorescent material) is suppressed. Therefore, the modified BVR having a mutation in E96A and Y97F can easily detect near-infrared fluorescence stably.
  • B-1 A nucleic acid encoding BVR that has been fluorescentized by modifying one or more amino acids present in the interaction region of wild-type BVR with NAD (P) H or BV.
  • B-2) The nucleic acid according to (B-1), wherein at least one region of the interaction region is modified.
  • B-3) The nucleic acid according to any one of (B-1) to (B-2), wherein there are ten interaction regions.
  • B-4) The above 10 interaction areas are the interaction area between 6 NAD (P) H and BVR, the interaction area between 3 BV and BVR, and 1 NAD (P) H.
  • nucleic acid according to (B-3) above which comprises an interaction region between BV and BVR.
  • the 10 interaction regions are any one of the above (B-3) to (B-4), which is a region having 10 to 3 consecutive amino acids shown in FIG.
  • the nucleic acid according to one is any one of the above (B-1) to (B-5), wherein two or more of the interaction regions described in FIG. 14 are modified.
  • Nucleic acid. (B-7) The nucleic acid according to (B-6), wherein the amino acid sequence is a combination of modified amino acid sequences described in Table 9 or 10.
  • DHODH Dihydroorotate dehydrogenase gene was isolated from a Bacillus strain which is a green fluorescent soil bacterium by wild-type Bacillus DHODH , and the base sequence was determined (FIG. 1, SEQ ID NO: 1).
  • the amino acid sequence obtained from the nucleotide sequence shows about 81% homology with DHODH of Bacillus methanolicus MGA3 and Bacillus cereus G9241. (FIG. 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively).
  • the Bacillus DHODH (bDHODH) gene was subcloned into the bacterial expression vector pGEX4T-1 and the eukaryotic expression vector pCS2 +, and expressed in E. coli DH5 ⁇ strain and human cell HEK293 strain, respectively. As a result, it was confirmed that when Bacillus DHODH was expressed in DH5 ⁇ strain and HEK293 strain, green fluorescence was emitted in both strains (FIG. 3). This result indicates that Bacillus DHODH is a protein that emits green fluorescence.
  • Non-green fluorescent wild-type human DHODH The following experiment was conducted for the purpose of confirming whether wild-type human DHODH (hDHODH) emits green fluorescence similarly to Bacillus DHODH.
  • Example 2 In order to investigate the difference in green fluorescence between Bacillus DHODH and hDHODH, the region of interaction with 12 FMNs or dihydroorotic acid present in DHODH, we compared the homology of the amino acid sequence of Bacillus DHODH with that of hDHODH. (Fig. 6). As a result, humans had 74 amino acids on the N-terminal side that were longer than Bacillus, and although there were few identical amino acids, there were many places where they were substituted with homologous amino acids.
  • FMN dihydroorotate
  • DHO dihydroorotate
  • Example 3 Green fluorescence by amino acid modification of wild-type hDHODH
  • Example 3A Wild-type hDHODH green fluorescence by amino acid modification of the interaction region with FMN or DHO
  • the structure of the interaction region is considered to be important and directly interacts with FMN or DHO.
  • site-directed random mutagenesis was performed one by one in 12 interaction areas. At that time, wild type hDHODH / pGEX4T-1 or ⁇ 1-74 wild type hDHODH / pGEX4T-1 prepared in Reference Example was used. The site-directed random mutagenesis used Takara's prime star mutagenesis basal kit.
  • PCR uses the following primers, and a part of the PCR product is confirmed on an agarose gel band.
  • the annealing temperature was raised by 5 ° C., and when the band did not appear or was thin, the reaction was carried out by lowering the temperature by 5 ° C.
  • the remaining PCR product was transformed into DH5 ⁇ competent cells and overnight at 37 ° C on AmpAL-Broth plates. Fluorescent colonies were screened with a stereoscopic fluorescence microscope, green fluorescent colonies were grown and miniprepped, and plasmid DNA was extracted.
  • Primers used for 12 site-directed random mutagenesis are as follows. Primer for interaction region No.1 HDH-118mu120-U1 (SEQ ID NO: 12), HDH-118mu120-D1 (SEQ ID NO: 13) Primer for interaction region No.2 HDH-143mu145-U1 (SEQ ID NO: 14), HDH-143mu145-D1 (SEQ ID NO: 15) Primer for interaction region No.3 HDH-210mu212-U11 (SEQ ID NO: 16), HDH-210mu212-D1 (SEQ ID NO: 17) Primer for interaction region No.4 HDH-253mu255-U1 (SEQ ID NO: 18), HDH-253mu255-D1 (SEQ ID NO: 19) Primer for interaction region No.5 HDH-282mu284-U1 (SEQ ID NO: 20), HDH-282mu284-D1 (SEQ ID NO: 21) Primer for interaction region No.6 HDH-304mu30
  • Interaction region No.” indicates the site of the interaction region with FMN or DHO shown in FIG. 9, and “Directly interacting amino acids (positions)” indicates FMN shown in FIG. 7 and FIG. The amino acid directly interacting with DHO and the position of the amino acid are shown.
  • the “amino acid sequence of the interaction region” shows the amino acid sequence of each interaction region shown in FIG. 9, and the “amino acid sequence of the interaction region after modification” is The amino acid sequence of the interaction region of the clone that was green-fluorescent by amino acid modification of one interaction region is shown.
  • Table 2 it is shown that any amino acid sequence may be used for a plurality of interaction region amino acid sequences after modification in one interaction region No.
  • hDHODH emits green fluorescence by amino acid modification in at least one of the 12 interaction regions with the FMN or DHO.
  • the amino acid modification also indicates that hDHODH emits green fluorescence by modifying one or more amino acids out of 3 to 7 amino acids in each of the interaction regions.
  • hDHODH This fluorescence of hDHODH is closely related to fluorescence by cofactors or substrates, and the fluorescence mechanism is different from that of the conventionally known GFP family. Moreover, hDHODH has no homology with the GFP family. That is, the fluorescence mechanism found by the present inventors is classified into a category different from the fluorescence mechanism to which the GFP family belongs.
  • Example 3B In order to confirm whether the fluorescence intensity is enhanced by increasing the number of the interaction regions that perform the amino acid modification that enhances the fluorescence intensity, it was obtained from the colony emitting green fluorescence obtained in Example 3A. A mutant plasmid was extracted, and a second site-directed random mutagenesis was performed in the same manner as in Example 3A. The mutant plasmid was extracted from the clone with enhanced fluorescence intensity, and the process of performing the third site-directed random mutagenesis was repeated.
  • “Number of modification sites” indicates the number of interaction regions modified with amino acids
  • “Interaction region No.” indicates the site of the interaction region with FMN or DHO shown in FIG. ”Indicates the combination of the modified interaction region numbers
  • “ Amino acid sequence of the interaction region after modification ” indicates the amino acid sequence of each interaction region of the clone that has been green-fluorinated by amino acid modification of multiple interaction regions.
  • “Fluorescence intensity” indicates the intensity of fluorescence of fluorescent DHODH. Table 4 shows that any one of the interaction regions after modification in one interaction region No. may have any amino acid sequence.
  • interaction regions No. 6 and No. 8 are amino acids that interact with FMN
  • No. 2, No. 11, and No. 12 are amino acids that interact with DHO
  • No. 5 is Contains amino acids that interact with both. Since No.5, No.6, and No.8 interact directly with FMN, which is a green fluorescent substance, the mutated amino acid has strong interaction with FMN that does not naturally emit green fluorescence in hDHODH. It is thought that the FMN is stabilized and the green fluorescence intensity is enhanced.
  • No.2, No.3, No.11, and No.12 are amino acids that interact directly with DHO, not FMN, so they directly affect FMN and participate in green fluorescence. It is hard to think. However, when site-directed random mutagenesis of these 12 interaction regions was performed, mutants emitting green fluorescence with hDHODH could be obtained at all locations.
  • FMN dehydrogenates as a coenzyme to convert DHO to orotic acid FMN and DHO are closest in structure. From these data, the following four possibilities can be considered as the reason why the green fluorescence intensity is enhanced by introducing a mutation in the amino acid in the amino acid region that interacts with DHO. 1. Because FMN and DHO exist in the vicinity, when the amino acid in the amino acid region that interacts with DHO is mutated, the position of DHO in hDHODH is slightly shifted, so the interaction with FMN is strong. Thus, the enhancement of green fluorescence intensity can occur because the FMN is stabilized. 2.
  • Table 5 shows a list of modified amino acid sequences involved in green fluorescence identified by a series of experiments in Example 3.
  • Interaction region No.” indicates the site of the interaction region with FMN or DHO shown in FIG. 9, and “Directly interacting amino acids (positions)” indicates FMN shown in FIG. 7 and FIG. The amino acid directly interacting with DHO and the position of the amino acid are shown.
  • the “amino acid sequence of the interaction region” shows the amino acid sequence of each interaction region shown in FIG. 9, and the “amino acid sequence of the interaction region after modification” is The amino acid sequence of the interaction region of a clone that is green-fluorescent by amino acid modification of one or more interaction regions is shown. Table 5 shows that any one of the interaction regions after modification in one interaction region No. may have any amino acid sequence.
  • Example 3 From the results of Example 3 above, it was revealed that hDHODH emits green fluorescence by amino acid modification in at least one of the 12 interaction regions.
  • the amino acid modification revealed that hDHODH emits green fluorescence by modifying one or more amino acids out of 3 to 7 amino acids in each of the interaction regions. Furthermore, it has been clarified that the fluorescence intensity is enhanced when the number of interaction regions for amino acid modification is increased.
  • wild-type mouse DHODH® did not emit fluorescence.
  • fluorescence intensity was enhanced by increasing the number of interaction regions for green amino acid modification and amino acid modification by amino acid modification at the above 12 sites in mDHODH (Table 6).
  • “number of modified sites” indicates the number of sites of interaction regions modified with amino acids
  • “interaction region No.” indicates the site of the interaction region with FMN or DHO in mDHODH shown in FIG.
  • “Modified amino acid sequence” indicates an amino acid sequence obtained by modifying the amino acid sequence of the interaction region for green fluorescence
  • “Combination” indicates a combination of the modified interaction region numbers
  • “Fluorescence intensity” indicates fluorescence DHODH The intensity of fluorescence is shown.
  • N-terminal deletion region indicates the amino acid region deleted from the N-terminal side of the fluorescent hDHODH
  • Fluorescence intensity indicates the fluorescence intensity of the fluorescent DHODH.
  • the fluorescent hDHODH used was obtained by modifying the interaction region No. 2 with CDP, No. 5 with VGSNT, No. 6 with MVD, and No. 8 with LFSA.
  • full-length fluorescent hDHODH, ⁇ 1-28 fluorescent hDHODH, ⁇ 1-41 fluorescent hDHODH, ⁇ 1-50 fluorescent hDHODH, and ⁇ 1-74 fluorescent hDHODH produced by changing the vector to pCS2 + are expressed in HEK293 cells.
  • the same fluorescence intensity was obtained as when expressed in the DH5 ⁇ strain.
  • green fluorescence was expressed in a granular form in HEK293 cells (FIG. 11). The reason why it is expressed in a granular form is considered to be that this granular green fluorescence is expressed in the outer mitochondrial membrane because the hDHODH protein is originally expressed in the outer mitochondrial membrane.
  • Example 6 Cell selection and cell purification using fluorescent DHODH
  • fluorescent DHODH In order to verify whether target cells can be selected and purified using green fluorescent DHODH, only HEK293 cells and PCS2 + with green fluorescent hDHODH / PCS2 + were introduced. The mixed cells were analyzed using a FACS Calibur (Becton, Dickinson and Company), excitation wavelength 488 nm, fluorescence wavelength 530 ⁇ 15 nm (FL1).
  • fluorescent hDHODH can be used for cell sorting and purification applications.
  • fluorescent hDHODH / pcDNA3.1 myc-His A was prepared, and myc-His tag fusion fluorescent hDHODH was expressed in HEK293 cells. It was confirmed that there was no effect and fluorescence was emitted (FIG. 13).
  • fluorescent hDHODH / pGEX4T-1 was prepared, and GST fusion fluorescent DHODH was expressed with DH5 ⁇ . As a result, green fluorescence was confirmed (data Is not shown).
  • Example 8 As described above, it has been clarified that DHODH emits fluorescence that can be stably detected by modifying the interaction region with FMN or DHO. If this principle is used, it is considered that a fluorescent protein can be prepared for other proteins as long as the protein interacts with a cofactor or a substrate. Therefore, in the following examples, it was examined whether or not the fluorescence that can be stably detected would be generated by modifying the interaction region for the cofactor or substrate of another protein.
  • Example 9 Near-infrared fluorescence by amino acid modification of BVR-A By introducing mutation into the amino acid in the interaction region identified in Example 8, whether or not near-infrared fluorescence is emitted was examined.
  • E96A and Y97F mutant genes of BVR-A were prepared and cloned into pGEX4T-1 vector together with heme oxygenase1 (E96A_Y97F / pGEX4T-1).
  • heme oxygenase1 was introduced to synthesize BVR-A substrate BV from heme present in bacteria.
  • E96A_Y97F / pGEX4T-1 was transformed into bacteria DH5 ⁇ .
  • Random mutation was performed using the E96A_Y97F / pGEX4T-1 as a template. Random mutation was performed by site-directed random mutagenesis using the Takara prime star mutagenesis basal kit in the same manner as the green fluorescence of DHODH shown in Example 3. Random mutation PCR products were transformed into DH5 ⁇ competent cells, and single clones were cultured in L-Broth + Amp liquid medium until stationary phase. Resuspended in 5 mM Tris-HCl (pH 6.8).
  • This suspension was placed in a 96-well plate, and near-infrared fluorescence was detected at an excitation wavelength of 680 nm and a fluorescence wavelength of 720 nm using a near-infrared fluorescence detector ODYSSEY (manufactured by LI-COR).
  • ODYSSEY manufactured by LI-COR
  • “Number of modified sites” in Table 9 indicates the number of interaction regions modified with amino acids, and “Interaction region No.” indicates the site of the interaction region with BV or NAD (P) H shown in FIG.
  • “Modified amino acid sequence” indicates an amino acid sequence obtained by modifying the amino acid sequence of the interaction region for near-infrared fluorescence, and “combination” indicates the modified interaction region No.
  • the “fluorescence intensity” indicates the fluorescence intensity of the fluorescent BVR-A.
  • FIG. 15, and FIG. 16 show that BVR-A is near-infrared fluorescent by altering at least one of the interaction regions with substrate BV or coenzyme NAD (P) H. This indicates that increasing the number of interaction regions for amino acid modification increases the near-infrared fluorescence intensity.
  • Table 10 it is clear that near-infrared fluorescence of BVR-A is possible by modifying other interaction regions.
  • NAD (P) H a coenzyme
  • Tables 11A and B exemplify candidate proteins that can be fluorescentized, but these are merely examples, and are not limited thereto. Proteins that interact with FMN can be converted into fluorescent proteins by modifying amino acids that interact with FMN or cofactor / substrate, as FMN originally emits green fluorescence. Examples of such proteins that interact with FMN are listed in Table 11A or B as examples. FAD emits green fluorescence in the same way as FMN.
  • compounds having a porphyrin ring such as heme also emit fluorescence. Since vitamin A emits yellow-green fluorescence, it interacts with FAD, a compound having a porphyrin ring, and vitamin A.
  • the acting protein is also a candidate protein that can be fluorescentized. Examples of such candidate proteins are listed in Table 11A or B.
  • a protein that interacts with BV does not fluoresce, although BV itself has a porphyrin ring, but, like the near-infrared fluorescent BVR-A produced this time, alters the amino acid that interacts with BV or a cofactor / substrate. These can be converted into fluorescent proteins, and candidates for such proteins that interact with BV are listed in Table 11A or B as examples.
  • cobalamin has a porphyrin ring like BV, it does not fluoresce, but it can be converted into a fluorescent protein by altering the amino acid that interacts with cobalamin or cofactor / substrate. Examples of interacting protein candidates are listed in Table 11A or B.
  • a fluorescent gene derived from a living organism that is, a fluorescent gene derived from a subject can be provided by modifying a gene originally possessed by a living organism including human.
  • the fluorescent gene according to an embodiment of the present invention can be used for known uses used in fluorescent substances such as existing fluorescent genes and fluorescent compounds.
  • the fluorescent gene derived from the subject according to one embodiment of the present invention is very safe and less likely to cause immunotoxicity compared to existing fluorescent genes derived from other species, and is applied to living bodies. It is useful for the application. For example, it can be used as a marker gene when purifying cells such as stem cells.

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Abstract

L'invention concerne une substance fluorescente qui est hautement sûre quand elle est administrée à un corps vivant et est dérivée d'un acide nucléique. On utilise un matériau pour le test de fluorescence, qui comprend au moins une substance biologique sélectionnée dans le groupe consistant en : (a) une protéine modifiée qui est produite en modifiant un résidu d'acide aminé localisé dans une région d'interaction avec un cofacteur ou un substrat, ou une protéine de fusion de celle-ci ; (b) un acide nucléique qui code la protéine modifiée ou la protéine de fusion de celle-ci mentionnée dans le point (a) ; (c) un complexe de protéine à fluorescence conférée de la protéine modifiée ou de la protéine de fusion de celle-ci mentionnée dans le point (a) et un cofacteur ou un substrat ; et (d) une cellule qui porte au moins une substance sélectionnée parmi le groupe consistant en la protéine modifiée mentionnée dans le point (a), l'acide nucléique mentionné dans le point (b) et le complexe de protéine à fluorescence conférée mentionné dans le point (c).
PCT/JP2013/057206 2012-07-31 2013-03-14 Nouvelle substance fluorescente WO2014020933A1 (fr)

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CN108192877A (zh) * 2017-12-29 2018-06-22 天津市湖滨盘古基因科学发展有限公司 一种蛋氨酸亚砜氧化酶mical1突变蛋白及其应用
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CN105567578A (zh) * 2016-01-06 2016-05-11 昆明理工大学 一种灵芝酸高产工程菌株kmust-SE
CN105567578B (zh) * 2016-01-06 2019-02-19 昆明理工大学 一种灵芝酸高产工程菌株kmust-SE
CN108192877A (zh) * 2017-12-29 2018-06-22 天津市湖滨盘古基因科学发展有限公司 一种蛋氨酸亚砜氧化酶mical1突变蛋白及其应用
EP3854803A4 (fr) * 2018-09-07 2022-06-29 Keyangle Life Technology Co., Ltd. Polypeptide ayant une fonction de reconnaissance d'une cible de cellule immunitaire et utilisation associée

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