WO2019187218A1 - Nouvelle protéine, nouveau gène, transformant, procédé de production d'un transformant, et procédé de criblage d'une nouvelle protéine fluorescente - Google Patents

Nouvelle protéine, nouveau gène, transformant, procédé de production d'un transformant, et procédé de criblage d'une nouvelle protéine fluorescente Download PDF

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WO2019187218A1
WO2019187218A1 PCT/JP2018/034338 JP2018034338W WO2019187218A1 WO 2019187218 A1 WO2019187218 A1 WO 2019187218A1 JP 2018034338 W JP2018034338 W JP 2018034338W WO 2019187218 A1 WO2019187218 A1 WO 2019187218A1
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protein
amino acid
base
novel
polynucleotide
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PCT/JP2018/034338
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Japanese (ja)
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晃尚 清水
行大 白鳥
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Ｎｅｃソリューションイノベータ株式会社
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Priority to JP2020508958A priority Critical patent/JP6933409B2/ja
Publication of WO2019187218A1 publication Critical patent/WO2019187218A1/fr

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    • 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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a novel protein, a novel gene, a transformant, a method for producing the transformant, and a method for screening a novel fluorescent protein.
  • Fluorescent proteins such as GFP (Green Fluorescent Protein) and YFP (Yellow Fluorescent Protein) are generally used (Non-Patent Document 1).
  • the fluorescent protein is used for various purposes such as research.
  • fluorescent proteins suitable for various applications do not necessarily exist. For this reason, a new fluorescent protein is required.
  • the novel protein of the present invention is the following protein (F1) or (F2).
  • F1 In the amino acid sequence of SEQ ID NO: 1, A protein comprising an amino acid sequence in which the 170th amino acid is an amino acid other than N (F2)
  • the amino acid sequence comprising the 170th amino acid consists of an amino acid sequence of 80% or more of the amino acid sequence of (F1), and Protein with drought resistance
  • the transformant of the present invention includes the novel gene of the present invention.
  • the novel fluorescent protein screening method of the present invention corresponds to the 170th amino acid in the amino acid sequence of SEQ ID NO: 1 from the protein consisting of the amino acid sequence of SEQ ID NO: 1 or a candidate protein in which a mutation has been introduced into the novel protein of the present invention.
  • the method includes a selection step of selecting a protein having an amino acid other than N and having drying resistance.
  • a novel fluorescent protein can be provided.
  • FIG. 1 is a graph showing the fluorescence intensity of each protein in Example 2.
  • the novel protein of the present invention is the following protein (F1) or (F2).
  • F1 In the amino acid sequence of SEQ ID NO: 1, A protein comprising an amino acid sequence in which the 170th amino acid is an amino acid other than N (F2)
  • the amino acid sequence comprising the 170th amino acid consists of an amino acid sequence of 80% or more of the amino acid sequence of (F1), and Protein with drought resistance
  • the present inventors have found that the 170th amino acid in the protein having the amino acid sequence of SEQ ID NO: 1 is related to the drying resistance of the protein. For this reason, according to the novel protein of the present invention, by setting the 170th amino acid to any amino acid other than N (asparagine), the drying resistance of the protein can be adjusted. For example, the protein is strong even under dry conditions. A fluorescent protein can be obtained. The above estimation does not limit the present invention.
  • novel protein is also referred to as a novel fluorescent protein.
  • novel fluorescent protein is a protein newly prepared by the present inventors.
  • the novel fluorescent protein may include, for example, one type of protein or two or more types of proteins (hereinafter the same).
  • the amino acid sequence of SEQ ID NO: 1 is as follows. In the following amino acid sequence, the amino acid (N) surrounded by a square is the 170th amino acid (asparagine).
  • the protein having the amino acid sequence of SEQ ID NO: 1 is also referred to as CpYGFP, for example.
  • the 170th amino acid in SEQ ID NO: 1 is an amino acid other than N.
  • Amino acids other than N are A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y, preferably D or E.
  • Examples of the protein (F1) include proteins consisting of the amino acid sequences of SEQ ID NOs: 2 and 6.
  • the (F1) is also referred to as the YGFP N170D amino acid sequence and the YGFP N170E amino acid sequence, respectively.
  • amino acid sequences of SEQ ID NOs: 2 and 6 in (F1) are as follows.
  • the amino acid sequences of SEQ ID NOs: 2 and 6 correspond to the case where the 170th amino acid surrounded by a square in the amino acid sequence of SEQ ID NO: 1 is D and E, respectively.
  • the protein of (F2) is, for example, an amino acid sequence in which the 170th amino acid of (F1) is conserved and has 80% or more identity to the amino acid sequence of (F1), and has drought resistance. It can also be said to have a protein.
  • the “identity” may be, for example, a range in which the (F2) has the drying resistance.
  • “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “identity” can be calculated from default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the novel protein of the present invention may be the following protein (F3).
  • F3 A protein having a drought tolerance comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence other than the 170th amino acid of (F1)
  • the 170th amino acid of (F1) is conserved, and in the amino acid sequence of (F1), one or several amino acids are deleted, substituted, inserted and / or added. It can also be referred to as a protein comprising a sequence and having drought resistance.
  • the “identity” may be, for example, a range in which the (F3) has the drying resistance.
  • “1 or several” means, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, ⁇ 5, 1-3, 1 or 2.
  • the numerical range of numbers discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 5” means all disclosures of “1, 2, 3, 4, 5” (hereinafter the same).
  • the novel fluorescent protein of the present invention has drought resistance. Having dry resistance means, for example, that a protein has fluorescence activity even when dried. Drying is, for example, a state in which the hydration water of the protein is relatively reduced as compared with the case where the protein is present in an aqueous solvent.
  • the method for measuring the drying resistance is not particularly limited.
  • the fluorescence intensity is measured before and after drying, and the fluorescence intensity before drying is compared with the fluorescence intensity after drying.
  • the drying resistance of the fluorescent protein can be confirmed.
  • the method for measuring the dry resistance may be performed, for example, according to the measurement method of Example 3 described later.
  • the excitation wavelength of the YGFP N170D is, for example, 400 to 410 nm, and the fluorescence wavelength is, for example, 503 to 513 nm.
  • the excitation wavelength of YGFP N170E is, for example, 401 to 411 nm, and the fluorescence wavelength is, for example, 500 to 510 nm.
  • the measurement method of the excitation wavelength, the excitation maximum wavelength, the fluorescence wavelength, and the fluorescence maximum wavelength is not particularly limited, and can be performed based on, for example, JIS K0120.
  • the measurement method of the excitation wavelength and the fluorescence wavelength may be performed, for example, according to the measurement method of Example 2 described later.
  • the novel fluorescent protein only needs to have drying resistance and fluorescent activity, and may further have other activities, for example.
  • the novel gene of the present invention is characterized by comprising at least one of the following polynucleotides (f1) and (f2).
  • (f1) a polynucleotide in which the 508th base, the 509th base, and the 510th base are mutated to a codon encoding an amino acid other than N in the base sequence of SEQ ID NO: 3
  • (f1) A polynucleotide encoding a protein comprising a nucleotide sequence having an identity of 80% or more and containing 508th base, 509th base, and 510th base, and having drought resistance
  • novel gene is a gene encoding the novel fluorescent protein of the present invention, it is hereinafter also referred to as a novel fluorescent protein gene.
  • novel fluorescent protein gene may contain, for example, one kind of polynucleotide or two or more kinds of polynucleotides (hereinafter the same).
  • the base sequence of SEQ ID NO: 3 in the above (f1) is as follows. In the following base sequence, the base (A) enclosed by a square is the 508th base. The underlined base (G) is the 381st base.
  • the protein encoded by the base sequence of SEQ ID NO: 3 is also referred to as CpYGFP, for example.
  • the polynucleotide consisting of the base sequence of SEQ ID NO: 3 is also referred to as CpYGFP polynucleotide.
  • the 508th base, the 509th base, and the 510th base are codons encoding amino acids other than N.
  • Codons encoding amino acids other than N are A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y.
  • Codon encoding preferably codon encoding D or E.
  • the 508th base is substituted with G, for example.
  • polynucleotide (f1) examples include polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 4 and 7.
  • (f1) is a polynucleotide comprising the nucleotide sequences of SEQ ID NOs: 4 and 7, the (f1) is also referred to as YGFP N170D 'and YGFP N170E polynucleotide.
  • the YGFP N170D ′ and YGFP N170E polynucleotides are polynucleotides encoding the amino acid sequence of YGFP N170D and the amino acid sequence of YGFP N170E, respectively.
  • the base sequences of SEQ ID NOs: 4 and 7 are as follows.
  • the base sequences of SEQ ID NOs: 4 and 7 are the same as those in the base sequence of SEQ ID NO: 3, when the 508th base surrounded by a square is replaced with G, and the 508th base surrounded by a square, This corresponds to the case where the 509th base and the 510th position are substituted with G, A, and G.
  • the underlined base (G) is the 381st base.
  • the polynucleotide (f2) when the 508th base is substituted with G, the polynucleotide (f2) preferably has the 508th base of (f1) conserved.
  • the polynucleotide of (f2) comprises the base sequence having the identity of 80% or more to the base sequence of (f1) in which the 508th base of (f1) is conserved.
  • the base 509, the 509th base, and the 510th base are preferably conserved.
  • the polynucleotide (f2) for example, the 508th base, the 509th base, and the 510th position of the (f1) are conserved, and 80% or more of the base sequence of the (f1) A polynucleotide encoding a protein having a drought tolerance.
  • polynucleotide (f2) examples include a polynucleotide having the base sequence of SEQ ID NO: 5.
  • (f2) is a base sequence consisting of SEQ ID NO: 5
  • the (f2) is also referred to as a YGFP N170D polynucleotide.
  • the YGFP N170D polynucleotide is a polynucleotide encoding the amino acid sequence of YGFP N170D.
  • the base sequence of SEQ ID NO: 5 is as follows.
  • the base sequence of SEQ ID NO: 5 corresponds to the case where the 508th base surrounded by a square and the 381 base underlined in the base sequence of SEQ ID NO: 3 are replaced with G and A, respectively. To do.
  • the novel gene of the present invention may be the following polynucleotides (f3) to (f7).
  • F3 In the base sequence other than the 508th base, the 509th base, and the 510th base, one or several bases are deleted, substituted, inserted and / or Alternatively, a polynucleotide comprising an added base sequence and encoding a protein having drought resistance (f4) From a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (f1) In the complementary sequence, a polynucleotide (f5) encoding a protein having a drought resistance in which bases corresponding to the 508th base, the 509th base, and the 510th base of (f1) are conserved In the amino acid sequence of SEQ ID NO: 1, the 170th amino acid is an amino acid other than N
  • the 508th base, the 509th base, and the 510th base of the (f1) are conserved.
  • one or several bases are stored. It can also be referred to as a polynucleotide that encodes a protein having a base sequence with deletion, substitution, insertion and / or addition, and having drought resistance.
  • “1 or several” may be, for example, within a range in which the protein encoded by the polynucleotide of (f3) has drought tolerance.
  • the “one or several” in (f3) is, for example, 1 to 132, 1 to 99, 1 to 66, 1 to 33, 1 to 26, 1 to 20, 1 to 13, 1-7, 1, 2, 3, or 4.
  • the polynucleotide of (f3) is the above (f1) It is preferable that the 508th base, the 509th base, and the 510th base are conserved.
  • the 508th base, the 509th base, and the 510th base of the (f1) are conserved, and one or several in the base sequence of the (f1)
  • the “one or several”, for example, the above description can be used.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (f1).
  • the hybridization can be detected by, for example, various hybridization assays.
  • the hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
  • the “stringent conditions” may be, for example, any of low stringent conditions, medium stringent conditions, and high stringent conditions.
  • Low stringent conditions are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C.
  • Medium stringent conditions are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C.
  • “High stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
  • the degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like.
  • “Stringent conditions” are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press ( 1989)]] and the like.
  • the polynucleotide (f5) may be any protein as long as the protein encoded by the polynucleotide (f5) has a drought resistance.
  • the base sequence of the polynucleotide of (f5) can be designed, for example, by replacing with the corresponding codon based on the amino acid sequence of SEQ ID NO: 1 and the 170th amino acid.
  • the protein encoded by the polynucleotide (f5) can also be referred to as, for example, the protein (F1) in the novel protein of the present invention, and the description thereof can be used.
  • “one or several” relating to the amino acid sequence may be within a range in which the protein encoded by the polynucleotide of (f6) has a drought tolerance, for example.
  • the “one or several” in (f6) is, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, 1 to 5, One to three, one or two.
  • the protein encoded by the polynucleotide (f6) can also be referred to as the protein (F3) in the novel protein of the present invention, and the description thereof can be used.
  • the “identity” regarding the amino acid sequence may be, for example, within a range in which the protein encoded by the polynucleotide of (f7) has drought tolerance.
  • the identity of (f7) is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the protein encoded by the polynucleotide (f7) can also be referred to as the protein (F2) in the novel protein of the present invention, and the description thereof can be used.
  • the various polynucleotides can be produced by, for example, a known genetic engineering technique or a synthetic technique.
  • the expression vector of the present invention is characterized by including the novel gene of the present invention.
  • the novel protein of the present invention can be easily produced by introducing the expression vector into the host.
  • the description of the novel gene of the present invention can be used for the expression vector of the present invention.
  • the expression vector only needs to contain the polynucleotide functionally so that, for example, the novel protein encoded by the polynucleotide that is the novel gene of the present invention can be expressed. Not limited.
  • the expression vector can be prepared, for example, by inserting the polynucleotide into a backbone vector (hereinafter also referred to as “basic vector”).
  • the type of the vector is not particularly limited, and can be appropriately determined according to the type of the host. Examples of the vector include viral vectors and non-viral vectors.
  • the vector is preferably a binary vector, for example. When transforming a plant, the vector is preferably a T-DNA type binary vector.
  • the vectors include, for example, pBI121 vector, pPZP202 vector, pBINPLUS vector, pBIN19 vector, pBIG2113N, pBIG2113SF, pRI101 DNA vector (manufactured by TaKaRa), pRI201 DNA vector ( TaKaRa), pRI909 DNA vector (TaKaRa), pRI910 DNA vector (TaKaRa), and the like.
  • a microorganism such as E.
  • the vectors include, for example, pETDuet-1 vector (Novagen), pQE-80L vector (QIAGEN), pBluescript II SK vector, pET101 / D-TOPO vector (manufactured by Invitrogen). ), PGEX-6P-1 vector (Amersham Biosciences), pcDNA3.2 / V5-GW / D-TOPO vector (Invitrogen), pEGFP vector, pcDNA3.1-hygro (-) vector (Invitrogen) Etc.
  • pETDuet-1 vector Novagen
  • pQE-80L vector QIAGEN
  • pBluescript II SK vector pET101 / D-TOPO vector
  • PGEX-6P-1 vector Amersham Biosciences
  • pcDNA3.2 / V5-GW / D-TOPO vector Invitrogen
  • pEGFP vector pcDNA3.1-hygro (-) vector (Invitrogen) Etc.
  • the expression vector preferably has a regulatory sequence that regulates, for example, the expression of the polynucleotide and the expression of the novel protein of the present invention encoded by the polynucleotide.
  • the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ori) and the like.
  • the arrangement of the regulatory sequences is not particularly limited.
  • the regulatory sequence may be arranged based on a known method as long as it is functionally regulated, for example, the expression of the polynucleotide and the expression of the novel protein encoded by the polynucleotide. .
  • the regulatory sequence for example, a sequence provided in advance in the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, and the regulatory sequence provided in the basic vector It may be replaced with the regulatory sequence.
  • the expression vector may further include a selection marker coding sequence, for example.
  • a selection marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, and the like.
  • the method for producing the novel protein of the present invention is not particularly limited, and for example, it may be produced by a genetic engineering technique or may be produced by a known synthesis method. In the former case, the production method of the present invention includes an expression step of expressing the novel gene of the present invention. Thereby, the manufacturing method of the novel protein of this invention can manufacture the said novel fluorescent protein of this invention easily, for example.
  • the expression of the polynucleotide which is the novel gene of the present invention may be performed using, for example, the expression vector of the present invention.
  • the method for expressing the polynucleotide is not particularly limited, and a known method can be adopted.
  • a cell-free protein synthesis system or a host may be used.
  • the polynucleotide is preferably expressed in a cell-free protein synthesis system.
  • an expression vector may be used for the expression of the polynucleotide.
  • the cell-free protein synthesis system can be performed by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which the target polynucleotide has been introduced. Reagent kits can be used.
  • the host in which the polynucleotide is introduced it is preferable to use the host in which the polynucleotide is introduced and to express the polynucleotide in the host by culturing the host.
  • a transformant that synthesizes the novel protein of the present invention can be produced by introducing the polynucleotide into a host, and the novel protein of the present invention can be synthesized by culturing the transformant. .
  • Examples of the host include non-human hosts such as microorganisms, plants, animals, insects or cultured cells thereof, isolated human cells or cultured cells thereof, and preferably plants.
  • the plant When the host is a plant, the plant may be a plant body or a part thereof, for example.
  • Examples of the plant part include organs, tissues, cells, and vegetative propagation bodies.
  • Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like.
  • the tissue is, for example, a part of the organ.
  • Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus.
  • the origin of the plant is not particularly limited, and for example, Brassicaceae, Eggplant, Gramineae, Legumes, Rosaceae, Pteridomyceae, Asteraceae, Gentianaceae, Ganodermaceae, Oenaceae, Primula, Cactiaceae, Orchidaceae Department and so on.
  • Brassicaceae include the Arabidopsis genus such as Arabidopsis thaliana .
  • the family Solanaceae for example, tobacco (Nicotiana tabacum) Nicotiana such as petunia (Petunia ⁇ hybrida) petunias genus such as (Petunia), Nierembergia (Nierembergia hippoamanica) Amamodoki genus such as, Calibrachoa (Calibrachoa hybrid Cultivar) such Examples include the genus Calibrachoa.
  • Examples of the Gramineae include a genus of maize such as corn ( Zea mays ) and a genus of rice such as rice ( Oryza sativa ).
  • the legumes include soybean genus such as soybean ( Glycine max ).
  • the rose family for example, the genus Rosa such as roses (Rosa), and the like.
  • Examples of the Nadesico family include Nadesico genus such as carnation ( Dianthus caryophyllus ).
  • the Compositae family for example, chrysanthemum, such as cultivation chrysanthemum (Chrysantemum morifolium), Gerbera (Gerbera cvs.) Gerbera (Oosenbon'yari) genus, etc., and the like.
  • the Gentianaceae includes, for example, Eustoma genus such as Eustoma grandiflorum .
  • Examples of the genus Pleurotus include Torenia ( Torenia tenteri ) Torenia.
  • Examples of the family Oleaceae include Verbena genus such as Verbena ( Garden verbena ).
  • the Primulaceae for example, Cyclamen spp such as cyclamen (Cyclamen persicum) and the like.
  • the cactus family includes, for example, Austrokilondropuntia, Astrophytum, Echinocactus, Echinocereus, Echinopsis, Epiphylam, Opuntia, Crab Cactus, Kamaecereus, Kirindropuntia, Gymnocalycium Cactus cactus genus, Serenicerus genus, Teflocactus genus, Neobox baumia genus, Neoraimondia genus, Nopalea genus, Ferrocactus genus, Mamiglia genus, Melocatus genus, Lipsalis genus, Roseocactus genus, Lofophora genus
  • Phalaenopsis Phalaenopsis cvs.
  • Phalaenopsis Phalaenopsis
  • Cymbidium Cymbidium cvs.
  • Cymbidium Chunlan
  • Nobile system Dendrobium Dendrobium nobile hybrids
  • Denfare system Dendrobium D. phalaenopsis hybrids
  • Oncidium genus such as Oncidium cvs.
  • Cattleya genus such as Cattleya cvs .
  • the microorganism include eukaryotes and prokaryotes.
  • the prokaryotes for example, E.
  • Escherichia coli Escherichia genus such as Pseudomonas putida (Pseudomonas putida) Pseudomonas such as bacteria and the like.
  • the eukaryote include yeasts such as Saccharomyces cerevisiae .
  • the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.
  • a method for introducing the polynucleotide into the host that is, a method for transformation of the host is not particularly limited, and for example, a method using the expression vector may be used, or the expression vector is not used. It may be a known method introduced into. In the latter case, the introduction method can be appropriately set depending on, for example, the type of the host.
  • the introduction method examples include heat shock method, lithium acetate method, introduction method using gene gun such as particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposome, electroporation method, ultrasonic nucleic acid introduction method, DEAE -A dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method using Agrobacterium, and the like.
  • the liposome include lipofectamine and cationic liposome
  • the introduction aid include atelocollagen, nanoparticles, and polymers.
  • the introduction method is preferably an Agrobacterium-mediated method.
  • the polynucleotide which is the novel gene of the present invention may be introduced into the host by the expression vector of the present invention, for example.
  • the method for culturing the host is not particularly limited, and can be appropriately set according to the type of the host.
  • the medium used for culturing the host is not particularly limited, and can be appropriately determined according to the type of the host.
  • the form of the medium used for culturing the host is not particularly limited, and a conventionally known medium such as a solid medium, a liquid medium, or an agar medium can be used as appropriate.
  • the components contained in the medium are not particularly limited.
  • the medium may include a commercially available medium, for example.
  • the commercial medium of the plant is not particularly limited, and examples thereof include Murashige-Skoog (MS) medium.
  • MS Murashige-Skoog
  • the commercially available medium for the plant cell is not particularly limited, and examples thereof include hyponex medium, MS medium, Gamborg B5 (B5) medium, White medium and the like.
  • the commercially available medium for the microorganism is not particularly limited, and examples thereof include LB medium, super broth medium, M9 medium and the like.
  • examples thereof include LB medium, super broth medium, M9 medium and the like.
  • one type of the medium may be used alone, or two or more types may be used in combination.
  • the pH of the medium is not particularly limited, and is, for example, in the range of pH 6 to 8, or in the range of pH 6.5 to 7.5.
  • the method for culturing the host is not particularly limited, and can be appropriately determined according to, for example, the type of the host.
  • examples of the culture method include a method of cultivating the plant in soil and water.
  • examples of the culture method include callus culture, root culture, ovule culture, embryo culture, and the like.
  • the culture temperature at the time of culturing the host is not particularly limited and can be appropriately determined according to, for example, the type of the host.
  • examples of the culture temperature include a plant-growing temperature and an optimum growth temperature.
  • the culture temperature can be, for example, in the range of 15 to 40 ° C., in the range of 30 to 37 ° C.
  • examples of the culture temperature include a plant cell growth temperature and a growth optimum temperature.
  • the culture temperature can be, for example, in the range of 15 to 40 ° C., in the range of 30 to 37 ° C.
  • the host may be cultured under an aerobic condition or an anaerobic condition, for example.
  • the aerobic condition or the anaerobic condition is not particularly limited, and can be set using a conventionally known method.
  • the transformant of the present invention includes the novel gene of the present invention.
  • the transformant of the present invention is characterized by including the novel gene of the present invention, and other configurations and conditions are not particularly limited. Since the transformant of the present invention contains the novel gene of the present invention, it has, for example, drought tolerance.
  • the transformant of the present invention for example, the description of the novel protein of the present invention can be used.
  • the transformant is not particularly limited, and examples thereof include animals and plants, but plants are preferred.
  • examples of the transformant include cancer cells such as human colon cancer cells.
  • the origin of the plant and the plant is not particularly limited, and for example, the above description can be used.
  • the plant may be a plant body or a part thereof, for example.
  • the plant part include organs, tissues, cells, and vegetative propagation bodies.
  • the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like.
  • the tissue is, for example, a part of the organ.
  • Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus.
  • the transformant of the present invention when the transformant is a plant, the transformant of the present invention can be further propagated, for example. At this time, the transformant of the present invention can be used as the propagation material.
  • the propagation material is not particularly limited, and may be the whole or a part of the transformant, for example.
  • examples of the propagation material include seeds, fruits, shoots, stems such as tubers, roots such as tuberous roots, strains, protoplasts, and callus.
  • the propagation method of the transformant of the present invention is not particularly limited, and a known method can be adopted.
  • the breeding method may be, for example, sexual reproduction or asexual reproduction, preferably asexual reproduction.
  • the reproduction by asexual reproduction includes, for example, vegetative propagation and is also called vegetative reproduction.
  • the method of vegetative propagation is not particularly limited, and in the case of the plant body or a part thereof, for example, bud propagation, propagation by cuttings, subdividing from an organ to a plant individual, growth by callus, and the like can be mentioned.
  • the organ for example, the leaves, stems, roots and the like as described above can be used.
  • the vegetative propagation material of the present invention preferably has the same properties as the transformant of the present invention.
  • the vegetative propagation material of the present invention is not particularly limited as in the case of the transformant, and examples thereof include a plant or a part thereof.
  • progeny such as growths grown from the seeds can be obtained. It is preferable that the progeny of the present invention obtains the same properties as the transformant of the present invention.
  • the progeny of the present invention may be, for example, a plant or a part thereof, for example, like the transformant.
  • the transformant of the present invention may be further processed, for example.
  • the kind of the transformant to be processed is not particularly limited, and examples thereof include flowers, leaves, branches and the like.
  • the processed product of the transformant is not particularly limited, and examples thereof include potpourri, dried flowers, dried flowers, preserved flowers, and resin-sealed products obtained by drying flowers, leaves, branches and the like.
  • the processed product of the present invention may be, for example, a processed product of a vegetative propagation body, organ, tissue or cell of the transformant.
  • the method for producing a transformant of the present invention includes an introduction step of introducing the novel gene of the present invention into a host.
  • the method for producing a transformant of the present invention is characterized by including an introduction step for introducing the novel gene of the present invention into a host, and other steps and conditions are not particularly limited. According to the method for producing a transformant of the present invention, for example, the transformant of the present invention can be easily produced.
  • the method for producing a transformant of the present invention may further include an expression step for expressing the novel gene in the host.
  • the method for introducing the novel gene of the present invention into the host is not particularly limited.
  • the description of the method for introducing the polynucleotide into the host in the method for producing the novel protein of the present invention is used. it can.
  • the host is preferably a plant.
  • the expression step is, for example, a step of expressing the novel protein of the present invention from the novel gene of the present invention in a host.
  • the host is preferably, for example, a transformant introduced with the novel gene of the present invention or the expression vector, and the novel protein of the present invention is expressed in the host by culturing the transformant. It is preferable to express.
  • the method for producing a transformant of the present invention may further include a breeding step for breeding the transformant obtained by the introducing step.
  • a breeding method in the breeding process for example, the above description can be used.
  • the method for producing a transformant of the present invention may include a seeding step for seeding from the propagated transformant, and may further include a growth step for growing a growth body from the seed obtained in the seeding step. .
  • the novel fluorescent protein screening method of the present invention is the amino acid of SEQ ID NO: 1 from the candidate protein in which mutation is introduced into the novel protein of the present invention. It includes a selection step in which the 170th amino acid in the sequence is an amino acid other than N and a protein having drought resistance is included, and the other steps and conditions are not particularly limited.
  • a protein having fluorescence activity can be easily screened.
  • the description of the novel protein, novel gene, expression vector, production method and the like of the present invention can be used.
  • the candidate protein is a protein obtained by introducing a mutation into the novel protein of the present invention.
  • the type of mutation introduced into the novel protein is not particularly limited, and examples thereof include amino acid deletion, substitution, insertion and / or addition.
  • the candidate protein is, for example, a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of the novel protein, or the amino acid of the novel protein Examples of the protein include an amino acid sequence having 80% or more identity to the sequence.
  • “1 or several” means, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, 1 to Five, one to three, one or two.
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the candidate protein may be produced by a known protein synthesis method based on the amino acid sequence of the candidate protein, or may be produced by a genetic engineering technique.
  • the screening method of the present invention includes, for example, a nucleotide sequence encoding the novel protein, that is, a mutation step of introducing a mutation into the novel gene of the present invention to produce a mutant polynucleotide, An expression step of expressing the candidate protein.
  • the mutation to be introduced into the novel gene is not particularly limited, and is, for example, base deletion, substitution, insertion and / or addition.
  • the method for introducing a mutation into the novel gene is not particularly limited, and can be carried out, for example, by a known method for introducing a mutation into a polynucleotide. Specific examples include random fragmentation of the polynucleotide of the novel gene of the present invention. Examples include a method of reconstructing a gene by recombination using PCR (so-called DNA shuffling method).
  • the method for expressing the mutant polynucleotide is not particularly limited.
  • the description of the method for producing a novel protein of the present invention described above can be used, and a cell-free protein synthesis system may be used.
  • a host may be used.
  • the host is preferably, for example, a prokaryote, particularly Escherichia coli .
  • the screening method of the present invention may include, for example, a step of purifying the candidate protein obtained in the expression step.
  • the purification method is not particularly limited, and examples thereof include salting out and various column chromatography.
  • the solvent used in the various purification steps is not particularly limited, and for example, a buffer solution can be used.
  • the candidate protein obtained in the expression step may be used as it is in the selection step described later, or a partially purified candidate protein that is partially purified may be used. A single purified candidate protein may be used.
  • the selection step is a step of selecting, from the candidate proteins, a protein having an amino acid corresponding to the 170th amino acid in the amino acid sequence of SEQ ID NO: 1 other than N and having drought resistance.
  • the amino acid corresponding to the 170th amino acid of the selected candidate protein is an amino acid other than N.
  • Amino acids other than N are A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y, preferably D or E.
  • the selected candidate protein has the 170th amino acid substituted with, for example, D or E, and has drying resistance.
  • the method for identifying the amino acid sequence of the candidate protein is not particularly limited, and can be performed by, for example, a known amino acid identification method.
  • the amino acid sequence of the candidate protein may be identified, for example, by replacing the codon of the base sequence of the mutant polynucleotide with the corresponding amino acid sequence.
  • the method for confirming the drying resistance of the candidate protein is not particularly limited, and a known method for confirming drying resistance can be used. For example, it can be confirmed by the same method as the method for measuring the drying resistance.
  • the selection step it is preferable to select a protein having an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 1 and having drought tolerance.
  • the “identity” may be, for example, a range in which the selected candidate protein has the drought tolerance.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • Example 1 It was confirmed that a novel fluorescent protein can be screened by the screening method of the present invention.
  • a mutated polynucleotide was prepared, and the mutated polynucleotide was linked to a vector to construct a mutated polynucleotide expression vector. Specifically, it was performed as follows. First, a CpYGFP polynucleotide (SEQ ID NO: 3) was amplified by PCR (Polymerase Chain Reaction) using the following primer set to obtain a mutant polynucleotide into which mutations were randomly inserted. A PCR reaction solution was prepared according to the instructions using a kit of GeneMorph II Random Mutagenesis Kit (200550, manufactured by Agilent Technologies).
  • Primer set Primer sequence 1 (SEQ ID NO: 8) 5'-ATGCTTCCGGCTCGTATGTTG-3 '
  • Primer sequence 2 (SEQ ID NO: 9) 5'-GTACGGCCGACTAGTAGGCC-3 '
  • the mutant polynucleotide was cleaved with restriction enzymes HindIII and EcoRI and ligated to a pEGFP vector (manufactured by Clonetec) cleaved with the restriction enzyme.
  • a pEGFP vector a vector in which a sequence (CpYGFP vector insertion sequence (SEQ ID NO: 10)) having a His tag sequence added to the 5 'end of a CpYGFP polynucleotide (SEQ ID NO: 3) was used in the EGFP portion.
  • the base sequence of the vector insertion sequence (SEQ ID NO: 10) is as follows.
  • the base sequence enclosed in parentheses is the base sequence corresponding to the CpYGFP polynucleotide, and the underlined base sequence outside the parentheses is the His tag sequence.
  • the vector into which the mutant polynucleotide was inserted was used as a mutant polynucleotide expression vector. In this way, a large number of mutant polynucleotide expression vectors were obtained.
  • the obtained suspension was subjected to ultrasonic treatment using an ultrasonic homogenizer (QSONICA, manufactured by Wakken Pharmaceutical Co., Ltd.) under the conditions of Amp 30% and 1 minute. And the suspension after the said process was centrifuged for 10 minutes on the conditions of 1300 rpm and 4 degreeC, and the obtained supernatant was collect
  • 0.2 ml (50% slurry) of TALON (registered trademark) Metal Affinity Resin (Z5502N, manufactured by TAKARA) was added and reacted at room temperature for 1 hour.
  • the reaction solution was washed with a PBS solution, and then 0.5 ml of a PBS solution containing 300 mM imidazole was added to elute the protein. Then, using Amicon Ultra-0.5 (UFC5010, manufactured by Merck Millipore), the reaction solution was subjected to buffer exchange and replaced with a PBS solution.
  • the purified sample obtained from the transformant was designated as YGFP N170D protein and used in the following experiments.
  • the protein concentration in each purified sample was determined by diluting each sample with a 10% SDS solution and then developing the color using a Pierce BCA Protein Assay Kit (# 23225, manufactured by Pierce), and using a microplate reader (Infinite (registered) (Trademark) M1000Pro, manufactured by TECAN) was used to determine the absorbance at 562 nm.
  • a purified sample containing YGFP N170E protein was prepared from the microbial cells having the novel gene (SEQ ID NO: 7) of the present invention.
  • the YGFP N170D protein and the YGFP N170E protein are each diluted based on the protein concentration obtained in (1) so that the protein concentration in the sample is 2 ⁇ mol / L. did.
  • fluorescence intensity FI
  • 70 ⁇ L of the diluted sample fluorescence intensity (FI) was measured under the following measurement conditions using the microplate reader.
  • BK15 SEQ ID NO: 11
  • the amino acid sequence of SEQ ID NO: 11 is as follows. In the following amino acid sequence, the amino acid (N) surrounded by a square is the 170th amino acid (asparagine).
  • the excitation wavelength (Ex) is 405 nm and the fluorescence wavelength (Em) is 508 nm.
  • the excitation wavelength (Ex) is 406 nm and the fluorescence wavelength (Em) is 505 nm.
  • Table 1 shows the results and the results for the positive control (BK15) when the excitation wavelength (Ex) is 398 nm and the fluorescence wavelength (Em) is 512 nm. As shown in Table 1, YGFP N170D and YGFP N170E showed higher fluorescence activity than BK15. From this result, it was found that the novel fluorescent protein of the present invention has fluorescent activity.
  • Dry resistance was measured using YGFP N170D and YGFP N170E of Example 2. The measurement of drying resistance was performed as follows.
  • the YGFP N170D protein and YGFP N170E protein are diluted based on the protein concentration obtained in Example 2 (1) so that the protein concentration in the sample is 5 and 0.5 ⁇ g / mL, respectively. did.
  • the measurement of drought resistance was performed using a microfiltration apparatus (Bio-Dot (registered trademark), manufactured by Biorad). Three biodot SF filter papers (1620161, manufactured by Biorad) were arranged as filter paper, and a nitrocellulose membrane (162-0147, manufactured by Biorad) was used as a membrane. 500 ⁇ L of each diluted sample (equivalent to 2.5 and 0.25 ⁇ g of the protein, respectively) was dispensed into the wells of the device.
  • FIG. 1 shows the results of photographing the fluorescence of YGFP N170D in Day 1 and Day 7 immediately after blotting.
  • the results of adsorbing 2.5, 0.25 and 0 ⁇ g of the protein in order from the left are shown.
  • the results of YGFP N170D, YGFP N170E and BK15 are shown.
  • YGFP N170D, YGFP N170E, and BK15 bands were detected in the lane where 2.5 ⁇ g of protein was adsorbed.
  • YGFP N170D, YGFP N170E and BK15 bands were slightly detected.
  • Day 1 the bands of YGFP N170D and YGFP N170E were detected in the lane where 2.5 ⁇ g of protein was adsorbed.
  • the BK15 band was slightly detected.
  • Day 7 bands of YGFP N170D and YGFP N170E were detected in the lane where 2.5 ⁇ g of protein was adsorbed.
  • the BK15 band was slightly detected.
  • YGFP N170E had fluorescent activity even after 1 day and 7 days had passed since the start of drying. From the above, it was found that the novel fluorescent protein of the present invention has drought resistance compared to BK15.
  • a novel fluorescent protein can be provided.
  • the present invention can be said to be an extremely useful technique in fields such as the life science field, the medical field, and the agricultural field.
  • (Appendix 1) A novel protein characterized by being a protein of the following (F1) or (F2).
  • F1 In the amino acid sequence of SEQ ID NO: 1, A protein comprising an amino acid sequence in which the 170th amino acid is an amino acid other than N (F2)
  • the amino acid sequence comprising the 170th amino acid consists of an amino acid sequence of 80% or more of the amino acid sequence of (F1), and Protein with drought resistance
  • Appendix 2 The novel protein according to appendix 1, wherein the protein of (F1) is a protein comprising at least one amino acid sequence of SEQ ID NOs: 2 and 6.
  • (Appendix 3) A novel gene comprising at least one of the following polynucleotides (f1) and (f2): (F1) a polynucleotide in which the 508th base, the 509th base, and the 510th base are mutated to a codon encoding an amino acid other than N in the base sequence of SEQ ID NO: 3 (f1) A polynucleotide encoding a protein having a nucleotide sequence of 80% or more identity including the 508th base, the 509th base, and the 510th base, and having a drought resistance (Notes) 4) In the base sequence of SEQ ID NO: 3, The novel gene according to appendix 3, wherein the 508th base, the 509th base, and the 510th base are codons encoding at least one of D and E.
  • (Appendix 5) The novel gene according to appendix 3 or 4, wherein the 381st base is replaced with A.
  • (Appendix 6) The novel gene according to any one of appendices 3 to 5, wherein the polynucleotide of (f1) is a polynucleotide comprising at least one base sequence of SEQ ID NOs: 4 and 7.
  • (Appendix 7) The novel gene according to any one of appendices 3 to 6, wherein the polynucleotide (f2) is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5.
  • (Appendix 8) An expression vector comprising the novel gene according to any one of appendices 3 to 7.
  • (Appendix 9) A transformant comprising the novel gene according to any one of appendices 3 to 7.
  • Appendix 10) The transformant according to appendix 9, wherein the transformant is a plant.
  • (Appendix 11) A method for producing a transformant, comprising an introduction step of introducing the novel gene according to any one of appendices 3 to 7 into a host.
  • (Appendix 12) The method for producing a transformant according to appendix 11, further comprising an expression step of expressing the novel gene in the host.
  • (Appendix 13) The method for producing a transformant according to appendix 11 or 12, wherein the host is a plant.
  • An amino acid corresponding to the 170th amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than N, from a protein comprising the amino acid sequence of SEQ ID NO: 1 or a candidate protein in which a mutation has been introduced into the novel protein of Appendix 1 or 2;
  • the amino acid corresponding to the 170th amino acid in the amino acid sequence of SEQ ID NO: 1 is D or E; The screening method according to appendix 14, wherein a protein having drought resistance is selected.

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Abstract

La présente invention concerne une nouvelle protéine fluorescente. La nouvelle protéine selon la présente invention est caractérisée en ce qu'elle est une protéine (F1) ou (F2). (F1) Une protéine qui comprend une séquence d'acides aminés représentée par SEQ ID NO: 1, où le 170ème acide aminé est un acide aminé autre que N. (F2) Une protéine qui comprend une séquence d'acides aminés ayant 80 % d'identité ou plus comprenant le 170ème acide aminé avec la séquence d'acides aminés de (F1) et qui a une tolérance à la dessiccation.
PCT/JP2018/034338 2018-03-30 2018-09-18 Nouvelle protéine, nouveau gène, transformant, procédé de production d'un transformant, et procédé de criblage d'une nouvelle protéine fluorescente WO2019187218A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100565A1 (fr) * 2004-03-31 2005-10-27 Nec Soft Ltd. Nouvelle proteine fluorescente et gene codant pour cette proteine
WO2016204123A1 (fr) * 2015-06-19 2016-12-22 Ｎｅｃソリューションイノベータ株式会社 Nouvelle protéine, nouveau gène, vecteur d'expression, transformant, procédé de production d'un transformant, et procédé de criblage d'une nouvelle protéine fluorescente

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100565A1 (fr) * 2004-03-31 2005-10-27 Nec Soft Ltd. Nouvelle proteine fluorescente et gene codant pour cette proteine
WO2016204123A1 (fr) * 2015-06-19 2016-12-22 Ｎｅｃソリューションイノベータ株式会社 Nouvelle protéine, nouveau gène, vecteur d'expression, transformant, procédé de production d'un transformant, et procédé de criblage d'une nouvelle protéine fluorescente

Non-Patent Citations (2)

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Title
CHAKRABORTEE, SOHINI ET AL.: "Intrinsically disordered proteins as molecular shields", MOLECULAR BIOSYSTEMS, vol. 8, no. 1, 1 January 2012 (2012-01-01), pages 210 - 219, XP055642196 *
SASAKI， KATSUTOMO ET AL: "Development of "Gleaming Flowers"Focus on improvement of viewing method and practical application", SEIBUTSU-KOGAKU KAISHI, vol. 92, no. 10, pages 545 - 548 *

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