WO2013115482A1 - Fusion protein capable of controlling calcium ion concentration in cells by light, and use thereof - Google Patents

Fusion protein capable of controlling calcium ion concentration in cells by light, and use thereof Download PDF

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WO2013115482A1
WO2013115482A1 PCT/KR2012/010979 KR2012010979W WO2013115482A1 WO 2013115482 A1 WO2013115482 A1 WO 2013115482A1 KR 2012010979 W KR2012010979 W KR 2012010979W WO 2013115482 A1 WO2013115482 A1 WO 2013115482A1
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protein
terminus
stim1
fluorescent protein
fusion protein
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허원도
이상규
경태윤
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한국과학기술원
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to a fusion protein and its use, and more particularly, to a fusion protein capable of controlling intracellular calcium ion concentration by light and its use.
  • Calcium plays a variety of roles as intracellular signal transmitters that regulate numerous cellular functions, for example in many cellular processes such as fertilization, apoptosis, sensory signaling, muscle contraction, motility, exocytosis, and fluid secretion. It plays an important role.
  • the concentration of free calcium ions in the cell is maintained by the coordinated action of various calcium pumps, ion channels and calcium-buffered proteins.
  • the concentration of calcium ions (Ca 2+ ) in the cytoplasm may be due to the release of calcium ions from intracellular organs or calcium from the extracellular space that perform calcium ion accumulation functions such as ER (ER). It is rapidly increased by ion inflow.
  • calcium ionophores In order to regulate the concentration of calcium ions in cells, calcium ionophores have conventionally been used.
  • Representative material of the calcium ionophore is ionomycin, which is derived from Streptomyces conglobatus and increases the intracellular concentration of calcium ions and calcium ions through the biological membrane. It has been used as a research tool to understand the transport of and is used to cause the activation of immune cells such as T cells when used in conjunction with Fourbol 12-Mistrate 13- Acetate (PMA) (Davis, L. and PE Lipsky, J.). Immunol ., 136 (10): 3588-3596, 1986).
  • PMA Fourbol 12-Mistrate 13- Acetate
  • STIM1 stromal interaction molecule 1
  • Orai1 a cell membrane protein and calcium channel forming protein interacting with it
  • STIM1 forms a polymer and reacts with Orai1 present in the cell membrane to allow Orai1 to form a calcium channel to allow calcium ions to enter the cell (FIG. 1).
  • Ionomycin leads to polymer formation of STIM1, leading to calcium influx into cells (FIG. 2).
  • Luik et al. FRB (FKBP-rapamycin-binding) capable of forming heterodimers by rapalogue such as rapamycin in the ER lumen domain of the STIM1 protein complexed by ionomycin.
  • FRB FKBP-rapamycin-binding
  • rapalogue such as rapamycin in the ER lumen domain of the STIM1 protein complexed by ionomycin.
  • recombinant fusion proteins prepared by inserting the FKBP (FK506 binding protein) domain, respectively, into the cell, and then treated with the rapalog, the cytoplasm without calcium ionophore by heterologous complex formation by the rapalog. It has been demonstrated that calcium ion influx into is promoted (Luik et al ., Nature , 454 (7203): 538-542, 2008).
  • the present invention has been made to solve various problems including the above problems, and it is an object of the present invention to provide a fusion protein and its use which can be used in in vitro conditions as well as in vivo conditions and reversibly control intracellular calcium concentration. .
  • these problems are exemplary, and the scope of the present invention is not limited thereby.
  • STIM1 stromal interaction molecule 1
  • the STIM1 protein may be an animal or plant-derived STIM1 protein
  • the animal-derived STIM1 protein may be a mammal-derived STIM1 protein
  • the mammalian-derived STIM1 protein may be a tree, a carnivorous tree, It may be a STIM1 protein from carnivorous, rodent, rabbit, ragweed, base, or lumber.
  • the photoinduced dimer forming protein may be a photoinduced heterodimer forming protein and / or a photoinduced homodimer forming protein.
  • the photoinduced heterodimer-forming protein is CIB (cryptochrome-interacting basic-helix-loop-helix protein), CIBN (N-terminal domain of CIB), Phy (phytochrome), PIF (phytochrome interacting factor), FKF1 (Flavin-binding) , Kelch repeat, F-box 1), GIGANTEA, CRY (chryptochrome) or PHR (phytolyase homolgous region), the homodimer-forming protein may be CRY or PHR.
  • CRY or PHR is known to form homodimers irrespective of light irradiation, but it has been found by the present inventors to form homodimers by light irradiation. Therefore, CRY or PHR is a protein capable of forming not only heterodimers but also homodimers by light irradiation.
  • the fluorescent protein may be further linked.
  • the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced dimer-forming protein, or may be connected to the N-terminus or C-terminus of the photoinduced dimer-forming protein.
  • the fluorescent protein may be a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), an orange fluorescent protein (OFP) , Cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein or tetracysteine motif (tetracystein motif).
  • the green fluorescent protein is enhanced green fluorescent protein (EGFP), Emerald (Tsien, Annu. Rev.
  • red fluorescent protein is mRuby (Kredel et al ., PLoS ONE , 4). (2): e4391, 2009), mApple (Shaner et al ., Nat. Methods , 5 (6): 545-551, 2008), mStrawberry (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), AsRed 2 (Shanner et al ., Nat. Biotehcnol ., 22: 1567-1572, 2004) or mRFP (Campbell et al ., Proc. Natl. Acad. Sci.
  • the orange fluorescent protein is Kusabira Orange (Karawawa et al ., Biochem. J. , 381 (Pt 1): 307-312, 2004), Kusabira Orange 2 (MBL International Corp., Japan), mOrange (2002). Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), mOrange2 (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), dTomato (Shaner et al ., Nat.
  • DsRed2 (Clontech, USA), DsRed-Express (Clontech, USA), DsRed-Monomer (Clontech, USA) or mTangerine (Shaner et al ., Nat. Biotechnol ., 22: 1567 -1572, 2004), wherein the cyan fluorescent protein is ECFP (enhanced cyan fluorescent protein, Cubitt et al ., Trends Biochem. Sci ., 20: 448-455, 1995), mECFP (Ai et al ., Biochem. J.
  • tetracysteine motif has the sequence of Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1).
  • Xaa may be an amino acid other than cysteine.
  • a polynucleotide encoding the fusion protein is provided.
  • a recombinant comprising the polynucleotide.
  • the vector may be a recombinant expression vector capable of expressing the fusion protein.
  • a transformed host cell transformed with the host cell with the vector is provided.
  • the transformed host cell may express the fusion protein in the cytoplasm.
  • a non-human transgenic animal transformed with the vector to express the fusion protein.
  • the non-human transgenic animal may be an insect, a circular animal, a mollusk, a volcano, a linear animal, a tonic, a sponge, an axon, a vertebrate, and the vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal,
  • the insects may be Drosophila
  • the linear animal may be C. elegans
  • the fish may be zebrafish
  • the mammal is a primate, carnivorous, carnivorous, planting It may be a neck, a wood head, a base head or a equipment head, and the mounting neck may be a rat or a mouse.
  • the transgenic plant may be an external plant or a genus plant, the genus plant may be a monocotyledonous plant or a dicotyledonous plant, the monocotyledonous plant may be rice, Lilium or orchidaceae, the dicotyledonous plant is a legume, It may be asteraceae, eggplant, rosaceae or cruciferous, the legume may be soybean, mung bean, pea, or red beans, the gourd may be gourd, watermelon, pumpkin, cucumber, melon or melon, and the asteraceae is asteraceae, May be lettuce, dandelion, garland chrysanthemum or wormwood, the branch may be tobacco, pepper, eggplant, tomato, the rosaceae may be apple, pear, peach, rose or strawberry, the crucifer may be radish, cabbage, rape
  • a photoinduced homodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein Preparing a transformed host cell prepared by transforming the host cell with an expression vector comprising a gene construct in which the polynucleotide encoding the fusion protein to which the forming protein is linked is operably linked to a promoter; Culturing the transformed host cell in a medium containing calcium; And a light irradiation step of irradiating the cultured host cell in culture with light having a wavelength that can induce homodimer formation of the photoinduced homodimer-forming protein. do.
  • STIM1 stromal interaction molecule 1
  • the photoinduced homodimer forming protein may be Cry or PHR.
  • the host cell may be an animal cell or a plant cell, and the animal cell may be derived from an insect, a circular animal, a mollusk, a beetle, a linear animal, a tonic, a sponge, an axon, or a vertebrate.
  • the vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal, the insect may be a Drosophila , the linear animal may be a C. elegans , and the fish may be zebra. It may be a fish (zebrafish), the mammal may be a tree, carnivorous, carnivorous, rodent, lumberjack, base or equipment, the rod may be a rat or mouse.
  • the fusion protein may further comprise a fluorescent protein.
  • the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced homodimer-forming protein, or connected to the C-terminus or N-terminus of the photoinduced homodimer-forming protein.
  • the fluorescent protein is as described above.
  • a photoinduced homodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein Preparing a transgenic plant or a non-human transgenic animal expressing the fusion protein by transforming the polynucleotide encoding the fusion protein to which the forming protein is linked with an expression vector comprising a gene construct operably linked to a promoter; And irradiating a specific organ or tissue of the transgenic plant or a non-human transgenic animal with light having a wavelength that can induce homodimer formation of the photoinduced homodimer-forming protein.
  • a method of reversibly increasing the calcium ion concentration in the cytoplasm in an organ or tissue is provided.
  • the photoinduced homodimer forming protein may be Cry or PHR.
  • the transgenic plant may be an external plant or a genus plant
  • the genus plant may be a monocotyledonous plant or a dicotyledonous plant
  • the monocotyledonous plant may be rice, Lilium or orchidaceae
  • the dicotyledonous plant is a soybean
  • It may be a fruit, fruit, asteraceae, eggplant, rosaceae or cruciferous
  • the legume may be soybean, mung bean, pea, or red beans
  • the fruit may be watermelon, watermelon, pumpkin, cucumber, melon or melon
  • the Asteraceae may be a chrysanthemum, lettuce, dandelion, garland chrysanthemum or mugwort
  • the branch family may be tobacco, pepper, eggplant, tomato
  • the rosaceae may be apple, pear, peach, rose or strawberry
  • the cruciferous radish, Cabbage, rapeseed lampshade, horserad
  • the non-human transgenic animal may be an insect, a circular animal, a mollusk, a volcano, a linear animal, a tonic, a sponge, an axon, a vertebrate, and the vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal,
  • the mammal may be a tree, a carnivorous tree, a carnivorous tree, a planting tree, a lumberjack, a base tree or a tree of equipment.
  • the fusion protein may further comprise a fluorescent protein.
  • the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced homodimer-forming protein, or connected to the N-terminus of the photoinduced homodimer-forming protein.
  • the fluorescent protein is as described above.
  • a photoinduced heterodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A polynucleotide encoding a first fusion protein to which a forming protein is linked, a first expression vector comprising a first gene construct operably linked to a promoter and a N- of the STIM1 protein or of a stromal interaction molecule 1 (STIM1) protein Encoding a second fusion protein linked to the N- or C-terminus of the cytosolic fragment cleaved from the end to the transmembrane domain, with the photoinduced heterodimer-forming protein and a partner protein that forms a heterodimer by irradiation.
  • STIM1 stromal interaction molecule 1
  • a host cell with a second expression vector comprising a second gene construct in which the polynucleotide is operably linked to a promoter Preparing a transformant transformed host cell produced by conversion; Culturing the transformed host cell in a medium containing calcium; And irradiating the cultured host cell in culture with light having a wavelength that can induce heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein.
  • a method of increasing is provided.
  • a photoinduced heterodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A first expression vector and a stromal interaction molecule 1 (STIM1) protein or a N-terminus of the STIM1 protein, wherein the polynucleotide encoding the first fusion protein to which the forming protein is linked comprises a first gene construct operably linked to a promoter A polynucleotide encoding a second fusion protein linked to the N-terminus or C-terminus of the cytosolic fragment cleaved from the transmembrane domain to the transmembrane domain and the partner protein which forms a heterodimer by irradiation with light.
  • STIM1 stromal interaction molecule 1
  • Nucleotide is transformed with a second expression vector comprising a second gene construct operably linked to a promoter Preparing a transgenic plant or non-human transgenic animals; And a light irradiation step of irradiating a specific organ or tissue of the transgenic plant or non-human transgenic animal with light having a wavelength that can induce heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein. Or a method for reversibly increasing the concentration of calcium ions in the cytoplasm in certain organs or tissues of a non-human animal.
  • the first fusion protein and / or the second fusion protein may further comprise a fluorescent protein.
  • the fluorescent protein is inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced heterodimer forming protein and / or the paired protein, or the photoinduced heterodimer forming protein and / or the N-terminus of the paired protein. Or C-terminus.
  • the fluorescent protein included in each of the first fusion protein and the second fusion protein may emit light of different wavelengths, and may be used to determine whether a complex is formed.
  • the fluorescent protein is as described above.
  • the photoinduced heterodimer forming protein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY or PHR.
  • the partner protein is a protein capable of forming heterodimers by photoinduced heterodimer formation protein and light irradiation, and may be CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY or PHR.
  • the heterodimer-forming protein is CRY or PHR, it may be CIB or CIBN, if the photoinduced heterodimer-forming protein is PIF, it is PhyB, and if the photoinduced heterodimer-forming protein is GIGANTEA, it is FKF1.
  • the PIF may be PIF3 or PIF6.
  • the transgenic plant or non-human transgenic animal is as described above.
  • the present inventors prepared a gene construct encoding a fusion protein in which PHR, a photoinduced dimer-forming protein, was linked to the N-terminus of a cytosolic fragment from which a subcellular membrane transmembrane domain of STIM1 was removed (see FIG. 3).
  • PHR a photoinduced dimer-forming protein
  • FIG. 3 When transduced into animal cells, cultured in a medium containing calcium ions and irradiated with light having a wavelength of 450 to 520 nm to induce homodimer formation of PHR, clusters of STIM1 proteins are formed (FIGS. 4 to 4). 6, not only proved that calcium ions were introduced into the cell to increase the concentration of calcium ions in the cytoplasm (see FIGS. 7 and 8), but also by using this to confirm the change in the intracellular location of calcium-responsive proteins (FIG. 6). 9 and 10), the present invention has been completed.
  • the methods and kits of the present invention study the physiological functions of calcium activity, development, cell migration, immune responses and insulin secretion by controlling the concentration of calcium ions in cells in living cells and individuals in time and space. And very useful for preparing model plants and animals for these studies.
  • the calcium ion concentration in the cytoplasm of the cell, animal or plant can be adjusted in time and space reversibly.
  • the scope of the present invention is not limited.
  • FIG. 1 is a schematic diagram showing the mechanism by which the calcium concentration in the cell is regulated by the STIM1 protein and the Orai1 protein interacting with it.
  • Figure 2 is a photograph taken with a fluorescence microscope of the complex formation of STIM1 protein by ionomycin and a graph (b) measuring the number of STIM1 protein complex with time after ionomycin treatment.
  • Figure 3 is a schematic diagram showing the structure of the STIM1 protein (left) and the fusion protein (right) according to an embodiment of the present invention.
  • FIG. 4 is a graph illustrating whether the complex of STIM1 protein is formed by light irradiation after fusion protein expression according to an embodiment of the present invention through FRET (fluorescence resonance energy transfer) analysis.
  • FIG. 5 is a schematic diagram schematically illustrating a process in which calcium ions are introduced into a cell by light irradiation after expressing a fusion protein in a cell according to an embodiment of the present invention.
  • Figure 6 is a fluorescence micrograph comparing the formation of a complex of STIM1 protein before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
  • Figure 7 is a graph comparing the number of STIM1 protein complexes before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
  • FIG. 8 is a fluorescence microscope photograph of complex fusion protein formation (upper panel) and intracellular calcium (lower panel) before and after light irradiation after fusion protein expression according to an embodiment of the present invention.
  • Figure 9 is a graph recording the change in the concentration of calcium ions in the cells by light irradiation after expression of the fusion protein in accordance with an embodiment of the present invention over time.
  • FIG. 10 is a fluorescence microscope photograph of the change in the position of the PKC ⁇ -C2 domain before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
  • FIG. 11 is a graph showing the location of the PKC ⁇ -C2 domain before and after light irradiation after fusion protein expression according to an embodiment of the present invention by recording the fluorescence intensity in the cell membrane.
  • light-induced heterodimerized protein refers to a protein that forms homodimers or paired protein heterodimers when irradiated with light of a particular wavelength.
  • partner protein refers to a protein of interest that interacts with photoinduced heterodimer-forming proteins to form heterodimers when irradiated with light of a particular wavelength.
  • heterodimer means that two different proteins interact to form a complex by interaction.
  • homodimer means that two proteins interact with each other to form a complex.
  • operably linked to means that a particular polynucleotide is linked to another polynucleotide to perform its function.
  • the operably linked polynucleotide encoding a particular protein means that the polynucleotide encoding the specific protein is linked so that it can be transcribed into mRNA and translated into the protein by the action of the promoter.
  • operably linked to a polynucleotide encoding another protein it is meant that the particular protein is linked so that it can be expressed in the form of a fusion protein with another protein.
  • CIB refers to cryptochrome-interacting basic-helix-loop-helix protein and is representative of Arabidopsis CIB1 (GenBank No .: NM_119618). There is).
  • CIBN refers to a site that interacts with cryptochrome (CRY) upon irradiation with light as the N-terminus of the CIB.
  • CRY refers to a kryptochrome protein, typically CRY2 (GenBank No .: NM_100320) of Arabidopsis.
  • PHR refers to a phytolyase homolgous homologous region as the N-terminal portion of the CRY, and interacts with the CIB or CIBN upon irradiation with light (Kennedy et al ., Nat. Methods , 7 (12): 973-975, 2010).
  • Phy refers to a phytochrome protein, and are representative of the Arabidopsis PhyA (GenBank No .: NM_001123784), PhyB (GenBank No .: NM_127435), PIF (phytochrome interacting factor) (Min et al ., Nature, 400: 781-784, 1999)
  • PIF refers to a phytochrome interacting factor, which is representative of the Arabidopsis PIF1 (GenBank No .: NM_001202630), PIF3 (GenBank No .: NM_179295), PIF4 (GenBank No .: NM_180050), PIF5 (GenBank No .: NM_180690), PIF6 (GenBank No .: NM_001203231), or PIF7 (GenBank No .: NM_125520).
  • FKF refers to Flavin-binding, Kelch repeat, F-box proteins, typically FKF1 (GenBank No .: NM_105475) of Arabidopsis. And interact with GIGANTEA proteins upon irradiation with light (Sawa et al ., Science , 318 (5848): 261-265, 2007).
  • GAGANTEA is involved in phytochrome signaling and is known as a protein that regulates flowering time.
  • tetracystein motif is a polypeptide comprising the sequence of Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), Xaa is an amino acid except cysteine, wherein Depending on the type and length of the polypeptide, the fluorescence pattern varies (Adams et al ., J. Am. Chem. Soc ., 124: 6063-6077, 2002).
  • transgenic plant or “transgenic animal” refers to a genetically engineered plant or animal in which a foreign gene has been introduced into the genome to express the foreign gene or to have a specific gene deleted.
  • genetically engineered germ cells in general, but may be prepared by a method for producing a cloned animal by nuclear substitution after genetic manipulation of the somatic cells, in the case of transgenic plants more simply, somatic cells After infection with Agrobacteria containing a foreign gene can be prepared through a process of dedifferentiation and redifferentiation. Methods of making such transgenic animals and transgenic plants are well known in the art (Jaenisch, R and B. Mintz, Proc. Natl. Acad. Sci.
  • FIG. 1 is a schematic diagram showing the mechanism by which the calcium concentration in the cell is regulated by the STIM1 protein and the Orai1 protein interacting with it.
  • the STIM1 protein senses a change in calcium ion concentration in the endoplasmic reticulum (ER) and forms a complex when the calcium ion concentration is lowered.
  • the complexed STIM1 protein interacts with the Orai1 protein, a pore-forming subunit of the calcium channel present in the cell membrane (PM), thereby activating Orai1 to form calcium channels. It will flow into.
  • Figure 2 is a photograph taken with a fluorescence microscope of the complex formation of STIM1 protein by ionomycin and a graph (b) measuring the number of STIM1 protein complex with time after ionomycin treatment.
  • Ionomycin is known to form calcium channels and induce calcium influx by forming complexes of the STIM1 protein.
  • a gene construct encoding a fusion protein linked to a fluorescent protein yellow fluorescent protein (YFP) to the STIM1 protein was prepared and transduced into cells, and then treated with ionomycin, before treatment with ionomycin.
  • YFP fluorescent protein yellow fluorescent protein
  • the fluorescent pattern dispersed in the cytoplasm was changed into the form of the point with strong intensity after the ionomycin treatment, and the formation of the complex of STIM1 protein was confirmed, and the number of strong fluorescent points was rapidly increased immediately after the ionomycin treatment. It was confirmed that gradually decrease as time passes.
  • FIG. 3 is a schematic diagram showing the structure of the STIM1 protein (left) and the fusion protein (right) according to an embodiment of the present invention.
  • STIM1 protein is a single-pass vesicle (ER) membrane protein located on the N-terminus of the ER lumen and on the C-terminus of the cytosol. It contains the EF hand, sterile-alpha-motif (SAM), transmembrane domain (TM domain), two coiled coils (CC), serine / proline-rich regions and lysine-rich regions from the N-terminus (Fig.
  • SAM sterile-alpha-motif
  • TM domain transmembrane domain
  • CC two coiled coils
  • serine / proline-rich regions and lysine-rich regions from the N-terminus
  • the present inventors have removed the portion from the N-terminus to the transmembrane domain of the STIM1 protein, and the removed portion was replaced with PHR (N-terminal portion of Cryptochrome), a dimerizing protein linked to mCerulean, a fluorescent protein.
  • PHR N-terminal portion of Cryptochrome
  • mCerulean-PHR-STIM1-ct Gene constructs encoding fusion proteins (mCerulean-PHR-STIM1-ct) were prepared. When transgene is expressed by transducing animal cells, the expressed fusion protein is present in the cytoplasm because there is no transmembrane domain (to activate the STIM1 fusion protein at the desired time period as described in the fertilization column). is).
  • FIG. 4 is a graph illustrating whether the complex of STIM1 protein is formed by light irradiation after fusion protein expression according to an embodiment of the present invention through FRET (fluorescence resonance energy transfer) analysis.
  • MCerulean-PHR-STIM1 used above was further prepared by constructing a gene construct encoding the fusion protein removed from the N-terminus of STIM1 from the N-terminus of STIM1 and replaced by mCitrine-linked PHR. Cells were transduced with -ct gene constructs to express two fusion proteins and irradiated with light, and then the ratio of the intensity of mCitrine and mCerulean fluorescence was calculated.
  • the fluorescence ratio was 1 before light irradiation, but the value was about 1.1 to 1.2 after light irradiation.
  • FIG. 5 is a schematic diagram schematically illustrating a process in which calcium ions are introduced into a cell by light irradiation after expressing a fusion protein in a cell according to an embodiment of the present invention.
  • fusion proteins including the whole STIM1 protein form a complex
  • calcium ions are introduced into cells by Orai1 protein in cell membranes interacting with STIM1 protein to form pores of calcium channels.
  • Orai1 protein in cell membranes interacting with STIM1 protein to form pores of calcium channels.
  • Figure 6 is a fluorescence micrograph comparing the formation of a complex of STIM1 protein before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention. Fluorescence microscopy images were taken before and after irradiating the cells transformed with the mCerulean-PHR-STIM1-ct gene construct prepared above. As a result, it was found that the fluorescence pattern evenly distributed in the cytoplasm before light irradiation changed to a pattern in which many strong fluorescence points appeared after light irradiation. This shows that the fusion protein according to an embodiment of the present invention formed a complex by light irradiation.
  • Figure 7 is a graph comparing the number of STIM1 protein complexes before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
  • the number of strong fluorescence points shown in the experimental results was regarded as the number of clusters of STIM1 fusion protein and counted. As a result, the number of clusters of the fusion protein was close to zero before light irradiation, but the number of clusters of the fusion protein increased to 70 after light irradiation.
  • FIG. 8 is a fluorescence microscope photograph of complex fusion protein formation (upper panel) and intracellular calcium (lower panel) before and after light irradiation after fusion protein expression according to an embodiment of the present invention.
  • the fluorescent images were taken after treating the cells with Fluo3-AM reagent capable of directly imaging calcium ions.
  • the fluorescence appeared in the form of a strong point in the intracellular portion before light irradiation (lower panel left), but after the light irradiation, the intensity of fluorescence generally increased and the number of strong fluorescence points also increased (bottom).
  • the present inventors have demonstrated that the fusion protein according to an embodiment of the present invention not only forms a complex by light irradiation but also forms a calcium channel to introduce calcium ions into the cytoplasm.
  • Figure 9 is a graph recording the change in the concentration of calcium ions in the cells by light irradiation after expression of the fusion protein in accordance with an embodiment of the present invention over time.
  • the inventors of the present invention after irradiating cells transformed with mCerulean-PHR-STIM1-ct construct and cells transformed with the empty vector, measured intracellular calcium concentration by fluorescence intensity by Fluo3-AM.
  • Fluo3-AM measured intracellular calcium concentration by Fluo3-AM.
  • the fluorescence intensity increased 8-fold after light irradiation, but decreased to a slightly gentle curve with time, whereas mCerulean-PHR-STIM1-ct was expressed. For cells that do not, no change in fluorescence was observed.
  • FIG. 10 is a fluorescence microscope photograph of the change in the position of the PKC ⁇ -C2 domain before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
  • the inventors prepared the mCherry-PHR-STIM1-ct construct and the YFP-PKC ⁇ -C2 domain construct, respectively, and co-transduced the cells, and observed changes in the fluorescence pattern before and after light irradiation.
  • the change in fluorescence by STIM1 fusion protein was the same as the result of FIG. 8, and in the case of PKC ⁇ -C2 domain, the fluorescence pattern was generally dispersed in the cytoplasm before light irradiation (bottom left panel). Fluorescence near the cell membrane was stronger (lower panel right), indicating that the PKC ⁇ -C2 domain had migrated towards the cell membrane.
  • FIG. 11 is a graph showing the location of the PKC ⁇ -C2 domain before and after light irradiation after fusion protein expression according to an embodiment of the present invention by recording the fluorescence intensity in the cell membrane.
  • the migration of PKC ⁇ -C2 to the cell membrane after light irradiation was investigated by measuring the intensity of fluorescence of the yellow fluorescent protein (YFP) linked with PKC ⁇ -C2. As a result, after about 100 seconds after the light irradiation, the fluorescence intensity increased in the cell membrane, and it was found that it was maintained until 300 seconds.
  • YFP yellow fluorescent protein
  • the fusion protein and the method of increasing intracellular calcium ion concentration by light irradiation using the same according to an embodiment of the present invention can be useful for studying various calcium-dependent biochemical reactions dependent on intracellular calcium ion. Able to know.
  • the PHR-STIM1 polynucleotide prepared in Example 1-1 was inserted into the pmCitrine-C1 vector (Clontech, USA) to prepare an mCitrine-PHR-STIM1-ct construct.
  • the mCherry-PHR-STIM1-ct construct was prepared by inserting the PHR-STIM1 polynucleotide prepared in Example 1-1 into the pmCherry-C1 vector (Clonthch, USA).
  • a YFP-PKC ⁇ -C2 construct was prepared by fusing the C2 domain (a.a 156-284) of PKC ⁇ (GenBank No. NM-002739) in front of YFP in a pcDNA3 vector containing a YFP fluorescent protein.
  • HeLa cells were cotransformed with mCerulean-PHR-STIM1-ct and mCitrine-PHR-STIM1-ct prepared in Examples 1-1 and 1-2, respectively, and then in DMEM medium containing 500 ⁇ g / ml of G418. After culturing at 37 ° C. and 10% CO 2 , fluorescence by mCerulean and mCitrine was measured before and after irradiation with light having a wavelength of 450-520 nm, respectively, and the ratio of mCerulean fluorescence intensity / mCitrine fluorescence intensity was calculated (FIG. 4). .
  • the ratio of fluorescence intensity was about 1 before light irradiation, but the value increased after light irradiation, and it rose up and down with an amplitude between about 1.1 and 1.2.
  • This is a typical fluorescence resonance energy transfer (FRET) phenomenon that indirectly demonstrates that two fusion proteins form a complex by light irradiation.
  • FRET fluorescence resonance energy transfer
  • the PHR contained between the two fusion proteins formed a homodimer by irradiation with light, which is a completely different result than that previously reported to form a homodimer regardless of light irradiation.
  • the present inventors first identified that PHR not only forms a heterodimer with CIB when irradiated with light, but also forms a homodimer. Therefore, PHR can be very useful because it can induce complex formation of STIM1 and intracellular influx of calcium ions by itself without using other photoinduced dimer forming proteins.
  • the present inventors transformed HeLa cells with the mCherry-PHR-STIM1-ct construct prepared in Examples 1-3 to confirm that the fusion protein actually formed a complex, and then G500 500 ⁇ g / ml
  • fluorescence microscopy images were obtained before and after irradiation with light having a wavelength of 450-520 nm (FIG. 6).
  • fluorescence by mCherry was uniformly dispersed in the cytoplasm before light irradiation (left), and that fluorescence appeared strongly in the form of many dots after light irradiation (right).
  • the fusion proteins including the fluorescent protein interacted with each other to form a complex.
  • the present inventors regarded the number of increased fluorescence intensity as the number of complexes of the fusion protein and counted it. As a result, the number of complexes per cell near zero before irradiation increased to about 70 after irradiation. 7).
  • the present inventors transformed HeLa cells with mCherry-PHR-STIM1-ct constructs prepared in Examples 1-3 to confirm that the influx of calcium into the cells is induced as the complex of the fusion protein is induced. Then, incubated in DMEM medium containing 500 ⁇ g / ml of G418 at 37 °C, 5% CO 2 conditions, to obtain a fluorescence microscope image before and after irradiation of light of wavelength 450-520 nm (Fig. 8 upper panel), Fluo3-AM (Sigma, USA), which can be imaged, was treated with 4.52 ⁇ g / ml 30 minutes before light irradiation, and the change of the fluorescence pattern was photographed with a fluorescence microscope (Fig.
  • the fluorescence appeared in the form of a strong point in the intracellular portion before light irradiation (lower panel left), but after the light irradiation, the intensity of fluorescence generally increased and the number of strong fluorescence points also increased (bottom).
  • the present inventors have demonstrated that the fusion protein according to an embodiment of the present invention not only forms a complex by light irradiation but also forms a calcium channel to introduce calcium ions into the cytoplasm.
  • the present inventors irradiated the cells transformed with the mCherry-PHR-STIM1-ct construct and HeLa cells transformed with the empty vector (pmCherry-C1 vector), and then measured the intracellular calcium concentration by Fluo3-AM. Measured by fluorescence intensity.
  • fluorescence intensity As a result, as shown in FIG. 9, in the case of cells expressing mCherry-PHR-STIM1-ct, the fluorescence intensity increased about 8-fold after light irradiation, but decreased to a slightly gentle curve over time, whereas mCherry-only For expressing cells, no change in fluorescence was observed. This is to demonstrate that the influx of calcium ions into the cytoplasm according to an embodiment of the present invention is not by the fluorescent protein, but by a fusion protein including PHR and STIM1.
  • the present inventors investigated the behavior of PKC ⁇ -C2 activated by calcium in order to confirm whether intracellular biochemical changes can be investigated by intracellular influx of calcium by the light irradiation. Specifically, after transforming HeLa cells with the YFP-PKC ⁇ -C2 construct prepared in Examples 1-4 and the mCherry-PHR-STIM1-ct construct prepared in Examples 1-3, G418 500 ⁇ g Incubated in DMEM medium containing / ml at 37 °C, 10% CO 2 conditions, to obtain a fluorescence microscope image before and after the irradiation of light of wavelength 450-520 nm (Fig.
  • the fusion protein and the method of increasing intracellular calcium ion concentration by light irradiation using the same according to an embodiment of the present invention can be useful for studying various calcium-dependent biochemical reactions dependent on intracellular calcium ion. Able to know.
  • the concentration of calcium ions in the cytoplasm of a cell, animal or plant can be temporally and reversibly controlled.
  • the scope of the present invention is not limited.

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Abstract

The present invention relates to a method and a kit for reversibly inhibiting a protein function using light-induced nanocluster formation, and provides a fusion protein, wherein a light-induced heterodimerized protein is connected to the N-terminal or C-terminal of stromal interaction molecule 1 (STIM1) protein or to a cytosolic fraction cleaved from the N-terminal of the STIM1 protein to a transmembrane domain.

Description

빛에 의해 세포내 칼슘 이온 농도의 제어가 가능한 융합단백질 및 그의 용도Fusion protein capable of controlling intracellular calcium ion concentration by light and its use
본 발명은 융합단백질 및 그의 용도에 관한 것으로서, 더 상세하게는 빛에 의해 세포내 칼슘 이온 농도의 제어가 가능한 융합단백질 및 그의 용도에 관한 것이다.The present invention relates to a fusion protein and its use, and more particularly, to a fusion protein capable of controlling intracellular calcium ion concentration by light and its use.
칼슘은 수많은 세포 기능을 조절하는 세포내 신호 전달자로서 다양한 역할을 하는데, 예를 들어, 수정, 세포사멸, 감각신호전달, 근육수축, 운동성, 엑소사이토시스, 및 체액 분비와 같은 여러 가지 세포 과정에서 중요한 역할을 수행한다. 세포내 자유 칼슘 이온의 농도는 여러 가지 칼슘 펌프, 이온 채널 및 칼슘-버퍼링 단백질의 조화된 작용에 의해 유지된다. 작용제(agonist)로 자극되었을 때, 세포질 내의 칼슘 이온(Ca2+)의 농도는 소포체(ER)와 같은 칼슘 이온 축적 기능을 수행하는 세포내 기관으로부터의 칼슘 이온의 방출 또는 세포외 공간으로부터의 칼슘 이온 유입에 의해 급속하게 높아진다. Calcium plays a variety of roles as intracellular signal transmitters that regulate numerous cellular functions, for example in many cellular processes such as fertilization, apoptosis, sensory signaling, muscle contraction, motility, exocytosis, and fluid secretion. It plays an important role. The concentration of free calcium ions in the cell is maintained by the coordinated action of various calcium pumps, ion channels and calcium-buffered proteins. When stimulated with an agonist, the concentration of calcium ions (Ca 2+ ) in the cytoplasm may be due to the release of calcium ions from intracellular organs or calcium from the extracellular space that perform calcium ion accumulation functions such as ER (ER). It is rapidly increased by ion inflow.
세포 내 칼슘 이온의 농도를 조절하기 위해, 종래에는 칼슘 이오노포어(ionophore)를 이용하여 왔다. 상기 칼슘 이오노포어로 대표적인 물질은 이오노마이신(ionomycin)이 있는데, 이오노마이신은 스트렙토마이세스 콩글로바투스(Streptomyces conglobatus)에서 유래한 물질로서 칼슘 이온의 세포내 농도를 증가시키고 생물학적 막을 통한 칼슘 이온의 수송을 이해하기 위한 연구수단으로 사용되고 있고, 포볼 12-미리스트레이트 13-아세테이트(PMA)과 함께 사용될 경우 T 세포와 같은 면역세포의 활성화를 야기하는 것으로 있다(Davis, L. and P. E. Lipsky, J. Immunol., 136(10): 3588-3596, 1986). In order to regulate the concentration of calcium ions in cells, calcium ionophores have conventionally been used. Representative material of the calcium ionophore is ionomycin, which is derived from Streptomyces conglobatus and increases the intracellular concentration of calcium ions and calcium ions through the biological membrane. It has been used as a research tool to understand the transport of and is used to cause the activation of immune cells such as T cells when used in conjunction with Fourbol 12-Mistrate 13- Acetate (PMA) (Davis, L. and PE Lipsky, J.). Immunol ., 136 (10): 3588-3596, 1986).
세포 내의 칼슘 이온 농도의 조절은 세포의 소포체 막에 존재하는 막투과 단백질인 STIM1(stromal interaction molecule 1) 그리고 이와 상호작용하는 세포막 단백질이자 칼슘 채널 형성 단백질인 Orai1에 의해 조절된다(Soboloff et al., J. Biol. Chem., 281(30): 20661-20665, 2006). 구체적으로, 세포의 ER 내부의 칼슘 이온의 농도가 낮아지면 STIM1은 중합체를 형성하여 세포막에 존재하는 Orai1과 반응하여 Orai1으로 하여금 칼슘채널을 형성하여 칼슘 이온이 세포 내부로 유입되도록 한다(도 1). 이오노마이신은 STIM1의 중합체 형성을 유발하여, 세포 내로의 칼슘 유입을 유도한다(도 2).Regulation of calcium ion concentration in cells is regulated by stromal interaction molecule 1 (STIM1), a transmembrane protein present in the endoplasmic reticulum membrane of cells, and Orai1, a cell membrane protein and calcium channel forming protein interacting with it (Soboloff et al ., J. Biol. Chem ., 281 (30): 20661-20665, 2006). Specifically, when the concentration of calcium ions in the ER of the cell is lowered, STIM1 forms a polymer and reacts with Orai1 present in the cell membrane to allow Orai1 to form a calcium channel to allow calcium ions to enter the cell (FIG. 1). . Ionomycin leads to polymer formation of STIM1, leading to calcium influx into cells (FIG. 2).
한편, Luik 등은 이오노마이신에 의해 복합체를 형성하는 STIM1 단백질의 ER 루멘 내 도메인에 라파마이신(rapamycin)과 같은 라팔로그(rapalogue)에 의해 헤테로 이합체를 형성할 수 있는 FRB(FKBP-rapamycin-binding) 도메인과 FKBP(FK506 binding protein) 도메인을 각각 삽입하여 제조한 재조합 융합단백질들을 세포 내에서 발현시킨 뒤, 상기 라팔로그를 처리할 경우, 라팔로그에 의한 이형 복합체 형성에 의해 칼슘 이오노포어 없이도 세포질로의 칼슘 이온 유입이 촉진됨을 입증한 바 있다(Luik et al., Nature, 454(7203): 538-542, 2008).On the other hand, Luik et al., FRB (FKBP-rapamycin-binding) capable of forming heterodimers by rapalogue such as rapamycin in the ER lumen domain of the STIM1 protein complexed by ionomycin. ) And recombinant fusion proteins prepared by inserting the FKBP (FK506 binding protein) domain, respectively, into the cell, and then treated with the rapalog, the cytoplasm without calcium ionophore by heterologous complex formation by the rapalog. It has been demonstrated that calcium ion influx into is promoted (Luik et al ., Nature , 454 (7203): 538-542, 2008).
그러나, 종래 방법들은 특정 항생제를 처리하는 방법으로서, 시험관내 조건에서만 사용될 수 있고, 항생제 자체가 비가역적 반응을 유발하거나 가역역인 반응을 유발하더라도 세포에 독성을 미치기 때문에 반복적인 실험이 불가능하다는 단점을 가지고 있다.However, conventional methods are a method for treating specific antibiotics, and can be used only in vitro conditions, and repeated experiments are not possible because antibiotics are toxic to cells even if they cause irreversible or reversible reactions. Have.
본 발명은 상기 문제점을 포함한 다양한 문제점들을 해결하기 위한 것으로서, 시험관 내 조건은 물론 생체 내 조건에서도 사용가능하고, 세포 내 칼슘 농도를 가역적으로 조절할 수 있는 융합 단백질 및 그의 용도를 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.Disclosure of Invention The present invention has been made to solve various problems including the above problems, and it is an object of the present invention to provide a fusion protein and its use which can be used in in vitro conditions as well as in vivo conditions and reversibly control intracellular calcium concentration. . However, these problems are exemplary, and the scope of the present invention is not limited thereby.
본 발명의 일 관점에 따르면, STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이합체 형성 단백질이 연결된 융합단백질이 제공된다.According to one aspect of the present invention, a photoinduced dimer-forming protein at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein. This linked fusion protein is provided.
상기 융합단백질에 있어서, 상기 STIM1 단백질은 동물 또는 식물 유래의 STIM1 단백질일 수 있고, 상기 동물 유래의 STIM1 단백질은 포유동물 유래의 STIM1 단백질일 수 있으며, 상기 포유동물 유래의 STIM1 단백질은 영장목, 식육목, 식충목, 설치목, 토끼목, 우제목, 기제목, 장비목 유래의 STIM1 단백질일 수 있다.In the fusion protein, the STIM1 protein may be an animal or plant-derived STIM1 protein, the animal-derived STIM1 protein may be a mammal-derived STIM1 protein, and the mammalian-derived STIM1 protein may be a tree, a carnivorous tree, It may be a STIM1 protein from carnivorous, rodent, rabbit, ragweed, base, or lumber.
상기 융합단백질에 있어서, 상기 광유도 이합체 형성 단백질은 광유도 이형이합체 형성 단백질 및/또는 광유도 동형이합체 형성단백질일 수 있다. 상기 광유도 이형이합체 형성 단백질은 CIB(cryptochrome-interacting basic-helix-loop-helix protein), CIBN(N-terminal domain of CIB), Phy(phytochrome), PIF(phytochrome interacting factor), FKF1(Flavin-binding, Kelch repeat, F-box 1), GIGANTEA, CRY(chryptochrome) 또는 PHR(phytolyase homolgous region)일 수 있고, 상기 동형이합체 형성 단백질은 CRY 또는 PHR일 수 있다. 종래에는 CRY 또는 PHR은 광조사와 무관하게 동형이합체를 형성하는 것으로 알려졌으나, 본 발명자에 의해 광조사에 의해 동형이합체를 형성하는 것으로 밝혀졌다. 따라서, CRY 또는 PHR은 광조사에 의해 이형이합체를 형성할 뿐만 아니라 동형이합체도 형성할 수 있는 단백질이다.In the fusion protein, the photoinduced dimer forming protein may be a photoinduced heterodimer forming protein and / or a photoinduced homodimer forming protein. The photoinduced heterodimer-forming protein is CIB (cryptochrome-interacting basic-helix-loop-helix protein), CIBN (N-terminal domain of CIB), Phy (phytochrome), PIF (phytochrome interacting factor), FKF1 (Flavin-binding) , Kelch repeat, F-box 1), GIGANTEA, CRY (chryptochrome) or PHR (phytolyase homolgous region), the homodimer-forming protein may be CRY or PHR. Conventionally, CRY or PHR is known to form homodimers irrespective of light irradiation, but it has been found by the present inventors to form homodimers by light irradiation. Therefore, CRY or PHR is a protein capable of forming not only heterodimers but also homodimers by light irradiation.
아울러, 상기 융합단백질에 있어서, 형광단백질이 추가로 연결될 수 있다. 이때, 상기 형광단백질은 상기 STIM1 단백질 또는 상기 세포질성 단편과 상기 광유도 이합체 형성 단백질 사이에 삽입되거나, 상기 광유도 이합체 형성 단백질의 N-말단 또는 C-말단에 연결될 수 있다. 이 때, 상기 형광단백질은 녹색형광단백질(green fluorescent protein, GFP), 황색형광단백질(yellow fluorescent protein, YFP), 적색형광단백질(red fluorescent protein, RFP), 주황형광단백질(orange fluorescent protein, OFP), 청록색형광단백질(cyan fluorescent protein, CFP), 청색형광단백질(blue fluorescent protein, BFP), 원적색형광단백질(far-red fluorescent protein) 또는 테트라시스테인 모티프(tetracystein motif)일 수 있다. 여기서, 상기 녹색형광단백질은 EGFP(enhanced green fluorescent protein), Emerald(Tsien, Annu. Rev. Biochem., 67: 509-544, 1998), Superfolder(Pedelacq et al., Nat. Biotech., 24: 79-88, 2006), GFP(Prendergast et al., Biochem., 17(17): 3448-3453, 1978), Azami Green(Karasawa, et al., J. Biol. Chem., 278: 34167-34171, 2003), TagGFP(Evrogen, Russia), TurboGFP(Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), ZsGreen(Matz et al., Nat. Biotechnol., 17: 969-973, 1999) 또는 T-Sapphire(Zapata-Hommer et al., BMC Biotechnol., 3:5, 2003)일 수 있고, 상기 황색형광단백질은 EYFP(enhanced yellow fluorescent protein, Tsien, Annu. Rev. Biochem., 67: 509-544, 1998), Topaz(Hat et al., Ann. N. Y. Acad. Sci., 1: 627-633, 2002), Venus(Nagai et al., Nat. Biotechnol., 20(1): 87-90, 2002), mCitrine(Griesbeck et al., J. Biol. Chem., 276: 29188-29194, 2001), Ypet(Nguyet and Daugherty, Nat. Biotechnol., 23(3): 355-360, 2005), TagYFP(Evrogen, Russia), PhiYFP(Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), ZsYellow1(Matz et al., Nat. Biotechnol., 17: 969-973, 1999) 또는 mBanana(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004)일 수 있으며, 상기 적색형광단백질은 mRuby(Kredel et al., PLoS ONE, 4(2):e4391, 2009), mApple(Shaner et al., Nat. Methods, 5(6): 545-551, 2008), mStrawberry(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), AsRed2(Shanner et al., Nat. Biotehcnol., 22: 1567-1572, 2004) 또는 mRFP(Campbell et al., Proc. Natl. Acad. Sci. USA, 99(12): 7877-7882, 2002)일 수 있고, 상기 주황형광단백질은 Kusabira Orange(Karawawa et al., Biochem. J., 381(Pt 1): 307-312, 2004), Kusabira Orange2(MBL International Corp., Japan), mOrange(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), mOrange2(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), dTomato(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), dTomato-Tandem(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), TagRFP(Merzlyak et al., Nat. Methods, 4(7): 555-557, 2007), TagRFP-T(Shaner et al., Nat. Methods, 5(6): 545-551, 2008), DsRed(Baird et al., Proc. Natl. Acad. Sci. USA, 97: 11984-11989, 1999) DsRed2(Clontech, USA), DsRed-Express(Clontech, USA), DsRed-Monomer(Clontech, USA) 또는 mTangerine(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004)일 수 있으며, 상기 청록색형광단백질은 ECFP(enhanced cyan fluorescent protein, Cubitt et al., Trends Biochem. Sci., 20: 448-455, 1995), mECFP(Ai et al., Biochem. J., 400(3): 531-540, 2006), mCerulean(Koushik et al., Biophys. J., 91(12): L99-L101, 2006), CyPet(Nguyet and Daugherty, Nat. Biotechnol., 23(3): 355-360, 2005), AmCyan1(Matz et al., Nat. Biotechnol., 17: 969-973, 1999), Midori-Ishi Cyan(Karawawa et al., Biochem. J., 381(Pt 1): 307-312, 2004), TagCFP(Evrogen, Russia) 또는 mTFP1(Ai et al., Biochem. J., 400(3): 531-540, 2006)일 수 있고, 상기 청색형광단백질은 EBFP(enhanced blue fluorescent protein, Clontech, USA), EBFP2(Ai et al., Biochemistry, 46(20): 5904-5910, 2007), Azurite(Mena et al., Nat. Biotechnol., 24: 1569-1571, 2006) 또는 mTagBFP(Subach et al., Chem. Biol., 15(10: 1116-1124, 2008)일 수 있고, 상기 원적색형광단백질은 mPlum(Wang et al., Proc. Natl. Acad. Sci. USA, 101: 16745-16749, 2004), mCherry(Shanner et al., Nat. Biotehcnol., 22: 1567-1572, 2004), dKeima-Tandem(Kogure et al., Methods, 45(3): 223-226, 2008), JRed(Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), mRaspberry(Shanner et al., Nat. Biotehcnol., 22: 1567-1572, 2004), HcRed1(Fradkov et al., Biochem. J., 368(Pt 1): 17-21, 2002), HcRed-Tandem(Fradkov et al., Nat. Biotechnol., 22(3): 289-296, 2004) 또는 AQ143(Shkrob et al., Biochem. J., 392: 649-654, 2005)일 수 있으며, 상기 테트라시스테인 모티프는 Cys-Cys-Xaa-Xaa-Cys-Cys(서열번호 1)의 서열을 포함하는 폴리펩티드로서, 상기 Xaa는 시스테인을 제외한 아미노산일 수 있다.In addition, in the fusion protein, the fluorescent protein may be further linked. In this case, the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced dimer-forming protein, or may be connected to the N-terminus or C-terminus of the photoinduced dimer-forming protein. In this case, the fluorescent protein may be a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), an orange fluorescent protein (OFP) , Cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein or tetracysteine motif (tetracystein motif). Here, the green fluorescent protein is enhanced green fluorescent protein (EGFP), Emerald (Tsien, Annu. Rev. Biochem ., 67: 509-544, 1998), Superfolder (Pedelacq et al ., Nat. Biotech ., 24: 79 -88, 2006), GFP (Prendergast et al ., Biochem ., 17 (17): 3448-3453, 1978), Azami Green (Karasawa, et al ., J. Biol. Chem ., 278: 34167-34171, 2003), TagGFP (Evrogen, Russia), TurboGFP (Shagin et al ., Mol. Biol. Evol ., 21 (5): 841-850, 2004), ZsGreen (Matz et al ., Nat. Biotechnol ., 17: 969-973, 1999) or T-Sapphire (Zapata-Hommer et al ., BMC Biotechnol ., 3: 5, 2003), wherein the yellow fluorescent protein is an enhanced yellow fluorescent protein, Tsien, Annu. Rev. Biochem ., 67: 509-544, 1998), Topaz (Hat et al ., Ann. NY Acad. Sci ., 1: 627-633, 2002), Venus (Nagai et al ., Nat. Biotechnol ., 20 ( 1): 87-90, 2002), mCitrine (Griesbeck et al ., J. Biol. Chem ., 276: 29188-29194, 2001), Ypet (Nguyet and Daugherty, Nat. Biotechnol ., 23 (3): 355 -360, 2005), TagYFP (Evrogen, Russia), PhiYFP (Shagin e t al ., Mol. Biol. Evol ., 21 (5): 841-850, 2004), Zs Yellow 1 (Matz et al ., Nat. Biotechnol. , 17: 969-973, 1999) or mBanana (Shaner et al., Nat. Biotechnol ., 22: 1567-1572, 2004), wherein the red fluorescent protein is mRuby (Kredel et al ., PLoS ONE , 4). (2): e4391, 2009), mApple (Shaner et al ., Nat. Methods , 5 (6): 545-551, 2008), mStrawberry (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), AsRed 2 (Shanner et al ., Nat. Biotehcnol ., 22: 1567-1572, 2004) or mRFP (Campbell et al ., Proc. Natl. Acad. Sci. USA , 99 (12): 7877-7882, 2002), wherein the orange fluorescent protein is Kusabira Orange (Karawawa et al ., Biochem. J. , 381 (Pt 1): 307-312, 2004), Kusabira Orange 2 (MBL International Corp., Japan), mOrange (2002). Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), mOrange2 (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), dTomato (Shaner et al ., Nat. Biotechnol , 22: 1567-1572, 2004), dTomato-Tandem (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), TagRFP (Merzlyak et al ., Nat. Methods , 4 (7): 555-557, 2007), TagRFP-T from Shaner et al ., Nat . Methods , 5 (6): 545-551, 2008), DsRed (Baird et al ., Proc. Natl. Acad. Sci. USA , 97: 11984-11989, 1999) DsRed2 (Clontech, USA), DsRed-Express (Clontech, USA), DsRed-Monomer (Clontech, USA) or mTangerine (Shaner et al ., Nat. Biotechnol ., 22: 1567 -1572, 2004), wherein the cyan fluorescent protein is ECFP (enhanced cyan fluorescent protein, Cubitt et al ., Trends Biochem. Sci ., 20: 448-455, 1995), mECFP (Ai et al ., Biochem. J. , 400 (3): 531-540, 2006), mCerulean (Koushik et al ., Biophys. J. , 91 (12): L99-L101, 2006), CyPet (Nguyet and Daugherty, Nat. Biotechnol ., 23 (3): 355-360, 2005), AmCyan 1 (Matz et al ., Nat. Biotechnol ., 17: 969-973, 1999), Midori-Ishi Cyan (Karawawa et al ., Biochem. J. , 381 ( Pt 1): 307-312, 2004), TagCFP (Evrogen, Russia) or mTFP1 (Ai et al ., Biochem. J. , 400 (3): 531-540, 2006), wherein the blue fluorescent protein is Enhanced blue fluorescent protein (EBFP), Clontech, USA), EBFP2 (Ai et al ., Biochemistry , 46 (20): 5904-5910, 2007), Azurite (Mena et al ., Nat. Biotechnol ., 24: 1569-1571 , 2006) or mTagBFP (Subach et al ., Chem. Biol ., 15 (10: 1116-1124, 2008), wherein the primary red fluorescent protein is mPlum (Wang et al ., Proc. Natl. Acad. Sci. USA , 101: 16745-16749, 2004), mCherry (Shanner et al ., Nat. Biotehcnol ., 22: 1567-1572, 2004), d Keima-Tandem (Kogure et al ., Methods, 45 (3): 223- 226, 2008), JRed (Shagin et al ., Mol. Biol. Evol ., 21 (5): 841-850, 2004), mRaspberry (Shanner et al ., Nat. Biotehcnol ., 22: 1567-1572, 2004 ), HcRed 1 (Fradkov et al ., Biochem. J. , 368 (Pt 1): 17-21, 2002), HcRed-Tandem (Fradkov et al ., Nat. Biotechnol ., 22 (3): 289-296, 2004) or AQ143 (Shkrob et al ., Biochem. J. , 392: 649-654, 2005), wherein the tetracysteine motif has the sequence of Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1). As a polypeptide comprising, Xaa may be an amino acid other than cysteine.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 암호화하는 폴리뉴클레오티드가 제공된다. According to another aspect of the present invention, a polynucleotide encoding the fusion protein is provided.
본 발명의 다른 일 관점에 따르면, 상기 폴리뉴클레오티드를 포함하는 재조합 제공된다. 상기 벡터는 상기 융합단백질을 발현할 수 있는 재조합 발현벡터일 수 있다.According to another aspect of the invention, there is provided a recombinant comprising the polynucleotide. The vector may be a recombinant expression vector capable of expressing the fusion protein.
본 발명의 다른 일 관점에 따르면, 상기 벡터로 숙주 세포를 형질전환시킨 형질전환 숙주세포가 제공된다. 상기 형질전환 숙주세포는 상기 융합단백질을 세포질 내에 발현할 수 있다.According to another aspect of the present invention, a transformed host cell transformed with the host cell with the vector is provided. The transformed host cell may express the fusion protein in the cytoplasm.
본 발명의 다른 일 관점에 따르면, 상기 벡터로 형질전환되어 상기 융합단백질을 발현하는 비인간 형질전환 동물이 제공된다.According to another aspect of the invention, there is provided a non-human transgenic animal transformed with the vector to express the fusion protein.
상기 비인간 형질전환 동물은 곤충류, 환형동물, 연체동물, 완족류, 선형동물, 강장동물, 해면류, 축색류, 척추동물일 수 있고 상기 척추동물은 어류, 양서류, 파충류, 조류 또는 포유류일 수 있으며, 상기 곤충류는 초파리(Drosophila)일 수 있고, 상기 선형동물은 예쁜꼬마선충(C. elegans)일 수 있으며, 상기 어류는 지브라피쉬(zebrafish)일 수 있고, 상기 포유류는 영장목, 식육목, 식충목, 설치목, 우제목, 기제목 또는 장비목일 수 있고, 상기 설치목은 래트 또는 마우스일 수 있다.The non-human transgenic animal may be an insect, a circular animal, a mollusk, a volcano, a linear animal, a tonic, a sponge, an axon, a vertebrate, and the vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal, The insects may be Drosophila , the linear animal may be C. elegans , the fish may be zebrafish, the mammal is a primate, carnivorous, carnivorous, planting It may be a neck, a wood head, a base head or a equipment head, and the mounting neck may be a rat or a mouse.
본 발명의 다른 일 관점에 따르면, 상기 벡터로 형질전환되어 상기 융합단백질을 발현하는 형질전환 식물이 제공된다. 상기 형질전환 식물은 겉씨식물 또는 속씨식물일 수 있고, 상기 속씨식물은 외떡잎 식물 또는 쌍떡잎 식물일 수 있으며, 상기 외떡잎 식물은 벼과, 나리과 또는 난초과일 수 있고, 상기 쌍떡잎 식물은 콩과, 박과, 국화과, 가지과, 장미과 또는 십자화과일 수 있으며, 상기 콩과는 대두, 녹두, 완두, 또는 팥일 수 있고, 상기 박과는 박, 수박, 호박, 오이, 멜론 또는 참외일 수 있으며, 상기 국화과는 국화, 상추, 민들레, 쑥갓 또는 쑥일 수 있고, 상기 가지과는 담배, 고추, 가지, 토마토일 수 있으며, 상기 장미과는 사과, 배, 복숭아, 장미 또는 딸기일 수 있고, 상기 십자화과는 무우, 배추, 유채, 갓, 고추냉이(horseradish) 또는 애기장대(Arabidopsis thaliana)일 수 있다.According to another aspect of the invention, there is provided a transgenic plant transformed with the vector to express the fusion protein. The transgenic plant may be an external plant or a genus plant, the genus plant may be a monocotyledonous plant or a dicotyledonous plant, the monocotyledonous plant may be rice, Lilium or orchidaceae, the dicotyledonous plant is a legume, It may be asteraceae, eggplant, rosaceae or cruciferous, the legume may be soybean, mung bean, pea, or red beans, the gourd may be gourd, watermelon, pumpkin, cucumber, melon or melon, and the asteraceae is asteraceae, May be lettuce, dandelion, garland chrysanthemum or wormwood, the branch may be tobacco, pepper, eggplant, tomato, the rosaceae may be apple, pear, peach, rose or strawberry, the crucifer may be radish, cabbage, rapeseed, fresh , Horseradish or Arabidopsis thaliana .
본 발명의 다른 일 관점에 따르면, STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 동형이합체 형성 단백질이 연결된 융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 유전자컨스트럭트를 포함하는 발현벡터로 숙주세포를 형질전환하여 제조된 형질전환 숙주세포를 준비하는 단계; 상기 형질전환 숙주세포를 칼슘을 포함하는 배지에서 배양하는 배양단계; 및 상기 배양중인 형질전환 숙주세포에 상기 광유도 동형이합체 형성 단백질의 동형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 세포질 내의 칼슘 이온 농도를 가역적으로 증가시키는 방법이 제공된다.According to another aspect of the present invention, a photoinduced homodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein Preparing a transformed host cell prepared by transforming the host cell with an expression vector comprising a gene construct in which the polynucleotide encoding the fusion protein to which the forming protein is linked is operably linked to a promoter; Culturing the transformed host cell in a medium containing calcium; And a light irradiation step of irradiating the cultured host cell in culture with light having a wavelength that can induce homodimer formation of the photoinduced homodimer-forming protein. do.
상기 방법에 있어서, 상기 광유도 동형이합체 형성 단백질은 Cry 또는 PHR일 수 있다.In the method, the photoinduced homodimer forming protein may be Cry or PHR.
상기 방법에 있어서, 상기 숙주세포는 동물세포 또는 식물세포일 수 있고, 상기 동물세포는 곤충류, 환형동물, 연체동물, 완족류, 선형동물, 강장동물, 해면류, 축색류, 척추동물 유래의 것일 수 있고, 상기 척추동물은 어류, 양서류, 파충류, 조류 또는 포유류일 수 있으며, 상기 곤충류는 초파리(Drosophila)일 수 있고, 상기 선형동물은 예쁜꼬마선충(C. elegans)일 수 있으며, 상기 어류는 지브라피쉬(zebrafish)일 수 있고, 상기 포유류는 영장목, 식육목, 식충목, 설치목, 우제목, 기제목 또는 장비목일 수 있고, 상기 설치목은 래트 또는 마우스일 수 있다.. In the above method, the host cell may be an animal cell or a plant cell, and the animal cell may be derived from an insect, a circular animal, a mollusk, a beetle, a linear animal, a tonic, a sponge, an axon, or a vertebrate. The vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal, the insect may be a Drosophila , the linear animal may be a C. elegans , and the fish may be zebra. It may be a fish (zebrafish), the mammal may be a tree, carnivorous, carnivorous, rodent, lumberjack, base or equipment, the rod may be a rat or mouse.
상기 방법에 있어서, 상기 융합단백질은 추가로 형광단백질을 포함할 수 있다. 이때, 상기 형광단백질은 상기 STIM1 단백질 또는 상기 세포질성 단편과 상기 광유도 동형이합체 형성 단백질 사이에 삽입되거나, 상기 광유도 동형이합체 형성 단백질의 C-말단 또는 N-말단에 연결될 수 있다. 상기 형광단백질은 상술한 바와 같다.In the method, the fusion protein may further comprise a fluorescent protein. In this case, the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced homodimer-forming protein, or connected to the C-terminus or N-terminus of the photoinduced homodimer-forming protein. The fluorescent protein is as described above.
본 발명의 다른 일 관점에 따르면, STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 동형이합체 형성 단백질이 연결된 융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 유전자컨스트럭트를 포함하는 발현벡터로 형질전환되어 상기 융합단백질을 발현하는 형질전환 식물 또는 비인간 형질전환 동물를 준비하는 단계; 및 상기 형질전환 식물 또는 비인간 형질전환 동물의 특정 기관 또는 조직에 상기 광유도 동형이합체 형성 단백질의 동형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 식물 또는 비인간 동물의 특정 기관 또는 조직에서의 세포질 내 칼슘 이온 농도를 가역적으로 증가시키는 방법이 제공된다.According to another aspect of the present invention, a photoinduced homodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein Preparing a transgenic plant or a non-human transgenic animal expressing the fusion protein by transforming the polynucleotide encoding the fusion protein to which the forming protein is linked with an expression vector comprising a gene construct operably linked to a promoter; And irradiating a specific organ or tissue of the transgenic plant or a non-human transgenic animal with light having a wavelength that can induce homodimer formation of the photoinduced homodimer-forming protein. A method of reversibly increasing the calcium ion concentration in the cytoplasm in an organ or tissue is provided.
상기 방법에 있어서, 상기 광유도 동형이합체 형성 단백질은 Cry 또는 PHR일 수 있다.In the method, the photoinduced homodimer forming protein may be Cry or PHR.
상기 방법에 있어서, 상기 형질전환 식물은 겉씨식물 또는 속씨식물일 수 있고, 상기 속씨식물은 외떡잎 식물 또는 쌍떡잎 식물일 수 있으며, 상기 외떡잎 식물은 벼과, 나리과 또는 난초과일 수 있고, 상기 쌍떡잎 식물은 콩과, 박과, 국화과, 가지과, 장미과 또는 십자화과일 수 있으며, 상기 콩과는 대두, 녹두, 완두, 또는 팥일 수 있고, 상기 박과는 박, 수박, 호박, 오이, 멜론 또는 참외일 수 있으며, 상기 국화과는 국화, 상추, 민들레, 쑥갓 또는 쑥일 수 있고, 상기 가지과는 담배, 고추, 가지, 토마토일 수 있으며, 상기 장미과는 사과, 배, 복숭아, 장미 또는 딸기일 수 있고, 상기 십자화과는 무우, 배추, 유채, 갓, 고추냉이(horseradish) 또는 애기장대(Arabidopsis thaliana)일 수 있다. 상기 비인간 형질전환 동물은 곤충류, 환형동물, 연체동물, 완족류, 선형동물, 강장동물, 해면류, 축색류, 척추동물일 수 있고, 척추동물은 어류, 양서류, 파충류, 조류 또는 포유류일 수 있으며, 상기 포유류는 영장목, 식육목, 식충목, 설치목, 우제목, 기제목 또는 장비목일 수 있다. In the method, the transgenic plant may be an external plant or a genus plant, the genus plant may be a monocotyledonous plant or a dicotyledonous plant, the monocotyledonous plant may be rice, Lilium or orchidaceae, and the dicotyledonous plant is a soybean It may be a fruit, fruit, asteraceae, eggplant, rosaceae or cruciferous, the legume may be soybean, mung bean, pea, or red beans, the fruit may be watermelon, watermelon, pumpkin, cucumber, melon or melon, The Asteraceae may be a chrysanthemum, lettuce, dandelion, garland chrysanthemum or mugwort, the branch family may be tobacco, pepper, eggplant, tomato, the rosaceae may be apple, pear, peach, rose or strawberry, the cruciferous radish, Cabbage, rapeseed, lampshade, horseradish or Arabidopsis thaliana . The non-human transgenic animal may be an insect, a circular animal, a mollusk, a volcano, a linear animal, a tonic, a sponge, an axon, a vertebrate, and the vertebrate may be a fish, an amphibian, a reptile, a bird or a mammal, The mammal may be a tree, a carnivorous tree, a carnivorous tree, a planting tree, a lumberjack, a base tree or a tree of equipment.
상기 방법에 있어서, 상기 융합단백질은 추가로 형광단백질을 포함할 수 있다. 이때, 상기 형광단백질은 상기 STIM1 단백질 또는 상기 세포질성 단편과 상기 광유도 동형이합체 형성 단백질 사이에 삽입되거나, 상기 광유도 동형이합체 형성 단백질의 N-말단에 연결될 수 있다. 상기 형광단백질은 상술한 바와 같다.In the method, the fusion protein may further comprise a fluorescent protein. In this case, the fluorescent protein may be inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced homodimer-forming protein, or connected to the N-terminus of the photoinduced homodimer-forming protein. The fluorescent protein is as described above.
본 발명의 다른 일 관점에 따르면, STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이형이합체 형성 단백질이 연결된 제1융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제1유전자컨스트럭트를 포함하는 제1발현벡터 및 STIM1(stromal interaction molecule 1) 단백질의 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 상기 광유도 이형이합체 형성 단백질과 광조사에 의해 이형이합체를 형성하는 짝 단백질이 연결된 제2융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제2유전자컨스트럭트를 포함하는 제2발현벡터로 숙주세포를 형질전환하여 제조된 형질전환 숙주세포를 준비하는 단계; 상기 형질전환 숙주세포를 칼슘을 포함하는 배지에서 배양하는 배양단계; 및 상기 배양중인 형질전환 숙주세포에 상기 광유도 이형이합체 형성 단백질과 상기 짝 단백질 사이의 이형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 세포질 내의 칼슘 이온 농도를 가역적으로 증가시키는 방법이 제공된다.According to another aspect of the invention, a photoinduced heterodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A polynucleotide encoding a first fusion protein to which a forming protein is linked, a first expression vector comprising a first gene construct operably linked to a promoter and a N- of the STIM1 protein or of a stromal interaction molecule 1 (STIM1) protein Encoding a second fusion protein linked to the N- or C-terminus of the cytosolic fragment cleaved from the end to the transmembrane domain, with the photoinduced heterodimer-forming protein and a partner protein that forms a heterodimer by irradiation. A host cell with a second expression vector comprising a second gene construct in which the polynucleotide is operably linked to a promoter Preparing a transformant transformed host cell produced by conversion; Culturing the transformed host cell in a medium containing calcium; And irradiating the cultured host cell in culture with light having a wavelength that can induce heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein. A method of increasing is provided.
본 발명의 다른 일 관점에 따르면, STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이형이합체 형성 단백질이 연결된 제1융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제1유전자컨스트럭트를 포함하는 제1발현벡터 및 STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 상기 광유도 이형이합체 형성 단백질과 광조사에 의해 이형이합체를 형성하는 짝 단백질이 연결된 제2융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제2유전자컨스트럭트를 포함하는 제2발현벡터로 형질전환된 형질전환 식물 또는 비인간 형질전환 동물을 준비하는 단계; 및 상기 형질전환 식물 또는 비인간 형질전환 동물의 특정 기관 또는 조직에 상기 광유도 이형이합체 형성 단백질과 상기 짝 단백질 사이의 이형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 식물 또는 비인간 동물의 특정 기관 또는 조직에서의 세포질 내 칼슘 이온 농도를 가역적으로 증가시키는 방법이 제공된다.According to another aspect of the invention, a photoinduced heterodimer at the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A first expression vector and a stromal interaction molecule 1 (STIM1) protein or a N-terminus of the STIM1 protein, wherein the polynucleotide encoding the first fusion protein to which the forming protein is linked comprises a first gene construct operably linked to a promoter A polynucleotide encoding a second fusion protein linked to the N-terminus or C-terminus of the cytosolic fragment cleaved from the transmembrane domain to the transmembrane domain and the partner protein which forms a heterodimer by irradiation with light. Nucleotide is transformed with a second expression vector comprising a second gene construct operably linked to a promoter Preparing a transgenic plant or non-human transgenic animals; And a light irradiation step of irradiating a specific organ or tissue of the transgenic plant or non-human transgenic animal with light having a wavelength that can induce heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein. Or a method for reversibly increasing the concentration of calcium ions in the cytoplasm in certain organs or tissues of a non-human animal.
상기 방법에 있어서, 상기 제1융합단백질 및/또는 제2융합단백질은 추가로 형광단백질을 포함할 수 있다. 이때, 상기 형광단백질은 상기 STIM1 단백질 또는 상기 세포질성 단편과 상기 광유도 이형이합체 형성 단백질 및/또는 상기 짝 단백질 사이에 삽입되거나, 상기 광유도 이형이합체 형성 단백질 및/또는 상기 짝 단백질의 N-말단 또는 C-말단에 연결될 수 있다. 아울러, 상기 제1융합단백질과 상기 제2융합단백질에 각각 포함되는 형광단백질은 서로 다른 파장의 빛을 방출하여, 복합체 형성 여부를 확인하는데 사용될 수 있다. 상기 형광단백질은 상술한 바와 같다. In the method, the first fusion protein and / or the second fusion protein may further comprise a fluorescent protein. In this case, the fluorescent protein is inserted between the STIM1 protein or the cytoplasmic fragment and the photoinduced heterodimer forming protein and / or the paired protein, or the photoinduced heterodimer forming protein and / or the N-terminus of the paired protein. Or C-terminus. In addition, the fluorescent protein included in each of the first fusion protein and the second fusion protein may emit light of different wavelengths, and may be used to determine whether a complex is formed. The fluorescent protein is as described above.
상기 방법에 있어서, 상기 광유도 이형이합체 형성 단백질은 CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY 또는 PHR일 수 있다. In the method, the photoinduced heterodimer forming protein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY or PHR.
상기 방법에 있어서, 상기 짝 단백질은 상기 광유도 이형 이합체 형성 단배질과 광조사에 의해 이형 이합체를 형성할 수 있는 단백질로서, CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY 또는 PHR일 수 있고, 상기 광유도 이형 이합체 형성 단백질이 CIB 또는 CIBN일 경우 CRY 또는 PHR이며, 상기 광유도 이형 이합체 형성 단백질이 PhyB일 경우 PIF이고, 상기 광유도 이형 이합체 형성 단백질이 FKF1일 경우 GIGANTEA이며, 반대로 상기 광유도 이형 이합체 형성 단백질이 CRY 또는 PHR일 경우 CIB 또는 CIBN일 수 있고, 상기 광유도 이형 이합체 형성 단백질이 PIF일 경우 PhyB이며, 상기 광유도 이형 이합체 형성 단백질이 GIGANTEA일 경우 FKF1이다. 상기 PIF는 PIF3 또는 PIF6일 수 있다. In the method, the partner protein is a protein capable of forming heterodimers by photoinduced heterodimer formation protein and light irradiation, and may be CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY or PHR. , CRY or PHR when the photoinduced heterodimer forming protein is CIB or CIBN, PIF when the photoinduced heterodimer forming protein is PhyB, and GIGANTEA when the photoinduced heterodimer forming protein is FKF1, and vice versa If the heterodimer-forming protein is CRY or PHR, it may be CIB or CIBN, if the photoinduced heterodimer-forming protein is PIF, it is PhyB, and if the photoinduced heterodimer-forming protein is GIGANTEA, it is FKF1. The PIF may be PIF3 or PIF6.
상기 형질전환 식물 또는 비인간 형질전환 동물은 상술한 바와 같다.The transgenic plant or non-human transgenic animal is as described above.
본 발명자들은 광유도 이합체 형성 단백질인 PHR을 STIM1의 세포막 통과 도메인 이하를 제거한 세포질성 단편(cytosolic fragment)의 N-말단에 연결한 융합단백질을 암호화하는 유전자 컨스트럭트를 제조하여(도 3 참조), 이를 동물세포에 형질도입한 후, 칼슘 이온 포함 배지에서 배양하다가 PHR의 동형이합체 형성을 유도하는 450~520 nm의 파장을 갖는 빛을 조사할 경우, STIM1 단백질의 클러스터가 형성되고(도 4 내지 6 참조), 칼슘 이온이 세포내로 유입되어 세포질내 칼슘이온의 농도가 증가함을 입증하였을 뿐만 아니라(도 7 및 8 참조), 이를 이용하여 칼슘 반응 단백질의 세포 내 위치의 변화를 확인함으로써(도 9 및 10 참조), 본 발명을 완성하였다.The present inventors prepared a gene construct encoding a fusion protein in which PHR, a photoinduced dimer-forming protein, was linked to the N-terminus of a cytosolic fragment from which a subcellular membrane transmembrane domain of STIM1 was removed (see FIG. 3). When transduced into animal cells, cultured in a medium containing calcium ions and irradiated with light having a wavelength of 450 to 520 nm to induce homodimer formation of PHR, clusters of STIM1 proteins are formed (FIGS. 4 to 4). 6, not only proved that calcium ions were introduced into the cell to increase the concentration of calcium ions in the cytoplasm (see FIGS. 7 and 8), but also by using this to confirm the change in the intracellular location of calcium-responsive proteins (FIG. 6). 9 and 10), the present invention has been completed.
따라서, 본 발명의 방법 및 키트는 살아 있는 세포 및 개체 내에서 세포 내의 칼슘 이온의 농도를 시공간적으로 조절함으로써, 칼슘에 의한 신경 활성, 발생, 세포이동, 면역반응 및 인슐린 분비 등 생리학적 기능을 연구 및 이들 연구를 위한 모델 동식물을 제조하는 매우 유용하게 사용될 수 있다.Thus, the methods and kits of the present invention study the physiological functions of calcium activity, development, cell migration, immune responses and insulin secretion by controlling the concentration of calcium ions in cells in living cells and individuals in time and space. And very useful for preparing model plants and animals for these studies.
상기한 바와 같이 이루어진 본 발명의 일 실시예에 따르면, 세포, 동물 또는 식물의 세포질 내 칼슘이온 농도를 시공간적으로 그리고 가역적으로 조절할 수 있다. 물론 본 발명의 범위가 한정되는 것은 아니다.According to one embodiment of the present invention made as described above, the calcium ion concentration in the cytoplasm of the cell, animal or plant can be adjusted in time and space reversibly. Of course, the scope of the present invention is not limited.
도 1은 STIM1 단백질과 그와 상호작용하는 Orai1 단백질에 의해 세포 내 칼슘 농도가 조절되는 기전을 개략적으로 도시한 개요도이다.1 is a schematic diagram showing the mechanism by which the calcium concentration in the cell is regulated by the STIM1 protein and the Orai1 protein interacting with it.
ER: 소포체(endoplasmic reticulum); 및ER: endoplasmic reticulum; And
PM: 세포막(plasma membrane).PM: plasma membrane.
도 2는 이오노마이신에 의해 STIM1 단백질의 복합체 형성되는 것을 형광현미경으로 촬영한 사진(a) 및 이오노마이신 처리 후 시간에 따른 STIM1 단백질 복합체의 수를 측정한 그래프(b)이다.Figure 2 is a photograph taken with a fluorescence microscope of the complex formation of STIM1 protein by ionomycin and a graph (b) measuring the number of STIM1 protein complex with time after ionomycin treatment.
도 3은 STIM1 단백질(좌측)과 본 발명의 일실시예에 따른 융합단백질(우측)의 구조를 개략적으로 도시한 개요도이다.Figure 3 is a schematic diagram showing the structure of the STIM1 protein (left) and the fusion protein (right) according to an embodiment of the present invention.
도 4는 본 발명의 일실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의해 STIM1 단백질의 복합체 형성 여부를 FRET(fluorescence resonance energy transfer) 분석을 통해 조사한 그래프이다.4 is a graph illustrating whether the complex of STIM1 protein is formed by light irradiation after fusion protein expression according to an embodiment of the present invention through FRET (fluorescence resonance energy transfer) analysis.
도 5는 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의해 칼슘 이온이 세포내로 유입되는 과정을 개략적으로 도시한 개요도이다.5 is a schematic diagram schematically illustrating a process in which calcium ions are introduced into a cell by light irradiation after expressing a fusion protein in a cell according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 STIM1 단백질의 복합체가 형성여부를 비교한 형광현미경 사진이다.Figure 6 is a fluorescence micrograph comparing the formation of a complex of STIM1 protein before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전 후의 STIM1 단백질 복합체의 수를 비교한 그래프이다.Figure 7 is a graph comparing the number of STIM1 protein complexes before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
도 8은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 융합단백질의 복합체 형성(상부 판넬) 및 세포 내 칼슘(하부 판넬)을 이미징한 형광현미경 사진이다.FIG. 8 is a fluorescence microscope photograph of complex fusion protein formation (upper panel) and intracellular calcium (lower panel) before and after light irradiation after fusion protein expression according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의한 세포 내 칼슘 이온의 농도 변화를 시간의 경과에 따라 기록한 그래프이다.Figure 9 is a graph recording the change in the concentration of calcium ions in the cells by light irradiation after expression of the fusion protein in accordance with an embodiment of the present invention over time.
도 10은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 PKCγ-C2 도메인의 위치의 변화를 촬영한 형광현미경 사진이다. 10 is a fluorescence microscope photograph of the change in the position of the PKCγ-C2 domain before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 PKCγ-C2 도메인의 위치를 세포막에서의 형광강도를 기록함으로써 나타낸 그래프이다.11 is a graph showing the location of the PKCγ-C2 domain before and after light irradiation after fusion protein expression according to an embodiment of the present invention by recording the fluorescence intensity in the cell membrane.
본 문서에서 사용되는 용어를 정의하면 하기와 같다.The terms used in this document are defined as follows.
본 문서에서 사용되는 "광유도 이합체 형성 단백질(light-induced heterodimerized protein)"는 특정 파장의 빛을 조사할 경우 동형 이합체를 형성하거나 짝 단백질 이형이합체(heterodimer)를 형성하는 단백질을 의미한다.As used herein, "light-induced heterodimerized protein" refers to a protein that forms homodimers or paired protein heterodimers when irradiated with light of a particular wavelength.
본 문서에서 사용되는 "짝 단백질(partner protein)"은 특정 파장의 빛을 조사할 경우 광유도 이형이합체 형성 단백질과 상호작용하여 이형이합체를 형성하는 대상 단백질을 의미한다.As used herein, "partner protein" refers to a protein of interest that interacts with photoinduced heterodimer-forming proteins to form heterodimers when irradiated with light of a particular wavelength.
본 문서에서 사용되는 "이형이합체(heterodimer)"는 서로 다른 두 가지 단백질이 상호작용에 의해 하나의 복합체(complex)를 형성한 것을 의미한다.As used herein, "heterodimer" means that two different proteins interact to form a complex by interaction.
본 문서에서 사용되는 "동형이합체(homodimer)"는 같은 단백질이 서로 두 개가 상호작용하여 하나의 복합체(complex)를 형성한 것을 의미한다.As used herein, "homodimer" means that two proteins interact with each other to form a complex.
본 문서에서 사용되는 "작동 가능하게 연결된(operably linked to)"는 특정 폴리뉴클레오티드가 그 기능을 발휘할 수 있게 다른 폴리뉴클레오티드에 연결된 것을 의미한다. 즉, 특정 단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결되었다는 것은 당해 프로모터의 작용에 의해 mRNA로 전사되고 당해 단백질로 번역까지 될 수 있게 연결되었다는 것을 의미하고, 특정 단백질을 암호화하는 폴리뉴클레오티드가 다른 단백질을 암호화하는 폴리뉴클레오티드에 작동 가능하게 연결되었다는 것은 당해 특정 단백질이 다른 단백질과 융합단백질의 형태로 발현될 수 있게 연결되었다는 것을 의미한다.As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide to perform its function. In other words, the operably linked polynucleotide encoding a particular protein means that the polynucleotide encoding the specific protein is linked so that it can be transcribed into mRNA and translated into the protein by the action of the promoter. By operably linked to a polynucleotide encoding another protein, it is meant that the particular protein is linked so that it can be expressed in the form of a fusion protein with another protein.
본 문서에서 사용되는 "CIB"는 크립토크롬-상호작용 염기성 헬릭스-루프-헬릭스 단백질(cryptochrome-interacting basic-helix-loop-helix protein)을 의미하며, 대표적으로 애기장대의 CIB1(GenBank No.: NM_119618)가 있다.As used herein, "CIB" refers to cryptochrome-interacting basic-helix-loop-helix protein and is representative of Arabidopsis CIB1 (GenBank No .: NM_119618). There is).
본 문서에서 사용되는 "CIBN"은 상기 CIB의 N-말단으로서 광조사시 크립토크롬(cryptochrome, CRY)와 상호작용하는 부위를 의미한다."CIBN" as used herein refers to a site that interacts with cryptochrome (CRY) upon irradiation with light as the N-terminus of the CIB.
본 문서에서 사용되는 "CRY"는 크립토크롬(chryptochrome) 단백질을 의미하며, 대표적으로 애기장대의 CRY2(GenBank No.: NM_100320)가 있다.As used herein, "CRY" refers to a kryptochrome protein, typically CRY2 (GenBank No .: NM_100320) of Arabidopsis.
본 문서에서 사용되는 "PHR"은 상기 CRY의 N-말단 부위로서 파이토라이아제 상동성 지역(phytolyase homolgous homologous region)을 의미하며, 광조사시 상기 CIB 또는 CIBN과 상호작용한다(Kennedy et al., Nat. Methods, 7(12): 973-975, 2010).As used herein, "PHR" refers to a phytolyase homolgous homologous region as the N-terminal portion of the CRY, and interacts with the CIB or CIBN upon irradiation with light (Kennedy et al ., Nat. Methods , 7 (12): 973-975, 2010).
본 문서에서 사용되는 "Phy"는 파이토크롬(phytochrome) 단백질을 의미하고, 대표적으로 애기장대의 PhyA(GenBank No.: NM_001123784), PhyB(GenBank No.: NM_127435) 등이 있으며, PIF(phytochrome interacting factor)와 상호작용하는 것으로 알려져 있다(Min et al., Nature, 400: 781-784, 1999)As used herein, "Phy" refers to a phytochrome protein, and are representative of the Arabidopsis PhyA (GenBank No .: NM_001123784), PhyB (GenBank No .: NM_127435), PIF (phytochrome interacting factor) (Min et al ., Nature, 400: 781-784, 1999)
본 문서에서 사용되는 "PIF"는 파이토크롬 상호작용 인자(phytochrome interacting factor)을 의미하며, 대표적으로 애기장대의 PIF1(GenBank No.: NM_001202630), PIF3(GenBank No.: NM_179295), PIF4(GenBank No.: NM_180050), PIF5(GenBank No.: NM_180690), PIF6(GenBank No.: NM_001203231) 또는 PIF7(GenBank No.: NM_125520)이 있다.As used herein, "PIF" refers to a phytochrome interacting factor, which is representative of the Arabidopsis PIF1 (GenBank No .: NM_001202630), PIF3 (GenBank No .: NM_179295), PIF4 (GenBank No .: NM_180050), PIF5 (GenBank No .: NM_180690), PIF6 (GenBank No .: NM_001203231), or PIF7 (GenBank No .: NM_125520).
본 문서에서 사용되는 "FKF"는 플라빈-결합, 켈치 반복, F-박스(Flavin-binding, Kelch repeat, F-box) 단백질을 의미하고, 대표적으로 애기장대의 FKF1(GenBank No.: NM_105475)이 있으며, 광조사시 GIGANTEA 단백질과 상호작용하는 것으로 알려져 있다(Sawa et al., Science, 318(5848): 261-265, 2007).As used herein, "FKF" refers to Flavin-binding, Kelch repeat, F-box proteins, typically FKF1 (GenBank No .: NM_105475) of Arabidopsis. And interact with GIGANTEA proteins upon irradiation with light (Sawa et al ., Science , 318 (5848): 261-265, 2007).
본 문서에서 사용되는 "GIGANTEA"는 파이토크롬 신호전달에 관련되어 있고, 꽃의 개화시기를 조절하는 단백질로 알려져 있다.As used herein, "GIGANTEA" is involved in phytochrome signaling and is known as a protein that regulates flowering time.
본 문서에서 사용되는 "테트라시스테인 모티프(tetracystein motif)"는 Cys-Cys-Xaa-Xaa-Cys-Cys(서열번호 1)의 서열을 포함하는 폴리펩티드로서, Xaa는 시스테인을 제외한 아미노산으로서, 상기 Xaa의 종류와 폴리펩티드의 길이에 따라, 형광패턴이 달라진다(Adams et al., J. Am. Chem. Soc., 124: 6063-6077, 2002).As used herein, the term "tetracystein motif" is a polypeptide comprising the sequence of Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), Xaa is an amino acid except cysteine, wherein Depending on the type and length of the polypeptide, the fluorescence pattern varies (Adams et al ., J. Am. Chem. Soc ., 124: 6063-6077, 2002).
본 문서에서 사용되는 "형질전환 식물" 또는 "형질전환 동물"은 외래 유전자를 게놈 내에 도입하여 상기 외래 유전자를 발현하거나 또는 특정 유전자가 발현되지 않도록 결손시킨 유전자 조작된 식물 또는 동물을 의미한다. 형질전환 동물의 경우 통상의 경우 생식세포를 유전자 조작하여 제조될 수 있으나, 체세포에 대한 유전자 조작 이후 핵치환에 의한 복제동물 제조방법으로 제조될 수 있고, 형질전환 식물의 경우 더 간단하게, 체세포를 외래 유전자를 포함하는 아그로박테리아로 감염시킨 후 탈분화 및 재분화의 과정을 거쳐서 제조될 수 있다. 상기 형질전환 동물 및 형질전환 식물의 제조방법은 당업계에 잘 알려져 있다(Jaenisch, R and B. Mintz, Proc. Natl. Acad. Sci. USA, 71(4): 1250-1254, 1974; Cho et al., Curr. Protoc. Cell Biol., 42: 19.11.1-19.11.22, 2009; Johnston, S. A. and D. C. Tang, Meth. Cell Biol., 43 Pt A: 353-365, 1994; Sasaki et al., Nature 459(7246): 523-527, 2009; Vaek et al., Nature, 328(6125): 33-37, 1987).As used herein, “transgenic plant” or “transgenic animal” refers to a genetically engineered plant or animal in which a foreign gene has been introduced into the genome to express the foreign gene or to have a specific gene deleted. In the case of transgenic animals, genetically engineered germ cells in general, but may be prepared by a method for producing a cloned animal by nuclear substitution after genetic manipulation of the somatic cells, in the case of transgenic plants more simply, somatic cells After infection with Agrobacteria containing a foreign gene can be prepared through a process of dedifferentiation and redifferentiation. Methods of making such transgenic animals and transgenic plants are well known in the art (Jaenisch, R and B. Mintz, Proc. Natl. Acad. Sci. USA , 71 (4): 1250-1254, 1974; Cho et. al ., Curr. Protoc. Cell Biol ., 42: 19.11.1-19.11.22, 2009; Johnston, SA and DC Tang, Meth.Cell Biol ., 43 Pt A: 353-365, 1994; Sasaki et al . Nature 459 (7246): 523-527, 2009; Vaek et al ., Nature , 328 (6125): 33-37, 1987).
이하, 첨부된 도면들을 참조하여 본 발명의 실시예를 상세히 설명하면 다음과 같다. 그러나 본 발명은 이하에서 개시되는 실시예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. 또한 설명의 편의를 위하여 도면에서는 구성 요소들이 그 크기가 과장 또는 축소될 수 있다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms, and the following embodiments are intended to complete the disclosure of the present invention, the scope of the invention to those skilled in the art It is provided to inform you completely. In addition, the components may be exaggerated or reduced in size in the drawings for convenience of description.
도 1은 STIM1 단백질과 그와 상호작용하는 Orai1 단백질에 의해 세포 내 칼슘 농도가 조절되는 기전을 개략적으로 도시한 개요도이다. STIM1 단백질은 소포체 (ER) 내의 칼슘 이온 농도의 변화를 감지하여 칼슘 이온 농도가 낮아지면 복합체를 형성한다. 복합체를 형성한 STIM1 단백질은 세포막(PM)에 존재하는 칼슘채널의 포어 형성 서브유닛인 Orai1 단백질과 상호작용을 하여 Orai1을 활성화시킴으로써 칼슘채널이 형성되게 되며, 이어 세포 외부에 존재하는 칼슘 이온이 세포질 내로 유입되게 된다.1 is a schematic diagram showing the mechanism by which the calcium concentration in the cell is regulated by the STIM1 protein and the Orai1 protein interacting with it. The STIM1 protein senses a change in calcium ion concentration in the endoplasmic reticulum (ER) and forms a complex when the calcium ion concentration is lowered. The complexed STIM1 protein interacts with the Orai1 protein, a pore-forming subunit of the calcium channel present in the cell membrane (PM), thereby activating Orai1 to form calcium channels. It will flow into.
도 2는 이오노마이신에 의해 STIM1 단백질의 복합체 형성되는 것을 형광현미경으로 촬영한 사진(a) 및 이오노마이신 처리 후 시간에 따른 STIM1 단백질 복합체의 수를 측정한 그래프(b)이다. 이오노마이신은 STIM1 단백질의 복합체를 형성시킴으로서 칼슘채널을 형성, 칼슘의 유입을 유도하는 것으로 알려져 있다. 실제, STIM1 단백질에 형광단백질인 황색형광단백질(YFP)을 연결한 융합단백질을 암호화하는 유전자컨스트럭트를 제조하고 이를 세포에 형질도입한 후, 이오노마이신을 처리한 결과, 이오노마이신 처리 전에는 세포질 내에 분산되어 있는 형광패턴이 이오노마이신 처리 후 강한 강도를 갖는 점의 형태로 변화하여 STIM1 단백질의 복합체 형성을 확인할 수 있었고, 강한 형광점의 수를 계수한 결과 이오노마이신 처리 직후 급격하게 증가하였다가 시간이 경과할수록 점차 감소함을 확인하였다. Figure 2 is a photograph taken with a fluorescence microscope of the complex formation of STIM1 protein by ionomycin and a graph (b) measuring the number of STIM1 protein complex with time after ionomycin treatment. Ionomycin is known to form calcium channels and induce calcium influx by forming complexes of the STIM1 protein. In fact, a gene construct encoding a fusion protein linked to a fluorescent protein yellow fluorescent protein (YFP) to the STIM1 protein was prepared and transduced into cells, and then treated with ionomycin, before treatment with ionomycin. The fluorescent pattern dispersed in the cytoplasm was changed into the form of the point with strong intensity after the ionomycin treatment, and the formation of the complex of STIM1 protein was confirmed, and the number of strong fluorescent points was rapidly increased immediately after the ionomycin treatment. It was confirmed that gradually decrease as time passes.
도 3은 STIM1 단백질(좌측)과 본 발명의 일실시예에 따른 융합단백질(우측)의 구조를 개략적으로 도시한 개요도이다. STIM1 단백질은 단일 통과 소포체(ER) 막 단백질로서 N-말단 쪽이 소포체강(ER lumen) 쪽에 위치하고 C-말단이 세포질(cytosol)에 위치한다. N-말단으로부터 EF hand, SAM(sterile-alpha-motif), 막통과 도메인(TM domain), 두 개의 이중나선(coiled coil, CC), 세린/프롤린 풍부지역 및 라이신 풍부지역을 포함하고 있다(도 3의 좌측). 본 발명자들은 상기 STIM1 단백질의 N-말단부터 막통과 도메인까지의 부분이 제거되고, 상기 제거된 부분이 형광단백질인 mCerulean에 연결된 광조사 이합체 형성 단백질인 PHR(Cryptochrome의 N-말단 부위)로 대체된 융합단백질을 암호화하는 유전자 컨스트럭트(mCerulean-PHR-STIM1-ct)를 제조하였다. 이렇게 제조된 유전자컨스트럭트를 동물세포에 형질도입하여 발현시킬 경우, 발현된 융합단백질은 막통과 도메인이 없어서 세포질에 존재하게 된다(수정란에 기재한 바와 같이 원하는 시간대에 STIM1 융합 단백질을 활성화 시키기 위함입니다).Figure 3 is a schematic diagram showing the structure of the STIM1 protein (left) and the fusion protein (right) according to an embodiment of the present invention. STIM1 protein is a single-pass vesicle (ER) membrane protein located on the N-terminus of the ER lumen and on the C-terminus of the cytosol. It contains the EF hand, sterile-alpha-motif (SAM), transmembrane domain (TM domain), two coiled coils (CC), serine / proline-rich regions and lysine-rich regions from the N-terminus (Fig. Left side of 3.) The present inventors have removed the portion from the N-terminus to the transmembrane domain of the STIM1 protein, and the removed portion was replaced with PHR (N-terminal portion of Cryptochrome), a dimerizing protein linked to mCerulean, a fluorescent protein. Gene constructs encoding fusion proteins (mCerulean-PHR-STIM1-ct) were prepared. When transgene is expressed by transducing animal cells, the expressed fusion protein is present in the cytoplasm because there is no transmembrane domain (to activate the STIM1 fusion protein at the desired time period as described in the fertilization column). is).
도 4는 본 발명의 일실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의해 STIM1 단백질의 복합체 형성 여부를 FRET(fluorescence resonance energy transfer) 분석을 통해 조사한 그래프이다. 상기 분석을 위해 STIM1의 N-말단으로부터 막통과 도메인까지 제거되고, 이 부분이 mCitrine이 연결된 PHR로 대체된 융합단백질을 암호화하는 유전자컨스트럭트를 추가로 제작하여, 상기에서 사용한 mCerulean-PHR-STIM1-ct 유전자컨스트럭트와 함께 세포에 형질도입하여 두 가지 융합단백질을 발현시킨 후 광조사를 한 후, mCitrine의 형광과 mCerulean의 형광의 강도의 비를 계산하였다. 그 결과, 광 조사 전에는 형광비가 1이었으나 광조사후 약 1.1 내지 1.2 사이의 값을 나타내게 되었다. 이는 두 융합단백질이 복합체를 형성하여 mCerulean의 형광에너지가 mCitrine로 전이되는 전형적인 형광 공명 에너지 전이현상(fluorescence resonance energy transfer)을 나타내는 것이다. 이로써, 본발명자들은 본 발명의 일실시예에서 사용한 PHR이 광조사와 무관하게 동형이합체를 형성한다는 기존의 보고와는 달리 광조사에 의해 동형 이합체를 형성한다는 놀라운 사실을 최초로 규명하였다.4 is a graph illustrating whether the complex of STIM1 protein is formed by light irradiation after fusion protein expression according to an embodiment of the present invention through FRET (fluorescence resonance energy transfer) analysis. MCerulean-PHR-STIM1 used above was further prepared by constructing a gene construct encoding the fusion protein removed from the N-terminus of STIM1 from the N-terminus of STIM1 and replaced by mCitrine-linked PHR. Cells were transduced with -ct gene constructs to express two fusion proteins and irradiated with light, and then the ratio of the intensity of mCitrine and mCerulean fluorescence was calculated. As a result, the fluorescence ratio was 1 before light irradiation, but the value was about 1.1 to 1.2 after light irradiation. This shows a typical fluorescence resonance energy transfer phenomenon in which two fusion proteins form a complex and fluorescence energy of mCerulean is transferred to mCitrine. Thus, the present inventors first identified the surprising fact that the PHR used in one embodiment of the present invention forms a homodimer by light irradiation, unlike conventional reports that the PHR forms a homodimer regardless of light irradiation.
도 5는 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의해 칼슘 이온이 세포내로 유입되는 과정을 개략적으로 도시한 개요도이다. 광조사에 의해 PHR 끼리 상호작용을 함으로써 결국 전체 STIM1 단백질을 포함한 융합단백질이 복합체를 형성하게 되고, STIM1 단백질과 상호작용하는 세포막의 Orai1 단백질이 칼슘채널의 포어를 형성함으로써 칼슘 이온이 세포 내로 유입될 수 있다.5 is a schematic diagram schematically illustrating a process in which calcium ions are introduced into a cell by light irradiation after expressing a fusion protein in a cell according to an embodiment of the present invention. By interacting with PHR by light irradiation, fusion proteins including the whole STIM1 protein form a complex, and calcium ions are introduced into cells by Orai1 protein in cell membranes interacting with STIM1 protein to form pores of calcium channels. Can be.
도 6은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 STIM1 단백질의 복합체가 형성여부를 비교한 형광현미경 사진이다. 상기에서 제조한 mCerulean-PHR-STIM1-ct 유전자컨스트럭트로 형질전환시킨 세포에 광조사하기 전과 후의 형광현미경 이미지를 촬영하였다. 그 결과 광조사전 세포질 내에 골고루 분산되어 있던 형광패턴이 광조사 후 강한 형광점이 다수 나타나는 패턴으로 변화함을 알 수 있었다. 이는 결국 광조사에 의해 본 발명의 일 실시예에 따른 융합단백질이 복합체를 형성하였음을 보여주는 것이다.Figure 6 is a fluorescence micrograph comparing the formation of a complex of STIM1 protein before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention. Fluorescence microscopy images were taken before and after irradiating the cells transformed with the mCerulean-PHR-STIM1-ct gene construct prepared above. As a result, it was found that the fluorescence pattern evenly distributed in the cytoplasm before light irradiation changed to a pattern in which many strong fluorescence points appeared after light irradiation. This shows that the fusion protein according to an embodiment of the present invention formed a complex by light irradiation.
도 7은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전 후의 STIM1 단백질 복합체의 수를 비교한 그래프이다. 상기 실험결과에서 나타난 강한 형광점의 갯수를 STIM1 융합단백질의 클러스터 수로 간주하여 이를 계수하였다. 그 결과 광조사 전에는 융합단백질의 클러스터 수가 0에 가까왔으나, 광조사 후 융합단백질의 클러스터의 수가 70여개로 증가하였다.Figure 7 is a graph comparing the number of STIM1 protein complexes before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention. The number of strong fluorescence points shown in the experimental results was regarded as the number of clusters of STIM1 fusion protein and counted. As a result, the number of clusters of the fusion protein was close to zero before light irradiation, but the number of clusters of the fusion protein increased to 70 after light irradiation.
도 8은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 융합단백질의 복합체 형성(상부 판넬) 및 세포 내 칼슘(하부 판넬)을 이미징한 형광현미경 사진이다. 형광단백질로 mCherry를 사용한 융합단백질을 암호화하는 유전자컨스트럭트 mCherry-PHR-STIM1-ct를 제조한 후, 이를 세포에 형질도입한 후 광조사 전과 후의 상기 형광단백질의 형광을 형광현미경으로 이미징하였다. 그 결과, 상기 도 7에서 관찰된 것과 마찬가지로 광조사 전에는 세포질 내에 고루 분산된 형광 패턴(상부 판넬 좌측)이 광조사 후 강한 형광점의 형태로 변화하였다(상부 판넬 우측). 이러한 STIM1 융합단백질의 복합체 형성에 의해 실제 세포내 칼슘 이온의 농도가 변화했는지 확인하기 위해, 칼슘이온을 직접 이미징할 수 있는 Fluo3-AM 시약을 세포에 처리한 후 이의 형광 이미지를 촬영하였다. 그 결과 광 조사 전에는 세포내 부분 부분 강한 점의 형태로 형광이 나타난 반면(하부 판넬 좌측), 광조사 후에는 전반적으로 형광의 강도가 증가하였고, 강한 형광점의 수도 증가함을 알 수 있었다(하부 판넬 우측). 따라서, 본 발명자들은 본 발명의 일 실시예에 따른 융합단백질이 광조사에 의해 복합체를 형성함은 물론 칼슘채널을 형성하여 칼슘 이온을 세포질 내로 유입함을 입증하였다.FIG. 8 is a fluorescence microscope photograph of complex fusion protein formation (upper panel) and intracellular calcium (lower panel) before and after light irradiation after fusion protein expression according to an embodiment of the present invention. After constructing the gene construct mCherry-PHR-STIM1-ct encoding a fusion protein using mCherry as a fluorescent protein, the cells were transduced and imaged by fluorescence microscopy of the fluorescent protein before and after light irradiation. As a result, as shown in FIG. 7, the fluorescent pattern (upper left panel) uniformly dispersed in the cytoplasm was changed to a strong fluorescent point after light irradiation (upper right panel) before light irradiation. In order to confirm whether the concentration of calcium ions in the cell was changed by the formation of the complex of the STIM1 fusion protein, the fluorescent images were taken after treating the cells with Fluo3-AM reagent capable of directly imaging calcium ions. As a result, the fluorescence appeared in the form of a strong point in the intracellular portion before light irradiation (lower panel left), but after the light irradiation, the intensity of fluorescence generally increased and the number of strong fluorescence points also increased (bottom). Right panel). Accordingly, the present inventors have demonstrated that the fusion protein according to an embodiment of the present invention not only forms a complex by light irradiation but also forms a calcium channel to introduce calcium ions into the cytoplasm.
도 9는 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사에 의한 세포 내 칼슘 이온의 농도 변화를 시간의 경과에 따라 기록한 그래프이다. 본 발명자들은 mCerulean-PHR-STIM1-ct 컨스트럭트로 형질전환된 세포와 공벡터로 형질전환된 세포에 광조사를 한 후 세포내 칼슘 농도를 Fluo3-AM에 의한 형광 강도로 측정하였다. 그 결과 mCerulean-PHR-STIM1-ct를 발현하는 세포의 경우 광조사 후 형광 강도가 8배 정도 증가하였다가, 시간의 경과에 따라 다소 완만한 곡선으로 감소한 반면, mCerulean-PHR-STIM1-ct를 발현하지 않는 세포의 경우 형광의 변화를 관찰할 수 없었다. Figure 9 is a graph recording the change in the concentration of calcium ions in the cells by light irradiation after expression of the fusion protein in accordance with an embodiment of the present invention over time. The inventors of the present invention, after irradiating cells transformed with mCerulean-PHR-STIM1-ct construct and cells transformed with the empty vector, measured intracellular calcium concentration by fluorescence intensity by Fluo3-AM. As a result, in the case of cells expressing mCerulean-PHR-STIM1-ct, the fluorescence intensity increased 8-fold after light irradiation, but decreased to a slightly gentle curve with time, whereas mCerulean-PHR-STIM1-ct was expressed. For cells that do not, no change in fluorescence was observed.
도 10은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 PKCγ-C2 도메인의 위치의 변화를 촬영한 형광현미경 사진이다. 본 발명자들은 mCherry-PHR-STIM1-ct 컨스트럭트와 YFP-PKCγ-C2 도메인 컨스트럭트를 각각 제조한 뒤 세포에 공형질도입한 후, 광조사 전후의 형광 패턴의 변화를 관찰하였다. 그 결과 STIM1 융합단백질에 의한 형광의 변화는 상기 도 8의 결과와 동일하였고, PKCγ-C2 도메인의 경우 광조사 전에는 세포질 내에 전반적으로 분산된 양상의 형광패턴이었으나(하부 판넬 좌측), 광조사후에는 세포막 부근의 형광이 더 강하게 나타나(하부 판넬 우측), PKCγ-C2 도메인이 세포막쪽으로 이동하였음을 나타냈다.10 is a fluorescence microscope photograph of the change in the position of the PKCγ-C2 domain before and after light irradiation after expressing the fusion protein in a cell according to an embodiment of the present invention. The inventors prepared the mCherry-PHR-STIM1-ct construct and the YFP-PKCγ-C2 domain construct, respectively, and co-transduced the cells, and observed changes in the fluorescence pattern before and after light irradiation. As a result, the change in fluorescence by STIM1 fusion protein was the same as the result of FIG. 8, and in the case of PKCγ-C2 domain, the fluorescence pattern was generally dispersed in the cytoplasm before light irradiation (bottom left panel). Fluorescence near the cell membrane was stronger (lower panel right), indicating that the PKCγ-C2 domain had migrated towards the cell membrane.
도 11은 본 발명의 일 실시예에 따른 융합단백질을 세포 내에서 발현시킨 후 광조사 전후의 PKCγ-C2 도메인의 위치를 세포막에서의 형광강도를 기록함으로써 나타낸 그래프이다. 광조사 후의 PKCγ-C2의 세포막으로의 이동 여부를 PKCγ-C2와 연결된 황색형광단백질(YFP)의 형광의 강도를 측정함으로써 조사하였다. 그 결과, 광조사 후 100 초 정도 경과 후에 세포막에서의 형광강도가 증가하였으며, 300초가 될 때까지 계속 유지됨을 알 수 있었다.11 is a graph showing the location of the PKCγ-C2 domain before and after light irradiation after fusion protein expression according to an embodiment of the present invention by recording the fluorescence intensity in the cell membrane. The migration of PKCγ-C2 to the cell membrane after light irradiation was investigated by measuring the intensity of fluorescence of the yellow fluorescent protein (YFP) linked with PKCγ-C2. As a result, after about 100 seconds after the light irradiation, the fluorescence intensity increased in the cell membrane, and it was found that it was maintained until 300 seconds.
따라서, 본 발명의 일 실시예에 따른 융합단백질 및 이를 이용한 광조사에 의한 세포내 칼슘 이온 농도의 증가방법은 세포 내에서의 칼슘 이온 의존적인 다양한 생화학적 반응을 연구하는데, 유용하게 사용될 수 있음을 알 수 있다.Therefore, the fusion protein and the method of increasing intracellular calcium ion concentration by light irradiation using the same according to an embodiment of the present invention can be useful for studying various calcium-dependent biochemical reactions dependent on intracellular calcium ion. Able to know.
이하, 실시예 및 실험예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예 및 실험예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예 및 실험예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but may be embodied in various different forms. The following examples and experimental examples are provided to make the disclosure of the present invention complete and the general knowledge. It is provided to fully inform those who have the scope of the invention.
실시예 1: 벡터의 제작Example 1 Construction of Vector
1-1: mCerulean-PHR-STIM1-ct 컨스트럭트의 제작1-1: Construction of mCerulean-PHR-STIM1-ct Construct
인간 STIM1 유전자(GenBank 등록번호: NM_003156)의 N-말단으로부터 막통과 도메인까지가 절단된 세포질성 단편(a.a. 238-685)의 N-말단에 CRY2 유전자(GenBank No.: NM_100320)의 PHR에 해당하는 1-498 아미노산이 연결된 융합단백질을 암호화하는 폴리뉴클레오티드(PHR-STIM1)를 제조한 후, 이를 pmCerulean-C1 벡터(Clontech, USA)에 삽입하여 mCerulean-PHR-STIM1-ct 컨스트럭트를 제작하였다(도 3).Corresponding to the PHR of the CRY2 gene (GenBank No .: NM_100320) at the N-terminus of the cytoplasmic fragment (aa 238-685) cleaved from the N-terminus to the transmembrane domain of the human STIM1 gene (GenBank Accession No .: NM_003156). After preparing a polynucleotide (PHR-STIM1) encoding a fusion protein linked to 1-498 amino acids, it was inserted into the pmCerulean-C1 vector (Clontech, USA) to prepare an mCerulean-PHR-STIM1-ct construct ( 3).
1-2: mCitrine-PHR-STIM1-ct 컨스트럭트의 제작1-2: Construction of the mCitrine-PHR-STIM1-ct Construct
상기 실시예 1-1에서 제조한 PHR-STIM1 폴리뉴클레오티드를 pmCitrine-C1 벡터(Clontech, USA)에 삽입하여 mCitrine-PHR-STIM1-ct 컨스트럭트를 제작하였다. The PHR-STIM1 polynucleotide prepared in Example 1-1 was inserted into the pmCitrine-C1 vector (Clontech, USA) to prepare an mCitrine-PHR-STIM1-ct construct.
1-3: mCherry-PHR-STIM1-ct 컨스트럭트의 제작1-3: Construction of the mCherry-PHR-STIM1-ct Construct
상기 실시예 1-1에서 제조한 PHR-STIM1 폴리뉴클레오티드를 pmCherry-C1 벡터(Clonthch, USA)에 삽입하여 mCherry-PHR-STIM1-ct 컨스트럭트를 제작하였다. The mCherry-PHR-STIM1-ct construct was prepared by inserting the PHR-STIM1 polynucleotide prepared in Example 1-1 into the pmCherry-C1 vector (Clonthch, USA).
1-4: YFP-PKCγ-C2 컨스트럭트의 제작1-4: Construction of the YFP-PKCγ-C2 Construct
YFP 형광단백질이 포함된 pcDNA3 벡터에서 YFP 앞에 PKCγ(GenBank No. NM-002739)의 C2 도메인(a.a 156-284)을 융합하여 YFP-PKCγ-C2 컨스트럭트를 제작하였다. A YFP-PKCγ-C2 construct was prepared by fusing the C2 domain (a.a 156-284) of PKCγ (GenBank No. NM-002739) in front of YFP in a pcDNA3 vector containing a YFP fluorescent protein.
실험예 1: 광유도에 의한 PHR 포함 STIM1 융합단백질의 동형이합체 형성 여부 확인Experimental Example 1: Confirmation of homodimer formation of PHR-containing STIM1 fusion protein by photoinduction
상기 실시예 1-1 및 1-2에서 각각 제조한 mCerulean-PHR-STIM1-ct와 mCitrine-PHR-STIM1-ct로 HeLa 세포를 공형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 10% CO2 조건에서 배양하다가, 파장 450-520 nm의 빛의 조사 전후로 mCerulean과 mCitrine에 의한 형광을 각각 측정한 후, mCerulean 형광강도/mCitrine 형광강도의 비를 구하였다(도 4). 그 결과, 광조사 전에는 형광강도의 비가 약 1이었으나, 광조사후 그 값이 상승하여 약 1.1 내지 1.2 사이에서 진폭을 가지고 오르락내리락하였다. 이는 전형적인 FRET(fluorescence resonance energy transfer) 현상으로서 두 융합단백질이 광조사에 의해 복합체를 형성함을 간접적으로 입증하는 것이다. 이는 두 융합단백질 사이에 포함된 PHR이 광조사에 의해 동형이합체를 형성하였음을 의미하는데, 종래에 PHR이 광조사와 무관하게 동형이합체를 형성한다고 보고된 것과 전혀 다른 결과이다. 이로써, 본 발명자들은 PHR이 광조사시 CIB와 이형이합체를 형성할 뿐만 아니라, 동형이합체를 형성함을 최초로 규명한 것이다. 따라서, PHR은 다른 광유도 이합체 형성 단백질을 사용하지 않고도 단독으로 STIM1의 복합체 형성 및 그에 의한 칼슘 이온의 세포내 유입을 유도할 수 있으므로, 매우 유용하게 사용될 수 있다.HeLa cells were cotransformed with mCerulean-PHR-STIM1-ct and mCitrine-PHR-STIM1-ct prepared in Examples 1-1 and 1-2, respectively, and then in DMEM medium containing 500 μg / ml of G418. After culturing at 37 ° C. and 10% CO 2 , fluorescence by mCerulean and mCitrine was measured before and after irradiation with light having a wavelength of 450-520 nm, respectively, and the ratio of mCerulean fluorescence intensity / mCitrine fluorescence intensity was calculated (FIG. 4). . As a result, the ratio of fluorescence intensity was about 1 before light irradiation, but the value increased after light irradiation, and it rose up and down with an amplitude between about 1.1 and 1.2. This is a typical fluorescence resonance energy transfer (FRET) phenomenon that indirectly demonstrates that two fusion proteins form a complex by light irradiation. This means that the PHR contained between the two fusion proteins formed a homodimer by irradiation with light, which is a completely different result than that previously reported to form a homodimer regardless of light irradiation. Thus, the present inventors first identified that PHR not only forms a heterodimer with CIB when irradiated with light, but also forms a homodimer. Therefore, PHR can be very useful because it can induce complex formation of STIM1 and intracellular influx of calcium ions by itself without using other photoinduced dimer forming proteins.
이어, 본 발명자들은 상기 융합단백질이 실제 복합체를 형성하는지 확인하기 위해, 실시예 1-3에서 제조한 mCherry-PHR-STIM1-ct 컨스트럭트로 HeLa 세포를 형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 10% CO2 조건에서 배양하다가, 파장 450-520 nm의 빛의 조사 전후로 형광현미경 이미지를 수득하였다(도 6). 그 결과 빛 조사전에는 세포질 내에 mCherry에 의한 형광이 골고루 분산되어 있는 양상을 나타내다가(좌측), 광조사 후에 형광이 다수의 점의 형태로 강하게 나타남을 발견하였다(우측). 이는 형광단백질을 포함하는 융합단백질이 서로 상호작용하여 복합체를 형성하였음을 의미한다. 본 발명자들은 상기 형광강도가 증가된 점의 개수를 융합단백질의 복합체의 수로 간주하고 이를 계수하였으며, 그 결과 광조사전에는 0에 가까운 세포당 복합체의 수가 광조사 후 70여개로 증가함을 확인하였다(도 7).Next, the present inventors transformed HeLa cells with the mCherry-PHR-STIM1-ct construct prepared in Examples 1-3 to confirm that the fusion protein actually formed a complex, and then G500 500 μg / ml After culturing at 37 ° C. and 10% CO 2 in DMEM medium, fluorescence microscopy images were obtained before and after irradiation with light having a wavelength of 450-520 nm (FIG. 6). As a result, it was found that fluorescence by mCherry was uniformly dispersed in the cytoplasm before light irradiation (left), and that fluorescence appeared strongly in the form of many dots after light irradiation (right). This means that the fusion proteins including the fluorescent protein interacted with each other to form a complex. The present inventors regarded the number of increased fluorescence intensity as the number of complexes of the fusion protein and counted it. As a result, the number of complexes per cell near zero before irradiation increased to about 70 after irradiation. 7).
실험예 2: 광유도에 의한 칼슘 유입 여부 확인Experimental Example 2: Check the influx of calcium by the light induction
본 발명자들은 상기 융합단백질의 복합체 형성에 따라, 칼슘의 세포내로의 유입이 유도되는지 확인하기 위해, 상기 실시예 1-3에서 제조한mCherry-PHR-STIM1-ct 컨스트럭트로 HeLa 세포를 형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 5% CO2 조건에서 배양하다가, 파장 450-520 nm의 빛의 조사 전후로 형광현미경 이미지를 수득하는 한편(도 8 상부 판넬), 칼슘을 이미징할 수 있는 형광물질인 Fluo3-AM(Sigma, USA)를 광조사 30분 전에 4.52 μg/ml로 처리한 후, 형광 패턴의 변화를 형광현미경으로 촬영하였다(도 8 하부 판넬). 그 결과 광 조사 전에는 세포내 부분 부분 강한 점의 형태로 형광이 나타난 반면(하부 판넬 좌측), 광조사 후에는 전반적으로 형광의 강도가 증가하였고, 강한 형광점의 수도 증가함을 알 수 있었다(하부 판넬 우측). 따라서, 본 발명자들은 본 발명의 일 실시예에 따른 융합단백질이 광조사에 의해 복합체를 형성함은 물론 칼슘채널을 형성하여 칼슘 이온을 세포질 내로 유입함을 입증하였다.The present inventors transformed HeLa cells with mCherry-PHR-STIM1-ct constructs prepared in Examples 1-3 to confirm that the influx of calcium into the cells is induced as the complex of the fusion protein is induced. Then, incubated in DMEM medium containing 500 μg / ml of G418 at 37 ℃, 5% CO 2 conditions, to obtain a fluorescence microscope image before and after irradiation of light of wavelength 450-520 nm (Fig. 8 upper panel), Fluo3-AM (Sigma, USA), which can be imaged, was treated with 4.52 μg / ml 30 minutes before light irradiation, and the change of the fluorescence pattern was photographed with a fluorescence microscope (Fig. 8 lower panel). As a result, the fluorescence appeared in the form of a strong point in the intracellular portion before light irradiation (lower panel left), but after the light irradiation, the intensity of fluorescence generally increased and the number of strong fluorescence points also increased (bottom). Right panel). Accordingly, the present inventors have demonstrated that the fusion protein according to an embodiment of the present invention not only forms a complex by light irradiation but also forms a calcium channel to introduce calcium ions into the cytoplasm.
아울러, 본 발명자들은 mCherry-PHR-STIM1-ct 컨스트럭트로 형질전환된 세포와 공벡터(pmCherry-C1 vector)로 형질전환된 HeLa 세포에 광조사를 한 후 세포내 칼슘 농도를 Fluo3-AM에 의한 형광 강도로 측정하였다. 그 결과 도 9에서 나타난 바와 같이, mCherry-PHR-STIM1-ct를 발현하는 세포의 경우 광조사 후 형광 강도가 8배 정도 증가하였다가, 시간의 경과에 따라 다소 완만한 곡선으로 감소한 반면, mCherry만 발현하는 세포의 경우 형광의 변화를 관찰할 수 없었다. 이는 본 발명의 일 실시예에 따른 칼슘 이온의 세포질 내 유입은 형광단백질에 의한 영향이 아니라, PHR 및 STIM1을 포함하는 융합단백질에 의한 것임을 입증하는 것이다.In addition, the present inventors irradiated the cells transformed with the mCherry-PHR-STIM1-ct construct and HeLa cells transformed with the empty vector (pmCherry-C1 vector), and then measured the intracellular calcium concentration by Fluo3-AM. Measured by fluorescence intensity. As a result, as shown in FIG. 9, in the case of cells expressing mCherry-PHR-STIM1-ct, the fluorescence intensity increased about 8-fold after light irradiation, but decreased to a slightly gentle curve over time, whereas mCherry-only For expressing cells, no change in fluorescence was observed. This is to demonstrate that the influx of calcium ions into the cytoplasm according to an embodiment of the present invention is not by the fluorescent protein, but by a fusion protein including PHR and STIM1.
실험예 3: 광유도에 의한 칼슘 의존성 생화학적 변화의 확인Experimental Example 3: Confirmation of calcium-dependent biochemical change by light induction
본 발명자들은 상기 광조사에 의한 칼슘의 세포질내 유입에 의해 세포내 생화학적 변화를 조사할 수 있는지 확인하기 위해, 칼슘에 의해 활성화되는 PKCγ-C2의 거동을 조사하였다. 구체적으로 상기 실시예 1-4에서 제조된 YFP-PKCγ-C2 컨스트럭트와 실시예 1-3에서 제조된 mCherry-PHR-STIM1-ct 컨스트럭트로 HeLa 세포를 공형질전환시킨 후, G418 500 μg/ml을 함유한 DMEM 배지에서 37℃, 10% CO2 조건에서 배양하다가, 파장 450-520 nm의 빛의 조사 전후로 형광현미경 이미지를 수득하는 한편(도 10 상부 판넬), PKCγ-C2의 위치를 융합파트너인 황새형광단백질(YFP)의 형광을 검출함으로써 분석하였다(도 10 하부 판넬). 그 결과 STIM1 융합단백질에 의한 형광의 변화는 상기 실험예 2의 결과와 동일하였고, PKCγ-C2 도메인의 경우 광조사 전에는 세포질 내에 전반적으로 분산된 양상의 형광패턴이었으나(하부 판넬 좌측), 광조사후에는 세포막 부근의 형광이 더 강하게 나타나(하부 판넬 우측), PKCγ-C2 도메인이 세포막쪽으로 이동하였음을 알 수 있었다. 본 발명자들은 PKCγ-C2의 위치의 변화를 세포막에서의 형광의 강도를 측정함으로서 분석하였다(도 11). 그 결과도 마찬가지로 광 조사후 일정 시간(약 100 초) 경과 후, 세포막에서의 형광 강도가 약 5배 정도 증가하는 것으로 나타났다. The present inventors investigated the behavior of PKCγ-C2 activated by calcium in order to confirm whether intracellular biochemical changes can be investigated by intracellular influx of calcium by the light irradiation. Specifically, after transforming HeLa cells with the YFP-PKCγ-C2 construct prepared in Examples 1-4 and the mCherry-PHR-STIM1-ct construct prepared in Examples 1-3, G418 500 μg Incubated in DMEM medium containing / ml at 37 ℃, 10% CO 2 conditions, to obtain a fluorescence microscope image before and after the irradiation of light of wavelength 450-520 nm (Fig. 10 upper panel), while the position of PKCγ-C2 It was analyzed by detecting the fluorescence of the fusion partner Stork fluorescent protein (YFP) (Fig. 10 lower panel). As a result, the change of fluorescence by STIM1 fusion protein was the same as the result of Experiment 2, and in the case of PKCγ-C2 domain, the fluorescence pattern was generally dispersed in the cytoplasm before light irradiation (bottom left panel). The fluorescence in the vicinity of the cell membrane was stronger (lower panel right), indicating that the PKCγ-C2 domain moved to the cell membrane. The inventors analyzed the change in the position of PKCγ-C2 by measuring the intensity of fluorescence in the cell membrane (FIG. 11). As a result, the fluorescence intensity in the cell membrane increased by about 5 times after a certain time (about 100 seconds) after light irradiation.
따라서, 본 발명의 일 실시예에 따른 융합단백질 및 이를 이용한 광조사에 의한 세포내 칼슘 이온 농도의 증가방법은 세포 내에서의 칼슘 이온 의존적인 다양한 생화학적 반응을 연구하는데, 유용하게 사용될 수 있음을 알 수 있다.Therefore, the fusion protein and the method of increasing intracellular calcium ion concentration by light irradiation using the same according to an embodiment of the present invention can be useful for studying various calcium-dependent biochemical reactions dependent on intracellular calcium ion. Able to know.
본 발명은 상술한 실시예 및 실험예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다. Although the present invention has been described with reference to the above-described examples and experimental examples, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.
본 발명의 일 실시예에 따르면, 세포, 동물 또는 식물의 세포질 내 칼슘이온 농도를 시공간적으로 그리고 가역적으로 조절할 수 있다. 물론 본 발명의 범위가 한정되는 것은 아니다.According to one embodiment of the invention, the concentration of calcium ions in the cytoplasm of a cell, animal or plant can be temporally and reversibly controlled. Of course, the scope of the present invention is not limited.
전자파일로 첨부하였음Attached as an electronic file

Claims (23)

  1. STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이합체 형성 단백질이 연결된 융합단백질.A fusion protein wherein a photoinduced dimer-forming protein is linked to an N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein.
  2. 제1항에 있어서,The method of claim 1,
    상기 광유도 이합체 형성 단백질은 광유도 이형이합체 형성 단백질 또는 광유도 동형이합체 형성단백질인, 융합단백질.The photoinduced dimer forming protein is a photoinduced heterodimer forming protein or a photoinduced homodimer forming protein.
  3. 제2항에 있어서,The method of claim 2,
    상기 광유도 이형이합체 형성 단백질은 CIB(cryptochrome-interacting basic-helix-loop-helix protein), CIBN(N-terminal domain of CIB), Phy(phytochrome), PIF(phytochrome interacting factor), FKF1(Flavin-binding, Kelch repeat, F-box 1), GIGANTEA, CRY(chryptochrome) 또는 PHR(phytolyase homolgous region)인, 융합단백질.The photoinduced heterodimer-forming protein is CIB (cryptochrome-interacting basic-helix-loop-helix protein), CIBN (N-terminal domain of CIB), Phy (phytochrome), PIF (phytochrome interacting factor), FKF1 (Flavin-binding) , Kelch repeat, F-box 1), GIGANTEA, CRY (chryptochrome) or PHR (phytolyase homolgous region).
  4. 제2항에 있어서,The method of claim 2,
    상기 광유도 동형이합체 형성 단백질은 CRY(chryptochrome) 또는 PHR(phytolyase homolgous region)인, 융합단백질.The photoinduced homodimer-forming protein is CRY (chryptochrome) or PHR (phytolyase homolgous region), fusion protein.
  5. 제1항에 있어서, The method of claim 1,
    형광단백질을 추가적으로 포함하는, 융합단백질.A fusion protein further comprising a fluorescent protein.
  6. 제5항에 있어서,The method of claim 5,
    상기 형광단백질은 녹색형광단백질(green fluorescent protein, GFP), 황색형광단백질(yellow fluorescent protein, YFP), 적색형광단백질(red fluorescent protein, RFP), 주황형광단백질(orange fluorescent protein, OFP), 청록색형광단백질(cyan fluorescent protein, CFP), 청색형광단백질(blue fluorescent protein, BFP), 원적색형광단백질(far-red fluorescent protein) 또는 테트라시스테인 모티프(tetracystein motif)인, 융합단백질.The fluorescent protein is a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent A fusion protein, which is a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracystein motif.
  7. 제1항 내지 제6항 중 어느 한 항의 융합단백질을 암호화하는 폴리뉴클레오티드.A polynucleotide encoding the fusion protein of any one of claims 1 to 6.
  8. 제7항의 폴리뉴클레오티드를 포함하는 벡터.A vector comprising the polynucleotide of claim 7.
  9. 제8항의 벡터로 숙주세포를 형질전환시킨 형질전환 숙주세포.A transformed host cell transformed with the host cell with the vector of claim 8.
  10. 제8항의 벡터로 형질전환되어 상기 융합단백질을 발현할 수 있는 비인간 형질전환 동물.A non-human transgenic animal which is transformed with the vector of claim 8 to express the fusion protein.
  11. 제8항의 벡터로 형질전환되어 상기 융합단백질을 발현할 수 있는 형질전환 식물.A transgenic plant transformed with the vector of claim 8 to express the fusion protein.
  12. STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 동형이합체 형성 단백질이 연결된 융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 유전자컨스트럭트를 포함하는 발현벡터로 숙주세포를 형질전환하여 제조된 형질전환 숙주세포를 준비하는 단계; A fusion protein encoding a photoinduced homodimer-forming protein connected to the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein. Preparing a transformed host cell prepared by transforming the host cell with an expression vector comprising a gene construct in which the polynucleotide is operably linked to a promoter;
    상기 형질전환 숙주세포를 칼슘을 포함하는 배지에서 배양하는 배양단계; 및Culturing the transformed host cell in a medium containing calcium; And
    상기 배양중인 형질전환 숙주세포에 상기 광유도 동형이합체 형성 단백질의 동형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 세포질 내의 칼슘 이온 농도를 가역적으로 증가시키는 방법.A method of reversibly increasing the concentration of calcium ions in the cytoplasm comprising a light irradiation step of irradiating the transformed host cell in culture with light of a wavelength capable of inducing homodimer formation of the photoinduced homodimer-forming protein.
  13. STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 동형이합체 형성 단백질이 연결된 융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 유전자컨스트럭트를 포함하는 발현벡터로 형질전환되어 상기 융합단백질을 발현하는 형질전환 식물 또는 비인간 형질전환 동물를 준비하는 단계; 및A fusion protein encoding a photoinduced homodimer-forming protein connected to the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein. Preparing a transgenic plant or a non-human transgenic animal expressing the fusion protein by transforming the polynucleotide with an expression vector comprising a gene construct operably linked to a promoter; And
    상기 형질전환 식물 또는 비인간 형질전환 동물의 특정 기관 또는 조직에 상기 광유도 동형이합체 형성 단백질의 동형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 식물 또는 비인간 동물의 특정 기관 또는 조직에서의 세포질 내 칼슘 이온 농도를 가역적으로 증가시키는 방법.A specific organ of a plant or a non-human animal, comprising a light irradiation step of irradiating a specific organ or tissue of the transgenic plant or a non-human transgenic animal with light having a wavelength capable of inducing homodimer formation of the photoinduced homodimer-forming protein Or reversibly increasing the concentration of calcium ions in the cytoplasm in tissues.
  14. 제12항 또는 제13항에 있어서, The method according to claim 12 or 13,
    상기 광유도 동형이합체 형성 단백질은 CRY 또는 PHR인, 방법.Wherein said photoinduced homodimer forming protein is CRY or PHR.
  15. 제12항에 있어서, The method of claim 12,
    상기 숙주세포는 동물세포 또는 식물세포인, 방법.The host cell is an animal cell or plant cell.
  16. 제12항 또는 제13항에 있어서,The method according to claim 12 or 13,
    상기 융합단백질은 형광단백질을 추가로 포함하는, 방법.The fusion protein further comprises a fluorescent protein.
  17. 제16항에 있어서,The method of claim 16,
    상기 형광단백질은 녹색형광단백질(green fluorescent protein, GFP), 황색형광단백질(yellow fluorescent protein, YFP), 적색형광단백질(red fluorescent protein, RFP), 주황형광단백질(orange fluorescent protein, OFP), 청록색형광단백질(cyan fluorescent protein, CFP), 청색형광단백질(blue fluorescent protein, BFP), 원적색형광단백질(far-red fluorescent protein) 또는 테트라시스테인 모티프(tetracystein motif)인, 방법.The fluorescent protein is a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent Protein (cyan fluorescent protein, CFP), blue fluorescent protein (BFP), far-red fluorescent protein, or tetracysteine motif.
  18. STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이형이합체 형성 단백질이 연결된 제1융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제1유전자컨스트럭트를 포함하는 제1발현벡터 및 STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 상기 광유도 이형이합체 형성 단백질과 광조사에 의해 이형이합체를 형성하는 짝 단백질이 연결된 제2융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제2유전자컨스트럭트를 포함하는 제2발현벡터로 숙주세포를 형질전환하여 제조된 형질전환 숙주세포를 준비하는 단계; A first fusion protein having a photoinduced heterodimer-forming protein linked to the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A first expression vector and a stromal interaction molecule 1 (STIM1) protein or a cytoplasm cleaved from the N-terminus to the transmembrane domain of the STIM1 protein, wherein the polynucleotide encoding comprises a first gene construct operably linked to a promoter. a polynucleotide encoding a second fusion protein linked to the promoter at the N-terminus or C-terminus of the cytosolic fragment and a partner protein which forms a heterodimer by irradiation with light. Transformation enzymes prepared by transforming host cells with a second expression vector comprising a second gene construct Preparing a cell;
    상기 형질전환 숙주세포를 칼슘을 포함하는 배지에서 배양하는 배양단계; 및 Culturing the transformed host cell in a medium containing calcium; And
    상기 배양중인 형질전환 숙주세포에 상기 광유도 이형이합체 형성 단백질과 상기 짝 단백질 사이의 이형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 세포질 내의 칼슘 이온 농도를 가역적으로 증가시키는 방법.Irreversibly increasing the concentration of calcium ions in the cytoplasm comprising a light irradiation step of irradiating the cultured host cell in culture with light having a wavelength capable of inducing heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein. How to let.
  19. STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 광유도 이형이합체 형성 단백질이 연결된 제1융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제1유전자컨스트럭트를 포함하는 제1발현벡터 및 STIM1(stromal interaction molecule 1) 단백질 또는 상기 STIM1 단백질의 N-말단으로부터 막통과 도메인까지 절단된 세포질(cytosolic) 단편의 N-말단 또는 C-말단에 상기 광유도 이형이합체 형성 단백질과 광조사에 의해 이형이합체를 형성하는 짝 단백질이 연결된 제2융합단백질을 암호화하는 폴리뉴클레오티드가 프로모터에 작동가능하게 연결된 제2유전자컨스트럭트를 포함하는 제2발현벡터로 형질전환된 형질전환 식물 또는 비인간 형질전환 동물을 준비하는 단계; 및A first fusion protein having a photoinduced heterodimer-forming protein linked to the N-terminus or C-terminus of a stromal interaction molecule 1 (STIM1) protein or a cytosolic fragment cleaved from the N-terminus to the transmembrane domain of the STIM1 protein A first expression vector and a stromal interaction molecule 1 (STIM1) protein or a cytoplasm cut from the N-terminus of the STIM1 protein to a transmembrane domain, wherein the polynucleotide encoding comprises a first gene construct operably linked to a promoter. a polynucleotide encoding a second fusion protein linked to the promoter at the N-terminus or C-terminus of the cytosolic fragment and a partner protein which forms a heterodimer by irradiation with light. Transformed plants or non-human transgenics transformed with a second expression vector comprising a gene construct Steps to prepare an animal; And
    상기 형질전환 식물 또는 비인간 형질전환 동물의 특정 기관 또는 조직에 상기 광유도 이형이합체 형성 단백질과 상기 짝 단백질 사이의 이형이합체 형성을 유도할 수 있는 파장의 빛을 조사하는 광조사단계를 포함하는 식물 또는 비인간 동물의 특정 기관 또는 조직에서의 세포질 내 칼슘 이온 농도를 가역적으로 증가시키는 방법.A plant comprising a light irradiation step of irradiating light of a wavelength capable of inducing heterodimer formation between the photoinduced heterodimer-forming protein and the partner protein to a specific organ or tissue of the transgenic plant or a non-human transgenic animal or A method of reversibly increasing the concentration of calcium ions in the cytoplasm in certain organs or tissues of non-human animals.
  20. 제18항 또는 제19항에 있어서,The method of claim 18 or 19,
    상기 광유도 이형이합체 형성 단백질은 CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY 또는 PHR인, 방법.The photoinduced heterodimer forming protein is CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY or PHR.
  21. 제18항 또는 제19항에 있어서,The method of claim 18 or 19,
    상기 짝 단백질은 CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY 또는 PHR인, 방법.And the partner protein is CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY or PHR.
  22. 제18항 또는 제19항에 있어서,The method of claim 18 or 19,
    상기 제1융합단백질 및/또는 제2융합단백질은 추가로 형광단백질을 포함하는, 방법.Wherein said first fusion protein and / or second fusion protein further comprise a fluorescent protein.
  23. 제23항에 있어서,The method of claim 23, wherein
    상기 형광단백질은 녹색형광단백질(green fluorescent protein, GFP), 황색형광단백질(yellow fluorescent protein, YFP), 적색형광단백질(red fluorescent protein, RFP), 주황형광단백질(orange fluorescent protein, OFP), 청록색형광단백질(cyan fluorescent protein, CFP), 청색형광단백질(blue fluorescent protein, BFP), 원적색형광단백질(far-red fluorescent protein) 또는 테트라시스테인 모티프(tetracystein motif)인, 방법.The fluorescent protein is a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent Protein (cyan fluorescent protein, CFP), blue fluorescent protein (BFP), far-red fluorescent protein, or tetracysteine motif.
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