KR101369853B1 - Fusion proteins capable of regulating intracellular calcium ion level by light and uses thereof - Google Patents

Abstract

The present invention relates to a method and kit for reversible inhibition of protein function using surface induced nanocluster formation, wherein the cytoplasmic substrate is cleaved from the stromal interaction molecule 1 (STIM1) protein or the N-terminus to the transmembrane domain of the STIM1 protein. Provided is a fusion protein linked to a photoinduced dimer forming protein at the N-terminus or C-terminus of the fragment.

Description

Fusion proteins capable of regulating intracellular calcium ion level by light and uses approximately

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. 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.

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).

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).

Luik et al. (FKBP-rapamycin-binding) can form 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).

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.

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 do not limit the scope of the present invention.

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.

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.

In the fusion protein, the light oil dimer-forming protein may be a light oil-releasing duplex-forming protein and / or a light-oil-atom duplex-forming protein. The light oil-type heterodimer-forming protein may be selected from the group consisting of cryptochrome-interacting basic-helix-loop-helix protein (CIB), N-terminal domain of CIB, phytochrome, phytochrome interacting factor, FKF1 , Kelch repeat, F-box 1), GIGANTEA, CRY (chryptochrome) or PHR (phytolyase homologous region), and the homodimerization protein may be CRY or PHR. Conventionally, it has been known that CRY or PHR forms a homodimer irrespective of light irradiation, but it has been found by the present inventors to form a homodimer by light irradiation. Therefore, CRY or PHR is a protein capable of not only forming a heterodimer by light irradiation but also forming a homodimer.

In addition, in the fusion protein, a 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.

According to another aspect of the present invention, there is provided a polynucleotide encoding said fusion protein.

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.

According to another aspect of the present invention, there is provided a transformed host cell transformed with said vector as said vector. The transfected host cell may express the fusion protein in the cytoplasm.

According to another aspect of the present invention there is provided a non-human transgenic animal that is transformed with said vector to express said 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.

According to another aspect of the present invention there is provided a transgenic plant which is transformed with said vector to express said fusion protein. The transgenic plant may be a germplasm plant or a herbaceous plant. The germplasm plant may be a monocot plant or a dicotyledon plant. The monocotyledonous plant may be a rice plant, a lycopene plant, or an orchid plant. And the beans may be soybeans, mung beans, peas, or beans, and the beans may be bee, watermelon, amber, cucumber, melon, or melon, and the asteraceae may be chrysanthemum, The rosacea may be an apple, a pear, a peach, a rose or a strawberry, and the crucifer may be a radish, a Chinese cabbage, a rape, a bean, , Horseradish or Arabidopsis thaliana .

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.

In the method, the photoinduced homodimer forming protein may be Cry or PHR.

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.

In the above 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.

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.

In the method, the photoinduced homodimer forming protein may be Cry or PHR.

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.

In the above 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.

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.

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.

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.

In the method, the photoinduced heterodimer forming protein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY or PHR.

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.

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). 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.

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.

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. The scope of the present invention is not limited thereto.

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: endoplasmic reticulum; And
PM: plasma membrane.
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.
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.
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.
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.
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 terms used in this document are defined as follows.

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.

As used herein, a " partner protein " refers to a protein that interacts with a light-oil heterodimer forming protein to form a heterodimer when light of a particular wavelength is irradiated.

As used herein, the term " heterodimer "means that two different proteins interact to form a complex.

As used herein, "homodimer" means that two proteins interact with each other to form a complex.

As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can perform its function. In other words, the fact that a polynucleotide encoding a specific protein is operatively linked to a promoter implies that it is transcribed into mRNA by the action of the promoter and is linked so as to be translated into the protein, and a polynucleotide encoding a specific protein Operatively linked to a polynucleotide encoding another protein means that the particular protein is linked to be expressed in the form of a fusion protein with another protein.

As used herein, "CIB" refers to cryptochrome-interacting basic-helix-loop-helix protein, and typically includes CIB1 (GenBank No .: NM_119618 ).

As used herein, "CIBN" refers to a site that interacts with cryptochrome (CRY) upon light irradiation as the N-terminus of the CIB.

As used herein, the term "CRY" refers to a chryptochrome protein, typically CRY2 (GenBank No .: NM_100320) of the Arabidopsis thaliana.

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).

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)

As used herein, the term "PIF" refers to a phytochrome interacting factor. Typical examples include Arabidopsis PIF1 (GenBank No .: NM_001202630), PIF3 (GenBank No .: NM_179295), PIF4 : NM_180050), PIF5 (GenBank No .: NM_180690), PIF6 (GenBank No .: NM_001203231), or PIF7 (GenBank No .: NM_125520).

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).

As used herein, "GIGANTEA" is associated with phytochrome signaling and is known as a protein that regulates flowering time of flowers.

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).

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. Generally, transgenic animals can be produced by genetically manipulating germ cells, but they can be prepared by cloning animal production methods by genetic modification of somatic cells and nuclear replacement. In the case of transgenic plants, somatic cells Can be produced by infecting with Agrobacterium containing foreign genes and then through the process of dedifferentiation and regeneration. 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).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user. Also, for convenience of explanation, the components may be exaggerated or reduced in size.

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. 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.

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).

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.

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. PHR interacts with each other by light irradiation, resulting in the formation of a complex of fusion proteins including the whole STIM1 protein, and calcium ions are introduced into the cell by Orai1 protein in the cell membrane interacting with the STIM1 protein to form pores of calcium channels. Can be.

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 was increased to about 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. 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). Therefore, 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. 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.

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.

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.

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.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.

Example 1: Construction of vector

1-1: Construction of mCerulean-PHR-STIM1-ct Construct

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: Construction of the mCitrine-PHR-STIM1-ct Construct

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: Construction of the mCherry-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).

1-4: Construction of the YFP-PKCγ-C2 Construct

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.

Experimental Example 1: Confirmation of homodimer formation of PHR-containing STIM1 fusion protein by photoinduction

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.

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).

Experimental Example 2: Check the influx of calcium by the light induction

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.

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.

Experimental Example 3: Confirmation of calcium-dependent biochemical change by light induction

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.

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.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

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Claims (23)

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.
The method of claim 1,
The photoinduced dimer forming protein is a photoinduced heterodimer forming protein or a photoinduced homodimer forming protein. 3. The method of claim 2,
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). 3. The method of claim 2,
The photoinduced homodimer-forming protein is CRY (chryptochrome) or PHR (phytolyase homolgous region), fusion protein. The method of claim 1,
A fusion protein additionally comprising a fluorescent protein. 6. The method of claim 5,
The fluorescent protein may be selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP) A fusion protein that is a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracystein motif. A polynucleotide encoding the fusion protein of any one of claims 1 to 6. 8. A vector comprising the polynucleotide of claim 7. A transformed host cell transformed with the vector of claim 8. A non-human transgenic animal capable of transforming with the vector of claim 8 to express said fusion protein. 9. A transgenic plant capable of being transformed with the vector of claim 8 to express said fusion protein. 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 the promoter;
Culturing the transformed host cell in a medium containing calcium; And
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. 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 transgenic plant or a non-human transgenic animal expressing the fusion protein by transforming the polynucleotide with an expression vector including a gene construct operably linked to a promoter; And
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 of the tissue. The method according to claim 12 or 13,
Wherein said photoinduced homodimer forming protein is CRY or PHR. The method of claim 12,
Wherein said host cell is an animal cell or a plant cell. The method according to claim 12 or 13,
Wherein said fusion protein further comprises a fluorescent protein. 17. The method of claim 16,
The fluorescent protein may be selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP) Wherein the fluorescent protein is a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracystein motif. 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;
Culturing the transformed host cell in a medium containing calcium; And
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. 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. Transformed plants or non-human transgenics transformed with a second expression vector comprising a gene construct Steps to prepare an animal; And
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. The method according to claim 18 or 19,
The photoinduced heterodimer forming protein is CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY or PHR 20. The method according to claim 18 or 19,
And the partner protein is CIB, CIBN, PhyB, PIF6, FKF1, GIGANTEA, CRY or PHR. 20. The method according to claim 18 or 19,
Wherein said first fusion protein and / or second fusion protein further comprise a fluorescent protein. The method of claim 22,
The fluorescent protein may be selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP) Wherein the fluorescent protein is a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), a far-red fluorescent protein, or a tetracystein motif.

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