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WO2012111772A1 - Polypeptide, acide nucléique isolé, vecteur recombinant, nécessaire de transfert de gènes, transformant et procédé de régulation de la signalisation calcique intracellulaire - Google Patents

Polypeptide, acide nucléique isolé, vecteur recombinant, nécessaire de transfert de gènes, transformant et procédé de régulation de la signalisation calcique intracellulaire Download PDF

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WO2012111772A1
WO2012111772A1 PCT/JP2012/053705 JP2012053705W WO2012111772A1 WO 2012111772 A1 WO2012111772 A1 WO 2012111772A1 JP 2012053705 W JP2012053705 W JP 2012053705W WO 2012111772 A1 WO2012111772 A1 WO 2012111772A1
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region
baccs
orai1
polypeptide
amino acid
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智浩 石井
隆夫 中田
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Tokyo Medical and Dental University NUC
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Tokyo Medical and Dental University NUC
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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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a polypeptide that optically controls a calcium signal, an isolated nucleic acid, a recombinant vector, a gene transfer kit, a transformant, and a method for regulating an intracellular calcium signal.
  • Non-Patent Document 1 a system that controls intracellular signals by light stimulation.
  • Non-Patent Document 2 channel rhodopsin 2 (bacterial-derived photoreceptive ion channel) that can change the membrane potential of nerve cells by light stimulation is used as such a system.
  • polypeptides produced by fusing a functional domain of a target cell signal protein and a photosensitive region contained in a protein derived from a plant or the like are also used.
  • the A specific example of such a polypeptide is a fusion protein of a photosensitive region LOV2-J ⁇ derived from phototropin 1, which is a photoreceptor protein of oats, and an animal cytoskeleton control protein Rac1.
  • the said fusion protein can change the form of a cell locally by irradiation of light (nonpatent literature 3).
  • Non-patent Document 4 establishment of a system that can control the calcium signal by light stimulation is eagerly desired (Non-Patent Document 5), and various attempts have been made to establish such a system.
  • a conventional system that controls calcium signals by light stimulation includes a system using caged calcium (Non-Patent Document 1; Non-Patent Document 2).
  • Caged compounds such as caged calcium are organic synthetic molecules that are degraded by light stimulation.
  • the calcium signal can be activated rapidly, but the caged calcium is a small molecule, so that it easily diffuses and the spatial control of the calcium signal is not easy.
  • the process of introducing caged calcium from the outside of the cell is difficult to operate, and there is a limit in application to animals and the like.
  • the present invention aims to provide a polypeptide capable of easily photocontrolling a calcium signal, an isolated nucleic acid, a recombinant vector, a gene transfer kit, a transformant, and a method for regulating an intracellular calcium signal. To do.
  • the present inventors have determined that a predetermined portion of the amino acid sequence of the LOV2-J ⁇ region of phototropin, which is a light-sensitive protein derived from oats, and STIM1, which is a regulatory protein of calcium channel Orai1 It has been found that the above problems can be solved by a polypeptide that combines a predetermined portion of the amino acid sequence of the CAD region.
  • the present invention LOV2 region consisting of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 1,
  • a J ⁇ region consisting of an amino acid sequence that maintains the function as an optical switch of SEQ ID NO: 2, SEQ ID NO: 3, or LOV2-J ⁇ , and in which one or more amino acids are substituted in the amino acid sequence of SEQ ID NO: 2 or 3,
  • a CAD region consisting of SEQ ID NO: 4 or a CAD region consisting of an amino acid sequence capable of activating calcium channel Orai1 and having one or more amino acids deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4.
  • a polypeptide comprising an amino acid sequence comprising: The LOV2 region, the J ⁇ region and the CAD region are arranged in the order of the LOV2 region, the J ⁇ region and the CAD region from the N-terminus toward the C-terminus, Provided is a polypeptide (BACCS) in which no amino acid sequence is interposed between the C-terminus of the J ⁇ region and the N-terminus of the CAD region.
  • BACCS polypeptide
  • the present inventors have also found that the above problem can be solved by a polypeptide combining the amino acid sequence of calcium channel Orai1 and BACCS.
  • the present invention The Orai1 region that can function as SEQ ID NO: 5 or an amino acid sequence that can function as a calcium channel and in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 5.
  • a polypeptide (Orai1-BACCS) is also provided.
  • a first linker may be interposed between the C-terminus of the Orai1 region and the N-terminus of the first BACCS region.
  • a second linker may be interposed between the C-terminal of the first BACCS region and the N-terminal of the second BACCS region.
  • the present inventors have also found that the above problem can be solved by a polypeptide in which BACCS is connected in two in tandem.
  • a first BACCS region; A second BACCS region, and A polypeptide (BACCS) that functions as SEQ ID NO: 5 or a calcium channel and that comprises an amino acid sequence that does not include the Orai1 region consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: X2) is also provided.
  • a linker may be interposed between the C-terminal of the first BACCS region and the N-terminal of the second BACCS region.
  • the present invention also provides an isolated nucleic acid encoding the polypeptide of the present invention.
  • the present invention also provides a recombinant vector containing the nucleic acid.
  • the present invention also provides a gene transfer kit containing the above recombinant vector.
  • the present invention also provides a transformant which is an isolated cell or non-human animal containing the above recombinant vector.
  • the present invention also provides a method for regulating an intracellular calcium signal, which regulates an intracellular calcium signal of the transformant by manipulating the intensity of light irradiated to the transformant.
  • the present invention also provides a method for regulating an intracellular calcium signal in which the polypeptide is BACCS and the cells express Orai1.
  • the present invention also provides a method for regulating an intracellular calcium signal, wherein the polypeptide is Orai1-BACCS.
  • a polypeptide capable of easily photocontrolling a calcium signal is provided.
  • (A) shows a schematic diagram of “BACCS”, and (B) shows a schematic diagram of “Orai1-BACCS”.
  • a comparison of the amino acid sequences of Orai1, Orai2 and Orai3 in various animal species is shown.
  • a comparison of the amino acid sequences of the CAD region of STIM1 in various animal species is shown.
  • a comparison of the amino acid sequences of the J ⁇ region in LOV2-J ⁇ in various plant species is shown.
  • (A) shows a schematic diagram of a protein (LOV lit-CAD) in which a CAD region is fused to a light mutant of LOV2-J ⁇ .
  • (B) shows imaging of the localization of Orai1 (Orai1-YFP) incorporating yellow fluorescent protein in HEK293 cells (left), LOV-lit-CAD (tdTomato-LOV-CAD) incorporating red fluorescent protein. Imaging of localization in HEK293 cells (middle panel) and the result of merging these two results (right panel).
  • (C) shows the results of the presence or absence of transfer of LOV lit-CAD to the cell membrane due to various deletions of the N-terminal side of the CAD region.
  • (A) shows a schematic diagram of a protein (LOV dark-CAD) in which a CAD region is fused to a dark variant of LOV2-J ⁇ .
  • (B) shows imaging of the localization of Orai1 (Orai1-YFP) incorporating yellow fluorescent protein in HEK293 cells (left), LOV dark-CAD (tdTomato-LOV dark-CAD) incorporating red fluorescent protein. ) Shows the imaging of the localization in HEK293 cells (middle figure) and the result of merging these two results (right figure).
  • (C) does not prevent inhibition of the function of the CAD region by the LOV2-J ⁇ dark mutant (ie, does not localize LOV dark-CAD to the cell membrane).
  • Four candidates for the amino acid sequence of the J ⁇ region to the membrane
  • the identification results of “none” for localization are shown.
  • FIG. 1 shows the location of the polypeptide when light is blocked in HEK293 cells co-expressed with the polypeptide (tdTomato-LOV (404-538) -CAD (347-448)), Orai1 and NFAT-GFP.
  • the present imaging (upper figure) and the imaging of polypeptide localization (lower figure) when irradiated with light of 470 nm (blue light) for 20 minutes are shown.
  • FIG. 1 shows the presence or absence of nuclear translocation of the NFAT transcription factor by the LOV2 region amino acid sequence, various J ⁇ region amino acid sequences, and a polypeptide (LOV-CAD) combined with the CAD region amino acid sequence, and LOV ⁇ The result of the presence or absence of transfer to the cell membrane of CAD is shown.
  • (A) expresses a polypeptide (mCherry-BACCS-2A-Orai1) in which red fluorescent protein, BACCS, 2A peptide and Orai1 are fused in order from the N-terminus to the C-terminus in HEK293 cells, and light at 470 nm (blue)
  • FIG. 1 shows the presence or absence of nuclear translocation of the NFAT transcription factor by the LOV2 region amino acid sequence, various J ⁇ region amino acid sequences, and a polypeptide (LOV-CAD) combined with the CAD region amino acid sequence, and LOV ⁇ The result of the presence or absence of transfer to the cell membrane of CAD.
  • FIG. 2 shows the calcium imaging results using Fluo-4, a calcium fluorescent probe, after continuous irradiation of (light) for the duration of the test.
  • the left figure shows the result of identification of the polypeptide-expressing cell by mCherry fluorescence.
  • the center and right diagrams show calcium imaging results before and after light stimulation, respectively.
  • (B) shows the time course of calcium concentration in 8 cells.
  • (A) shows that Orai1-BACCS (Orai1-mCherry-BACCS ⁇ 2) incorporating a red fluorescent protein is expressed in HEK293 cells, and 470 nm light (blue light) is irradiated continuously during the test period.
  • the results of calcium imaging using Fluo-4, a calcium fluorescent probe are shown.
  • the left figure shows the result of identification of the polypeptide-expressing cell by mCherry fluorescence.
  • the center and right diagrams show calcium imaging results before and after light stimulation, respectively.
  • (B) shows the time course of calcium concentration in HEK293 cells expressing Orai1-BACCS incorporating HA tag and / or fluorescent substance.
  • (C) shows that Orai1-BACCS (Orai1-HA-BACCS ⁇ 2-IRES-YFP) incorporating an HA tag and a fluorescent substance is expressed in HEK293 cells, and light irradiation and light blocking are performed three times.
  • 2 shows the time course of intracellular calcium concentration observed by calcium imaging using Rhod-3, a red fluorescent calcium sensor.
  • (A) is a polypeptide in which only one BACCS is fused to Orai1 and incorporates a red fluorescent protein (Orai1-tdTomato-BACCS ⁇ 1) and a polypeptide in which a yellow fluorescent protein is incorporated into the transcription factor NFAT (NFAT-YFP) Shows the result after co-expressing in HEK293 cells and irradiating with 470 nm light (blue light) for 20 minutes.
  • (B) As a control experiment, two BACCS were connected in tandem, and Orai1-tdTomato-BACCS ⁇ 2, NFAT-GFP incorporating a red fluorescent protein was coexpressed in HEK293 cells, and light at 470 nm (blue light) Shows the result after irradiation for 20 minutes.
  • (A) shows a schematic diagram of Orai1 (L273D) -BACCS ⁇ 2 in which Orai1 mutation (Orai1 (L273D)) is incorporated into Orai1-BACCS.
  • (B) irradiates HEK293 cells expressing Orai1 (L273D) -BACCS (Orai1 (L273D) -HA-BACCS ⁇ 2-igY) incorporating a HA tag and a fluorescent substance with 470 nm light (blue light). Calcium imaging using Rhod-3, a red fluorescent calcium sensor, before and after irradiation for 10 minutes.
  • (A) shows a schematic diagram of a polypeptide (Orai1-BACCS (347-630) ⁇ 2) obtained by extending the C-terminus of the CAD region so as to include the STIM1 CDI (Calcium dependent activation) region (the shaded area is CDI). Area).
  • (B) shows Orai1-BACCS (Orai1-HA-BACCS ⁇ 2-igY) and Orai1-BACCS (347-630) (Orai1-HA-BACCS (347-630) ⁇ 2) incorporating an HA tag and a fluorescent substance.
  • -IgY) shows calcium imaging results using Rhod-3, a red fluorescent calcium sensor, for cells expressing.
  • (A) shows a schematic diagram of “BACCS ⁇ 2”.
  • (B) shows calcium imaging results for cells expressing BACCS ⁇ 2-igY (B ⁇ 2) and Orai1-HA-BACCS ⁇ 2-igY (OB ⁇ 2). Calcium imaging results for cells expressing BACCS ⁇ 2-igY (B ⁇ 2) and YFP-BACCS ⁇ 2 (YB ⁇ 2) are shown.
  • BACCS Blue light-activated Calcium Channel Switch
  • endogenous Orai1 in the cells, or Orai1 expressed together with BACCS by gene transfer into the cells can be opened by irradiation with blue light.
  • Orai1 activated by the irradiation of blue light returns to an inactive state by blocking the blue light.
  • BACCS can be easily expressed in a cell-specific manner by a gene recombination technique known in the technical field to which the present invention belongs, the activity of Orai1 can be locally controlled. Therefore, according to BACCS, the spatial control of the calcium signal can be easily performed. Moreover, since the function of BACCS can be controlled only by the operation of the intensity of light to be irradiated, the calcium signal can be controlled by an easy operation.
  • LOV2-J ⁇ The photosensitivity of oat-derived phototropin 1 depends on the region of amino acids 404-543 in phototropin 1, which is called LOV2-J ⁇ (Harper et al., 2003). Since LOV2-J ⁇ has an absorption maximum wavelength of 447 nm that is a blue region (Salomon et al., 2000), the structure is changed by irradiation with blue light. Specifically, in a state where light is blocked, the LOV2 region and the J ⁇ region are bonded by a hydrophobic bond. On the other hand, when light is irradiated, the hydrophobic bonds of the LOV2 region and the J ⁇ region are removed. This is called “bright state”.
  • LOV2-J ⁇ When the signal protein is fused to LOV2-J ⁇ , LOV2-J ⁇ functions as a steric hindrance of the signal protein in a state where light is blocked. On the other hand, when light is irradiated, the structure of LOV2-J ⁇ changes, and the steric hindrance of the signal protein is eliminated. As a result, a desired intracellular signal can be generated.
  • the light for bringing LOV2-J ⁇ into a bright state may be in the blue region (510 nm or less).
  • the structure of LOV2-J ⁇ can be easily changed by using a commercially available blue LED.
  • the light irradiation time is not particularly limited, but LOV2-J ⁇ can be efficiently brought into a bright state by irradiation in milliseconds (Nakasone et al., 2008).
  • the wavelength of light to be irradiated may be longer than 510 nm or light may be blocked.
  • LOV2 area Among LOV2-J ⁇ s constituting the polypeptide of the present invention, the LOV2 region corresponds to positions 404 to 522 of the amino acid sequence of oat-derived phototropin.
  • the LOV2 region is conserved among many plants.
  • homology with the amino acid sequence of the oat LOV2 region is as follows: Asian rice phototropin 1; 81%, Arabidopsis phototropin 1; 87%, Arabidopsis photo Tropine 2; 83%, pea phototropin 1; 87%, broad bean phototropin 1; corn phototropin 1; 95%, tomato phototropin 1; 87%.
  • the LOV2 region of plant species in which the LOV2 region is functional has 80% or more sequence identity with the oat amino acid sequence.
  • BACCS can exert a light-sensitive action.
  • a region related to the function of the LOV2 region is included in the amino acid sequence can be identified, for example, by irradiating or blocking blue light on the obtained polypeptide and observing a circular dichroism spectrum. It is therefore possible to replace the oat LOV2 region with sequences from various plant species or synthetic sequences.
  • amino acid sequence corresponding to the LOV2 region constituting the polypeptide of the present invention positions 404 to 522 of the amino acid sequence of oat-derived phototropin, 80% or more, 85% or more, 90% or more, or 95% or more
  • amino acid sequence having the following sequence identity can be used.
  • J ⁇ region Of the LOV2-J ⁇ constituting the polypeptide of the present invention, the J ⁇ region corresponds to positions 523-535 or 523-538 of the amino acid sequence of phototropin derived from oats.
  • the J ⁇ region is an important region for the BACCS to perform a desired reaction (hereinafter referred to as “optical switch”) that opens Orai1 by light irradiation and closes Orai1 by blocking light. .
  • optical switch a desired reaction
  • the amino acid sequence of the J ⁇ region in BACCS is directly bound to the amino acid sequence of the CAD region, which is a regulatory protein of Orai1, without intervening the amino acid sequence.
  • the steric hindrance of the CAD region which is a signal protein, is eliminated.
  • the CAD region binds to Orai1, and Orai1 is activated, and the function of the CAD region as a control protein of Orai1 is not inhibited.
  • Orai1 binds to Orai1
  • Orai1 is activated, and the function of the CAD region as a control protein of Orai1 is not inhibited.
  • a light-sensitive polypeptide using positions 404-546 (Wu et al., 2009) and 404-543 (Stickland et al., 2008) of LOV2-J ⁇ has been produced,
  • the optical switch was not completely cut off and the signal was not blocked. This is thought to be due to insufficient steric hindrance at the signal functional site of the signal protein caused by the presence of the LOV2 region.
  • BACCS having the phototropin amino acid sequence 523-535 or 523-538 as the J ⁇ region shows a desired response to light.
  • BACCS having these sequences is preferable because calcium signals can be regulated by blue light irradiation in cells.
  • BACCS having the phototropin amino acid sequence 523-538 as the J ⁇ region is particularly preferred because it can strongly regulate the calcium signal in response to light.
  • BACCS having the phototropin amino acid sequence 523-535 as the J ⁇ region also induces a calcium signal in the light-blocked state, but the intensity of the signal can be adjusted by light irradiation.
  • This “L” is important even when the CAD region directly bound to the J ⁇ region activates Orai1. For this reason, in a state where light is blocked, this “L” in the J ⁇ region is used for hydrophobic binding to the LOV2 region, and as a result, the CAD region is inhibited from interacting with Orai1.
  • BACCS has a J ⁇ region corresponding to positions 523 to 535 or 523 to 538 of the amino acid sequence of phototropin
  • the activity of Orai1 depends on the operation of the intensity of the irradiated light.
  • control can be controlled.
  • BACCS having an amino acid sequence in which one or a plurality of amino acids are substituted in the amino acid sequence of the J ⁇ region and maintaining the function as an optical switch of LOV2-J ⁇ also has an intensity of irradiated light. Since a desired reaction can be caused similarly by exerting or inhibiting the function of the CAD region according to the operation, it is included in the scope of the present invention.
  • amino acid sequence in which one or a plurality of amino acid sequences are substituted means positions 523-535 or 523-538 of the amino acid sequence of phototropin derived from oats, which is a J ⁇ region constituting the polypeptide of the present invention. Refers to an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more. Whether or not the amino acid sequence contains an amino acid sequence that maintains the function of LOV2-J ⁇ as an optical switch can be identified by, for example, observing a circular dichroism spectrum of the obtained polypeptide.
  • the CAD region constituting the polypeptide of the present invention corresponds to positions 347 to 448 of the amino acid sequence of the CAD region of human STIM1.
  • the CAD region alone can control the activity of the calcium channel Orai1.
  • LOV2-J ⁇ in BACCS becomes bright by light irradiation, the CAD region is activated and binds to Orai1 and as a result activates Orai1.
  • the calcium channel Orai1 can be activated and the activity of the calcium channel Orai1 can also be controlled for BACCS having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of the CAD region.
  • the “amino acid sequence in which one or more amino acids are deleted, substituted, or added” means positions 347 to 448 of the amino acid sequence of the CAD region of human STIM1, which is the CAD region constituting the polypeptide of the present invention.
  • the amino acid sequence includes an amino acid sequence that activates the calcium channel Orai1, for example, the amino acid sequence is co-expressed with the Orai1 region, and whether or not there is an influx of calcium ions, the localization of NFAT is observed. Can be identified.
  • the present invention also provides a protein (hereinafter referred to as “Orai1-BACCS”) in which BACCS is connected in tandem with the C-terminus of the amino acid sequence of calcium channel Orai1.
  • BACCS can open and close Orai1, which is a calcium ion channel, in a light-dependent manner when endogenous Orai1 is expressed or coexpressed with the Orai1 gene in cells. Therefore, by combining Orai1 and BACCS and expressing them as a single polypeptide, the spatial distance between Orai1 and BACCS can be reduced, and more efficient light compared to a polypeptide consisting only of BACCS. Functions as an activated calcium channel.
  • Orai1 can be efficiently activated when Orai1 and CAD regions are present in a ratio of 1: 2, and Orai1 is constitutive when two CAD regions are connected in tandem to the C-terminus of Orai1. Is reported to be activated (Li et al., 2010). Therefore, the present inventors produced Orai1-BACCS having a structure in which BACCS is connected in tandem to the C-terminus of Orai1.
  • Orai1 is a store-operated calcium channel that is localized on the cell membrane and is involved in store-operated calcium influx. Orai1 activity is controlled by the CAD region of the endoplasmic reticulum protein STIM1, which is localized in the endoplasmic reticulum (Hogan et al., 2010). Orai1 has an important effect particularly in immune cells.
  • Regions within human STIM1 that activate Orai1 include CAD (amino acids 342-448; Park et al., 2009), SOAR (amino acids 344-442; Yuan et al., 2009), OASF (amino acids 233-450 or 474) Muik et al., 2009), CCB9 (amino acids 339-444; Kawasaki et al., 2009) have been identified.
  • the region at amino acid positions 342 to 440 in human STIM1 cannot activate Orai1 (Park et al., 2009).
  • Orai1 constituting the polypeptide of the present invention corresponds to position 1-301 of the amino acid sequence of human Orai1.
  • Orai1 not only has high sequence similarity to Orai2 and Orai3, but is conserved among various species such as nematodes and humans (Hogan et al., 2010). For this reason, the sequence of Orai1 constituting Orai1-BACCS is very likely to function as a calcium channel not only from humans but also from various species of homologues. Therefore, these homologues can be substituted as Orai1 constituting the polypeptide of the present invention. In addition, if a region for human Orai1 to function as a calcium channel is conserved, one or more amino acids may be deleted, substituted, or added at position 1-301 of the amino acid sequence of human Orai1. It can be used as Orai1 constituting the polypeptide of the present invention.
  • Orai1 which constitutes the polypeptide of the present invention, positions 1-301 of the amino acid sequence of human Orai1, 80% or more, 85% An amino acid sequence having a sequence identity of 90% or more, or 95% or more.
  • the amino acid sequence is coexpressed with the CAD region, and whether or not there is an influx of calcium ions, the localization of NFAT is observed Can be identified.
  • Linker A small amino acid sequence between the C-terminal of Orai1 and the N-terminal of the first BACCS, and between the C-terminal of the first BACCS and the N-terminal of the second BACCS, constituting the Orai1-BACCS, respectively. Fragments (hereinafter referred to as “linkers”) may intervene.
  • the linker may be a fluorescent protein (mCherry, YFP, etc.) and the like.
  • the linker interposed between the C-terminal of Orai1 and the N-terminal of the first BACCS may be a linker consisting of an amino acid sequence of 20 residues or more from the viewpoint of avoiding steric hindrance.
  • the linker interposed between the C-terminal of the first BACCS and the N-terminal of the second BACCS may be a linker consisting of an amino acid sequence of 25 residues or more from the viewpoint of avoiding steric hindrance.
  • BACCS ⁇ 2 The present invention also provides a protein in which BACCS is connected in two in tandem (hereinafter referred to as “BACCS ⁇ 2”).
  • CAD can interact with one Orai1 in the form of a dimer and efficiently activate Orai1 (Li et al., 2010). Therefore, according to BACCS (that is, BACCS ⁇ 2) connected in tandem, CAD can be efficiently made into a dimer-like structure, so that endogenous Orai1 can be activated efficiently.
  • a linker may be interposed between the C terminus of the first BACCS region and the N terminus of the second BACCS region constituting the BACCS ⁇ 2.
  • the linker may be added to the N-terminus of the first BACCS region or the C-terminus of the second BACCS region.
  • the N-terminal linker of the first BACCS region include fluorescent proteins (mCherry, YFP, etc.).
  • the C-terminal linker of the second BACCS region include 2A peptide.
  • the linker included in BACCS ⁇ 2 may be a linker consisting of an amino acid sequence of 25 residues or more from the viewpoint of avoiding steric hindrance.
  • the polypeptide of the present invention can be produced by a gene recombination technique known in the technical field to which the present invention belongs. For example, by incorporating a nucleic acid encoding the polypeptide of the present invention into a vector and introducing the vector into Escherichia coli or cells (such as HEK293 cells derived from human kidney), the polypeptide can be expressed in large quantities.
  • nucleic acid An isolated nucleic acid encoding the polypeptide of the present invention can be prepared by a genetic recombination technique known in the technical field to which the present invention belongs. For example, a nucleic acid encoding the polypeptide of the present invention by ligating a nucleic acid encoding a LOV2-J ⁇ region cloned by PCR or the like, or a nucleic acid encoding a CAD region by creating a primer from amino acid sequence information Can be isolated.
  • nucleic acid used in the present specification includes DNA, RNA and the like.
  • a recombinant vector containing an isolated nucleic acid encoding the polypeptide of the present invention can be prepared by a gene recombination technique known in the technical field to which the present invention belongs. For example, it can be prepared by cleaving any plasmid with any restriction enzyme (BglII, SalI, etc.) and ligating an isolated nucleic acid encoding the polypeptide of the present invention to the cleavage site.
  • any restriction enzyme BglII, SalI, etc.
  • the term “recombinant vector” refers to DNA used to transport heterologous DNA in recombinant DNA experiments and the like.
  • the recombinant vector of the present invention include, for example, a DNA fragment expressing the polypeptide of the present invention, a cloning vector having an arbitrary restriction enzyme recognition site, an arbitrary replication origin, etc., and a DNA fragment expressing the polypeptide of the present invention. And an expression vector having a transcription initiation point and the like.
  • the scope of the present invention also includes a gene transfer kit containing such a recombinant vector.
  • a gene transfer kit containing such a recombinant vector.
  • the kit includes, for example, the recombinant vector of the present invention, a reagent for transformation, an instruction manual, and the like, but is not limited thereto.
  • transformant A transformant containing the above recombinant vector can be prepared by a gene recombination technique known in the technical field to which the present invention belongs.
  • the term “transformant” as used herein is an isolated cell or non-human animal.
  • the cells are not particularly limited, and any cell line (for example, HEK293 cells derived from human kidney, HEK293T cells, etc.) can be used.
  • a non-human animal Escherichia coli, a mammal (a mouse, a rat, etc.), a model animal, etc. can be used.
  • the transformant of the present invention can be obtained, for example, by introducing a recombinant vector into an isolated cell by electroporation method, lipofection method, virus or the like.
  • a transformant of a non-human animal can be obtained by performing DNA microinjection into the pronucleus of a fertilized egg of a non-human animal, homologous recombination into an ES cell, infection with a recombinant virus, or the like.
  • the polypeptide of the present invention can be easily expressed in a cell-specific manner by the known techniques as described above, and its function can be achieved only by manipulating the intensity of irradiated light without using an additional drug. In particular, it can be easily and safely applied to non-human animals.
  • the intracellular calcium signal of the transformant can be regulated by manipulating the intensity of light applied to the transformant.
  • the manipulation of light intensity refers to adjustment of the wavelength of light irradiated to the transformant, the light irradiation time, the light irradiation amount, and the like.
  • an intracellular calcium signal can be activated by irradiating the transformant with light having a wavelength in a blue region.
  • intracellular calcium signals can be inactivated by blocking light having a wavelength of 510 nm or less from the transformant.
  • intracellular calcium signals in cells expressing Orai1 can be regulated.
  • intracellular calcium signals in cells expressing Orai1 can be regulated not only in cells expressing Orai1 but also in cells not expressing Orai1.
  • BACCS 1: 2 (only BACCS in tandem)
  • IRES internal ribosome entry site
  • IRES-dependent translation is often reported to be 20-50% more efficient than 5 'cap-dependent translation (Mizoguchi et al., 2000).
  • IRES-dependent Orai1 translation is predicted to be 20-50% more efficient than 5 ′ cap-dependent BACCS translation.
  • the expression level ratio of Orai1 and BACCS can be adjusted efficiently.
  • a fusion protein of Orai1 and BACCS ⁇ 2 (Orai1-BACCS ⁇ 2) can be preferably used.
  • the influx of intracellular calcium ions can be regulated
  • Intracellular calcium ion concentration and intracellular physiological phenomena activated by calcium ions for example, muscle contraction, synaptic transmission, secretion, cell differentiation, etc.
  • the polypeptide of the present invention can be advantageously used in research in the basic medicine or life science fields related to calcium signals and in the development of pharmaceuticals.
  • the field of basic medicine or life science using the polypeptide of the present invention is not particularly limited, and can be used in research in fields such as control of muscle contraction, neurotransmission signal, immune response, development, and secretion control.
  • it can be used in systems such as in vitro, in situ, or in vivo.
  • polypeptide of the present invention can also be used in the development of pharmaceuticals, for example, evaluation of drug discovery targets in diseases such as diabetes and hypertension, and screening of new drug candidate compounds.
  • the present invention will be described in detail based on the following examples, but the present invention is not limited only to these examples.
  • the LOV2-J ⁇ region of oat phototropin 1, the human Orai1, and the human STIM1 CAD region were used.
  • Example 1 Examination on optimization of binding site between LOV2-J ⁇ and CAD
  • LOV-CAD polypeptide in which LOV2-J ⁇ and CAD are combined
  • LOV-CAD was obtained as a fusion gene by recombinant PCR of a PCR fragment of LOV2 incorporating a restriction enzyme (BglII) recognition sequence and a PCR fragment of a CAD region incorporating a restriction enzyme (SalI) recognition sequence, This was amplified by a PCR thermal cycler.
  • the primers and templates used are shown below.
  • the obtained PCR product was treated with a restriction enzyme using BglII and SalI.
  • the restriction endonuclease-treated PCR product was ligated to the BglII-SalI site of the ptdTomato-C1 vector, which is a red fluorescent protein reporter vector, to recombine the polypeptide tdTomato-LOV (404-538) -CAD (347-448).
  • a vector (hereinafter referred to as “ptdTomato-BACCS”) was prepared.
  • the ptdTomato-C1 vector is obtained by replacing the GFP sequence of pEGFP-C1 (manufactured by Clontech) with tdTomato.
  • a recombinant vector in which a post-transcriptional control element WPRE derived from Woodchuck hepatitis virus, which can enhance the stability of mRNA transcribed from the recombinant vector and enhance the expression of the transgene, is inserted into the 3 ′ untranslated region was prepared.
  • the I539E mutation (I539E) was introduced into the J ⁇ region as a light mutant by recombinant PCR.
  • a C450A mutation (C450A) was introduced into the LOV2 region by recombinant PCR.
  • the light variant LOV2-J ⁇ is referred to as “LOV lit”
  • the dark variant LOV2-J ⁇ is referred to as “LOV dark”.
  • Each of the LOV lit and the LOV dark has a structure in which light is irradiated and a structure in which light is blocked regardless of whether light is irradiated.
  • LOV lit-CAD a protein in which a CAD region (342-448 to 350-448) with various deletions of the N-terminus was fused to LOV lit (404-546)
  • LOV lit-CAD LOV lit-CAD
  • FIG. 4A LOV lit-CAD was co-expressed in HEK293 cells together with Orai1 and the intracellular localization of LOV lit-CAD was observed by the following procedure.
  • a recombinant vector “ptdTomato-LOV lit-CAD-WPRE” was prepared so as to express tdTomato, which is a red fluorescent protein, as a polypeptide fused to the N-terminal side of LOV lit-CAD.
  • a recombinant vector “Orai1-YFP” was also prepared so as to express Orai1 incorporating a yellow fluorescent protein at the C-terminal side.
  • ptdTomato-LOV lit-CAD-WPRE and Orai1-YFP were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen), and the cells were transferred to MEM (Nacalai) containing 10% calf serum (Gibco). Tesque Co., Ltd.) (hereinafter referred to as “MEM + 10% FBS”) was cultured in a medium.
  • LOV dark-CAD C-terminal sequence of LOV2-J ⁇ Polypeptide
  • LOV dark 404-542
  • LOV dark 404-539
  • LOV dark 404-538
  • LOV dark 404-535
  • NFAT transcription factor NFAT known to be transferred to the nucleus by a calcium signal Verified using.
  • CAD 347-448
  • candidate wild-type LOVs 404-542, 404-539, 404-538, 404-535
  • Orai1 a nuclear translocation signal
  • ptdTomato-LOV-CAD-WPRE three plasmids, ptdTomato-LOV-CAD-WPRE, Orai1 (prepared by removing YFP from Orai1-YFP (manufactured by Addgene)) and NFAT-GFP were transfected into HEK293 cells, Cells were cultured in MEM + 10% FBS medium. 24-48 hours after gene transfer, cells were fixed with 4% paraformaldehyde in the dark. This was used as a sample under dark conditions, and shown as “dark” in FIG.
  • FIG. 6A shows the results of NFAT intracellular localization observed with a confocal microscope (trade name: Zeiss LSM510, manufactured by Carl Zeiss).
  • LOV (404-538) -CAD (347-448) was selected as a polypeptide candidate capable of optically controlling Orai1.
  • the polypeptide is hereinafter referred to as BACCS (Blue light-activated Calcium Channel Switch).
  • BACCS Blue light-activated Calcium Channel Switch
  • “L” located at the junction of LOV2-J ⁇ and the CAD region seems to play an important role.
  • the amino acid at this site needs to be hydrophobic (Harper et al., 2004). That is, it is considered that this “L” stabilizes the structure of the LOV2 region and the J ⁇ region in a state where light is blocked.
  • this “L” is also important when the CAD region activates Orai1. Therefore, only when the J ⁇ region is separated from the LOV2 region by exposure to light and “L” is exposed on the surface of the fusion protein, Orai1. It is speculated that can be activated.
  • ptdTomato-BACCS-2A-Orai1-WPRE has a structure in which 22 amino acids of 2A peptide (Felipe et al., 2006) derived from Thesea assigna virus and Orai1 are fused to the C-terminus of tdTomato-BACCS.
  • 2A peptide sequence is present during translation into a protein, the polypeptides before and after the 2A peptide are not connected and are expressed as two polypeptides. That is, ptdTomato-BACCS-2A-Orai1-WPRE is expressed as two separate polypeptides (tdTomato-BACCS and Orai1) by one translation.
  • a recombinant vector capable of simultaneously expressing Orai1 together with BACCS the expression of two proteins can be controlled simultaneously, so that the ratio of BACCS and Orai1 expressed in protein-expressing cells can be made constant.
  • Orai1-stop codon-SalI hereinafter referred to as “pOrai1-SalI”
  • pOrai1-SalI a recombinant vector Orai1-stop codon-SalI
  • BspEI-LOV 404-538
  • -CAD 347-448
  • -2A-BspEI fused with BACCS and 2A peptide was inserted into the BspEI site in ptdTomato-BspEI-Orai1-WPRE, and ptdTomato-BACCS-2A- Orai1-WPRE was produced.
  • a recombinant vector pCherry-BACCS-2A-Orai1-WPRE was prepared.
  • the recombinant vector was introduced into HEK293 cells, and BACCS and Orai1 were coexpressed in MEM + 10% FBS medium. 24 to 36 hours after gene transfer, the expressed cells were stimulated with blue light (470 nm), and a calcium ion fluorescent indicator Fluo-4 or Rhod-3 (manufactured by Invitrogen) was added to observe changes in calcium concentration. (FIG. 7A). Observation was performed with a confocal microscope (trade name; BioRad MRC1024, manufactured by Bio-Rad Laboratories Co., Ltd.) at a time lapse every 5 seconds.
  • ⁇ F amount of fluorescence change
  • F0 fluorescence intensity before light stimulation
  • BACCS can open and close the calcium ion channel Orai1 in a light-dependent manner when endogenous Orai1 is expressed or co-expressed with the Orai1 gene.
  • a polypeptide capable of controlling calcium influx was prepared by combining Orai1 and BACCS and expressing it as a single polypeptide.
  • Orai1 and CAD can be activated most efficiently when they are present in a ratio of 1: 2, and Orai1 is constitutively activated when 2 are connected in tandem to the C-terminus of Orai1.
  • a recombinant vector in which two BACCSs were connected in tandem to the C-terminus of Orai1 was prepared by the following procedure.
  • a recombinant vector ptdTomato-BACCS-WPRE was prepared, and the following linker having a restriction enzyme (XhoI) recognition site was added to the restriction enzyme recognition site NheI-AgeI site.
  • the recombinant vector (pXhoI-AgeI-tdTomato-BACCS-WPRE) was prepared by insertion.
  • pKZK-Orai1-YFP was treated with restriction enzymes (XhoI and AgeI), and the resulting DNA fragment was inserted into the XhoI-AgeI site in pXhoI-AgeI-tdTomato-BACCS-WPRE, and the recombinant vector (pKKK-Orai1- tdTomato-BACCS-WPRE) was produced.
  • pKKK-Orai1- tdTomato-BACCS-WPRE was produced between Orai1 and tdTomato in the recombinant vector includes a linker having an EcoRI-AgeI site, which is a restriction enzyme recognition site derived from Orai1-YFP.
  • the EcoRI-AgeI site was replaced with the following sequence.
  • the amino acid sequence of the obtained recombinant vector (pKZZ-Orai1-linker2-tdTomato-BACCS-WPRE) is not changed.
  • Amplification was performed with a PCR thermal cycler using the following primers and template.
  • the obtained PCR product (BglII-BamHI fragment) was inserted into the BglII site, which is the restriction enzyme recognition site between tdTomato and BACCS in pKKK-Orai1-linker2-tdTomato-BACCS-WPRE, and the recombinant vector (pKKK-Orai1) -Linker2-tdTomato-BACCS-L1-BACCS-WPRE, hereinafter referred to as “pOrai1-tdTomato-BACCS ⁇ 2-WPRE”).
  • “L1” was referred to the amino acid sequence of “L1” in Orai1-L2-CAD (336-485) -L1-CAD (336-485) prepared by Li et al. (2010).
  • tdTomato (AgeI-BglII fragment) in pOrai1-tdTomato-BACCS ⁇ 2-WPRE was replaced with the AgeI-BglII of the yellow fluorescent protein expression vector pEYFP-C1 to obtain a recombinant vector (pOrai1-YFP-BACCS ⁇ 2-WPRE). ) was produced.
  • a recombinant vector (pOrai1-BACCS ⁇ 2-WPRE) was prepared by replacing tdTomato (AgeI-BglII fragment) in pOrai1-tdTomato-BACCS ⁇ 2-WPRE with the following linker.
  • IRES- gapYFP Imai et al., 2006; hereinafter abbreviated as “igY”
  • IRES-membrane-tdTomato-myc IRES-membrane-tdTomato-myc
  • the IRES (internal ribosome entry site) sequence is a sequence that can start translation from the middle of the messenger RNA. For example, from Orai1-HA-BACCS ⁇ 2-igY messenger RNA, two polypeptides, Orai1-HA-BACCS ⁇ 2 and gapYFP, are translated.
  • the expression product by the above recombinant vector includes a 21 amino acid linker (Orai1-BACCS ⁇ 2), a 57 amino acid linker (Orai1-HA-BACCS ⁇ 2), a 258 amino acid linker (Orai1-mCherry-BACCS2) between Orai1 and BACCS,
  • This is a construct in which either a 498 amino acid linker (Orai1-tdTomato-BACCS ⁇ 2) or a 261 amino acid linker (Orai1-YFP-BACCS ⁇ 2) is inserted.
  • the calcium concentration was efficiently increased by irradiation with light of 470 nm (blue light) (FIG.
  • Example 3 Examination of the number of BACCS to be bound to Orai1
  • Orai1 and CAD can be efficiently activated when present in a 1: 2 ratio (Li et al., 2010). Therefore, in order to verify the effect of the ratio of Orai1 and CAD on the action of Orai1-BACCS, a polypeptide in which only one BACCS was bound to Orai1 was prepared, and as in Example 1 (4), a nucleus using NFAT was prepared. A migration assay was performed.
  • the pKZZ-Orai1-tdTomato-BACCS ⁇ 1-WPRE prepared in Example 2 (1) was used as a polypeptide (Orai1-tdTomato-BACCS ⁇ 1) in which only one BACCS was bound to Orai1.
  • NFAT was excised with restriction enzymes (AgeI and SacI) and inserted into the AgeI-SacI site which is the restriction enzyme recognition site of the fluorescent protein expression vector pEYFP-N1.
  • a recombinant vector (NFAT-YFP) was prepared.
  • Orai1-tdTomato-BACCS ⁇ 1 and NFAT-YFP were co-expressed in HEK293 cells and irradiated with 470 nm light (blue light) for 20 minutes.
  • NFAT-YFP fluorescence of NFAT-YFP
  • FIG. 9A the fluorescence of NFAT-YFP
  • NFAT-GFP NFAT-GFP was co-expressed in HEK293 cells.
  • NFAT was transferred to the nucleus by irradiation with light at 470 nm for 20 minutes (“NFAT-GFP” in FIG. 9B).
  • Example 4 Examination of photoresponse of BACCS using Orai1 mutant
  • Orai1 A mutation of Orai1 (Orai1 (L273D)), which suppresses the activation of Orai1 by the CAD region, was constitutively activated by a construct in which CAD1 was connected in tandem to the C-terminus of Orai1-CAD-CAD. The constitutive calcium influx is not observed (Li et al., 2010).
  • an inactive mutation L273D was introduced into Orai1 by recombinant PCR, and a recombinant vector (Orai1 (L273D) -HA-BACCS ⁇ 2-igY) in which an HA tag and a fluorescent substance were combined was prepared (FIG. 10A). This was introduced into HEK293 cells and the calcium signal was observed.
  • FIG. 10B shows the results of calcium imaging with Rhod-3, a red fluorescent calcium sensor, before irradiating Orai1 (L273D) -HA-BACCS ⁇ 2 with 470 nm light (blue light) and after irradiating for 10 minutes. . As shown in FIG.
  • the STIM1 CDI (Calcium dependent inactivation) region contains the region necessary for rapid inactivation of Orai1 (Mullins et al., 2009). Therefore, by incorporating the CDI region into Orai1-BACCS, a calcium channel that can immediately inactivate once activated Orai1 may be obtained. Therefore, the C-terminal of the CAD region in Orai1-BACCS is extended to include the CDI region (the extended CAD region is indicated as “CAD (347-630)”), and an HA tag and a fluorescent substance are incorporated therein.
  • a recombinant vector (Orai1-HA-BACCS (347-630) ⁇ 2-igY; FIG. 11A) was prepared.
  • the DNA fragment of CAD (347-630) was amplified from the plasmid STIM1-CFP by PCR, and BACCS (347-630) was prepared by the same procedure as in Example 1 above.
  • These recombinant vectors were expressed in HEK293 cells, and the cells were irradiated with light of 470 nm (blue light), and calcium imaging was performed using Rhod-3, a red fluorescent calcium sensor. After 50 seconds from the start of measurement, light of 470 nm (blue light) was irradiated for 10 minutes, and then observed under dark conditions for 15 minutes.
  • LOV2 as an optical switch is increased by double mutation of G528A mutation and N538E mutation (GVMLIKKTAEN to AVMLIKKTAEE) in LOV2-J ⁇ (Strickland et al., 2010).
  • double mutations were introduced into the four wild type LOVs (404-542, 404-539, 404-538, 404-535) used in the NFAT assay and irradiated with light at 470 nm.
  • NFAT was not transferred to the nucleus in any of the constructs, and the optical switch was not turned on (data not shown).
  • Example 6 Production of BACCS ⁇ 2
  • a recombinant vector in which two BACCSs were connected in tandem was prepared by the following procedure.
  • the effect of BACCS ⁇ 2 on endogenous Orai1 expressed in HEK293T was examined.
  • the expression product by the above recombinant vector has the same amino acid sequence as the BACCS ⁇ 2 region in the pOrai1-HA-BACCS ⁇ 2-WPRE expression product in Example 2, and has 10 amino acids (MGPVGGSGGS) at the N-terminus, 9 amino acids (GGSGGSLV) are added to the C-terminus. These are added so that they can be used as a linker when preparing a fusion protein.
  • FIG. 12B shows the result of plotting the value of ⁇ F / F0 on the vertical axis and time (seconds) on the horizontal axis.
  • calcium imaging was similarly performed on HEK293T cells expressing Orai1-HA-BACCS ⁇ 2-igY.
  • ⁇ F / F0 at each time point is shown as an average value for each cell together with a standard error. The slight increase in fluorescence intensity observed at the start of blue light irradiation is noise from the measuring instrument.
  • BACCS ⁇ 2 was able to activate endogenous Orai1 without using the Orai1 expression vector, and was found to increase the calcium concentration more efficiently than Orai1-HA-BACCS ⁇ 2.
  • Rhod-3 red fluorescent calcium sensor
  • the cells were stimulated by continuously irradiating an LED lamp having a wavelength of 470 nm.
  • ⁇ F / F0 was calculated in the same manner as described above, plotted on the vertical axis, and the results showing time (seconds) on the horizontal axis are shown in FIG.
  • ⁇ F / F0 at each time point is shown as an average value for each cell together with a standard error.
  • FIG. 13 it was confirmed that even when a protein was fused to the N-terminus, BACCS ⁇ 2 could activate endogenous Orai1 and efficiently increase the calcium concentration.
  • STIM1 has high sequence similarity to STIM2, and is conserved among various species such as nematodes and humans (FIG. 2B; modified from Yuan et al., 2009).
  • FIG. sap human; tau; cattle, P. a. tro; chimpanzee, E.I. cab; horse, G. et al. gal; mus; mouse, R.M. nor; rat, S. p. scr; pig, X. lae; Xenopus laevis, D. rer; zebrafish, D.R. mel; Drosophila, A. gam; mosquito, N. vit; jewel bee, C.I. ele; a nematode.
  • Orai1 has high sequence similarity to Orai2 and Orai3, and is conserved among various species such as nematodes and humans (FIG. 2A).
  • the abbreviations in FIG. 2A show h; human, m; mouse, z; zebrafish, x; Xenopus, d; Drosophila, ce;
  • the J ⁇ region is also highly conserved among various plant species (Fig. 3).
  • the abbreviations in FIG. 3 are respectively Os; rice phototropin 1, Zm; corn phototropin 1, At; Arabidopsis phototropin 1 and phototropin 2, Ps; pea phototropin 1, Vf; broad bean phototropin 1, Ac; Phytochrome 3, As; Oat wheat, phototropin 1, is shown (quoted from Harper et al., 2004).
  • the LOV2 region is also conserved among many plants as described above.
  • the homology with the amino acid sequence of the oat LOV2 region is as follows: phototropin 1 of Asian rice; 81%, phototropin 1 of Arabidopsis; 87%, Arabidopsis phototropin 2; 83%, pea phototropin 1; 87%, broad bean phototropin 1; corn phototropin 1; 95%, tomato phototropin 1; 87%.

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Abstract

La présente invention concerne un polypeptide permettant la régulation optique de la signalisation calcique, ledit polypeptide permettant une régulation et une manipulation spatiales aisées de la signalisation calcique. Ledit polypeptide est constitué d'une séquence d'acides aminés comportant un domaine LOV2 composé de SEQ ID NO : 1 ou d'une séquence d'acides aminés présentant au moins 80 % d'identité de séquence avec SEQ ID NO : 1 ; un domaine Jα composé de SEQ ID NO : 2, de SEQ ID NO : 3 ou d'une séquence d'acides aminés faisant office de commutateur optique LOV2-Jα et présentant une mutation prédéterminée dans la séquence d'acides aminés de SEQ ID NO : 2 ou 3 ; et SEQ ID NO : 4 ou un domaine CAD capable d'activer le canal calcique Orai1 et présentant une mutation prédéterminée dans la séquence d'acides aminés de SEQ ID NO : 4 ; le domaine LOV2, le domaine Jα et le domaine CAD étant disposés, depuis une extrémité N-terminale et jusqu'à une extrémité C-terminale dans l'ordre suivant : domaine LOV2, domaine Jα, domaine CAD ; et un polypeptide étant utilisé sans interposition d'une séquence d'acides aminés entre l'extrémité C-terminale du domaine Jα et l'extrémité N-terminale du domaine CAD.
PCT/JP2012/053705 2011-02-17 2012-02-16 Polypeptide, acide nucléique isolé, vecteur recombinant, nécessaire de transfert de gènes, transformant et procédé de régulation de la signalisation calcique intracellulaire Ceased WO2012111772A1 (fr)

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EP4266058A2 (fr) 2018-07-06 2023-10-25 The Regents of the University of Colorado, a body corporate Système génétiquement codé pour la construction et la détection d'agents biologiquement actifs
US11993635B2 (en) 2018-07-06 2024-05-28 The Regents Of The University Of Colorado, A Body Corporate Genetically encoded system for constructing and detecting biologically active agents
US12297234B2 (en) 2018-07-06 2025-05-13 The Regents Of The University Of Colorado, A Body Corporate Genetically encoded system for constructing and detecting biologically active agents
CN113321717A (zh) * 2021-06-16 2021-08-31 华中农业大学 Lov蛋白突变体及其应用

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