JPWO2012111772A1 - Polypeptide, isolated nucleic acid, recombinant vector, gene transfer kit, transformant, and method for regulating intracellular calcium signal - Google Patents

Abstract

To provide a polypeptide that optically regulates calcium signals, which facilitates spatial control and manipulation of calcium signals. Maintaining the function as an optical switch of SEQ ID NO: 1 or SEQ ID NO: 1 and an amino acid sequence having 80% or more sequence identity and SEQ ID NO: 2, SEQ ID NO: 3, or LOV2-Jα, and SEQ ID NO: A CAD region that can activate the Jα region consisting of an amino acid sequence having a predetermined mutation in the amino acid sequence of 2 or 3, and SEQ ID NO: 4 or calcium channel Orai1, and has the predetermined mutation in the amino acid sequence of SEQ ID NO: 4. A LOV2 region, a Jα region and a CAD region are arranged in the order of LOV2 region, Jα region and CAD region from the N-terminus toward the C-terminus, A polypeptide in which an amino acid sequence is not interposed between the C-terminus of and the N-terminus of the CAD region is used.

Description

  The present invention relates to polypeptides that optically control calcium signals, isolated nucleic acids, recombinant vectors, gene transfer kits, transformants, and methods for regulating intracellular calcium signals.

  In recent years, in basic medical research, in order to elucidate the function of intracellular signals and the like, systems that control intracellular signals by light stimulation have been used (Non-Patent Document 1; Non-Patent Document 2). For example, in the field of nerve function research, 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.

  In addition, as a system for controlling intracellular signals by light stimulation, 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 photosensitivity region LOV2-Jα 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).

  Among intracellular signals, calcium signals are known to have important actions in various cellular functions such as fertilization, cell proliferation, development, learning and memory, muscle contraction and secretion via calcium channels, It has important functions in almost all cells in the living body (Non-patent Document 4). Therefore, 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.

  Examples of conventional systems that control calcium signals by light stimulation include systems 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.

Kramer, R.A. H. Fortin, D .; L. & Trauner, D.A. New photochemical tools for controlling neuronal activity. Curr. Opin. Neurobiol. 19, 544-552 (2009) Rana, A .; & Dolmetsch, R.A. E. Use light to control signaling cascades in live neuros. Curr. Opin. Neurobiol. 20, 617-622 (2010) Wu, Y .; I. Et al., A genetically encoded photoactive Rac controls the mobility of living cells. Nature 461, 104-108 (2009) Berridge, M.M. J. et al. Lipp, P .; & Bootman, M .; D. The versatility and universality of calcium signaling. Nat. Rev. Mol. CellBiol. 1,11-21 (2000) Liu, X .; & Tonegawa, S .; Optogenetics 3.0. Cell 141, 22-24 (2010)

  According to the system using caged calcium, the calcium signal can be activated rapidly. However, caged calcium is a small molecule, so that it easily diffuses, and the spatial control of the calcium signal is not easy. In addition, in a system using caged calcium, 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.

  In view of the above situation, 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.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a predetermined portion of the amino acid sequence of the LOV2-Jα 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.

That is, 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 Jα 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-Jα, 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 Jα region and the CAD region are arranged in the order of the LOV2 region, the Jα 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 Jα region and the N-terminus of the CAD region.

  The present inventors have also found that the above problem can be solved by a polypeptide combining the amino acid sequence of the calcium channel Orai1 and BACCS.

That is, 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 first BACCS region;
A polypeptide comprising an amino acid sequence comprising a second BACCS region,
The Orai1 region, the first BACCS region, and the second BACCS region are arranged in the order of the Orai1 region, the first BACCS region, and the second BACCS region from the N-terminus toward the C-terminus. 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 in Orai1-BACCS.

  A second linker may be interposed between the C-terminal of the first BACCS region and the N-terminal of the second BACCS region in Orai1-BACCS.

  The present inventors have also found that the above problem can be solved by a polypeptide in which BACCS is connected in tandem.

That is, the present invention
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 terminus of the first BACCS region and the N terminus of the second BACCS region in BACCS × 2.

  The present invention also provides an isolated nucleic acid encoding a 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 comprising the above recombinant vector and being an isolated cell or non-human animal.

  The present invention also provides a method for regulating the intracellular calcium signal, which regulates the intracellular calcium signal of the transformant by manipulating the intensity of light applied 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 modulating an intracellular calcium signal, wherein the polypeptide is Orai1-BACCS.

  According to the present invention, 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 Jα region in LOV2-Jα 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-Jα. (B) Imaging of localization of Orai1 (Orai1-YFP) incorporating yellow fluorescent protein in HEK293 cells (left figure), 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-Jα. (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 dark variant of LOV2-Jα (ie, does not localize LOV dark-CAD to the cell membrane). Four candidates for the amino acid sequence of the Jα region (to the membrane) The identification results of “none” for localization are shown. (A) shows the position 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. (B) shows the presence or absence of nuclear transfer of the NFAT transcription factor by the amino acid sequence of the LOV2 region, the amino acid sequence of various Jα regions, and the polypeptide (LOV-CAD) combined with the amino acid sequence of the CAD region, 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 this order from N-terminal to C-terminal in HEK293 cells, and light at 470 nm (blue) The results of calcium imaging using Fluo-4, a calcium fluorescent probe, after continuous irradiation of light) during the test period. 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) expresses Orai1-BACCS (Orai1-mCherry-BACCS × 2) incorporating a red fluorescent protein in HEK293 cells, and after irradiating 470 nm light (blue light) continuously during the test period. The calcium imaging result using Fluo-4 which is a calcium fluorescent probe is 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 an HA tag and / or a 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. The time-dependent change of the intracellular calcium concentration observed by the calcium imaging using Rhod-3 which is a red fluorescent calcium sensor is shown. (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 BACCSs 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 light of 470 nm (blue light). Figure 5 shows 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) in which the C-terminal of the CAD region is extended so as to include the STIM1 CDI (Calcium dependent inactivation) region (the hatched portion is CDI). Area). (B) is 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). The calcium imaging result about the cell which expressed BACCSx2-igY (Bx2) and YFP-BACCSx2 (YBx2) is shown.

(BACCS)
The present invention relates to a polymorph containing an amino acid sequence of LOV2-Jα region, which is a light-sensitive region of phototropin 1, which is a photoreceptor protein of oats, and an amino acid sequence of a CAD region of STIM1, which is a regulatory protein of calcium channel Orai1. A peptide (Blue light-activated Calcium Channel Switch, hereinafter referred to as “BACCS”) is provided. In cells in which BACCS is expressed, 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. Moreover, since 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-Jα)
The photosensitivity of oat-derived phototropin 1 depends on the region of amino acids 404-543 in phototropin 1, which is called LOV2-Jα (Harper et al., 2003). Since LOV2-Jα 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 Jα region are bonded by a hydrophobic bond. On the other hand, when light is irradiated, the hydrophobic bonds of the LOV2 region and the Jα region are removed. This is called “bright state”. When the signal protein is fused to LOV2-Jα, LOV2-Jα functions as a steric hindrance of the signal protein in a state where light is blocked. On the other hand, in the state irradiated with light, the structure of LOV2-Jα changes, and the steric hindrance of the signal protein is eliminated. As a result, a desired intracellular signal can be generated.

  The light for setting LOV2-Jα to the bright state may be a wavelength in the blue region (510 nm or less). For example, the structure of LOV2-Jα can be easily changed by using a commercially available blue LED. In addition, although the irradiation time of light is not particularly limited, LOV2-Jα can be efficiently brightened by irradiation in milliseconds (Nakasone et al., 2008).

  In addition, in order to cancel the bright state of LOV2-Jα, the wavelength of light to be irradiated may be longer than 510 nm or light may be blocked.

[LOV2 area]
Among LOV2-Jαs constituting the polypeptide of the present invention, the LOV2 region corresponds to positions 404 to 522 in the amino acid sequence of oat-derived phototropin.

  The LOV2 region is conserved among many plants. For example, 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 at least 80% sequence identity with the oat amino acid sequence. By using such an amino acid sequence, it is considered that BACCS can exert a light-sensitive action. Whether or not 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. For example, as an amino acid sequence corresponding to the LOV2 region constituting the polypeptide of the present invention, positions 404 to 522 of the oat-derived phototropin amino acid sequence, 80% or more, 85% or more, 90% or more, or 95% or more An amino acid sequence having the following sequence identity can be used.

[Jα region]
Among LOV2-Jαs constituting the polypeptide of the present invention, the Jα region corresponds to positions 523-535 or 523-538 of the amino acid sequence of oat-derived phototropin.

  The Jα 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. . This is because the amino acid sequence of the Jα 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. In the blocked state, the steric hindrance of the CAD region, which is a signal protein, is eliminated. As a result, even in the light-blocked state, 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. For example, to date, a light-sensitive polypeptide using positions 404-546 (Wu et al., 2009) and 404-543 (Stickland et al., 2008) of LOV2-Jα has been produced, but when light is blocked. However, there was a problem that 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 Jα region shows the 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 Jα region is particularly preferable because it can strongly regulate the calcium signal in response to light. BACCS having the phototropin amino acid sequence 523-535 as the Jα region can induce a calcium signal in a blocked state, but the intensity of the signal can be adjusted by light irradiation.

  It has also been reported that in order for LOV2-Jα to function, the amino acids of I532, A536, and I539 in the Jα region must be hydrophobic (Harper et al., 2004). Therefore, when preparing BACCS, it is necessary to make these sites in the Jα region become hydrophobic amino acids. For example, in the amino acid sequence that constitutes BACCS, the hydrophobic amino acid “L (leucine)” located at the junction of LOV2-Jα (404-538) and the CAD region functions as an optical switch for LOV2-Jα. Plays an important role. That is, “L” is derived from the CAD region, but also functions as the 539th amino acid of LOV2-Jα. This “L” is important even when the CAD region directly bound to the Jα region activates Orai1. For this reason, in a state where light is blocked, this “L” in the Jα region is used for hydrophobic binding to the LOV2 region, and as a result, the CAD region is inhibited from interacting with Orai1.

  As a result of intensive studies by the present inventors, if BACCS has a Jα region corresponding to positions 523-535 or 523-538 of the amino acid sequence of phototropin, the activity of Orai1 depends on the operation of the intensity of the irradiated light. We found that control can be controlled. However, BACCS having an amino acid sequence in which one or a plurality of amino acids are substituted in the amino acid sequence of the Jα region and maintaining the function as an optical switch of LOV2-Jα also has the intensity of irradiation 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. Here, the “amino acid sequence in which one or a plurality of amino acid sequences are substituted” refers to positions 523-535 or 523-538 of the amino acid sequence of phototropin derived from oats, which is a Jα 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 the amino acid sequence contains an amino acid sequence that maintains the function as an optical switch of LOV2-Jα can be identified by, for example, observing a circular dichroism spectrum of the obtained polypeptide.

(CAD area)
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. When LOV2-Jα in BACCS becomes a bright state by irradiation with light, the CAD region is activated and binds to Orai1 and, as a result, activates Orai1.

  However, since 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. And within the scope of the present invention. Here, 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. An amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more. Whether 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.

(Orai1-BACCS)
The present invention also provides a protein (hereinafter referred to as “Orai1-BACCS”) in which BACCS is connected in tandem with the C-terminal 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)
Calcium channel 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. Note that 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. Moreover, if a region for human Orai1 to function as a calcium channel is conserved, even if one or more amino acids are 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. Here, “one or more amino acids are deleted, substituted, or added” means that Orai1 constituting the polypeptide of the present invention, position 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. Whether or not the region for Orai1 to function as a calcium channel is included in the amino acid sequence, for example, 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-terminus of Orai1 and the N-terminus of the first BACCS and between the C-terminus of the first BACCS and the N-terminus of the second BACCS, which constitutes the Orai1-BACCS Fragments (hereinafter referred to as “linkers”) may intervene. The linker may be a fluorescent protein (mCherry, YFP, etc.) and the like. By including such a linker in Orai1-BACCS, the expression of Orai1-BACCS in cells can be easily detected.

  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”).

  It has been reported that 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. In 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. For example, examples of the N-terminal linker of the first BACCS region include fluorescent proteins (mCherry, YFP, etc.). Examples of the C-terminal linker of the second BACCS region include 2A peptide. By including such a linker in BACCS × 2, the expression of BACCS × 2 in cells can be easily detected.

  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.

(Manufacturing method)
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 a polypeptide of the present invention by ligating a nucleic acid encoding a LOV2-Jα 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.

  As used herein, the term “nucleic acid” includes DNA, RNA, and the like.

(Recombinant vector)
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.

  As used herein, the term “recombinant vector” refers to DNA used to transport heterologous DNA in recombinant DNA experiments and the like. Examples of 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 introduction kit containing such a recombinant vector. By performing gene transfer using the kit, the polypeptide of the present invention can be easily expressed. 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. Moreover, it does not specifically limit as a non-human animal, Escherichia coli, mammals (a mouse | mouth, a rat, etc.), a model animal, etc. can be used.

  The transformant of the present invention can be obtained by introducing a recombinant vector into an isolated cell by electroporation method, lipofection method, virus or the like, for example. 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.

(Application using the polypeptide of the present invention)
Furthermore, according to the present invention, 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. For example, an intracellular calcium signal can be activated by irradiating the transformant with light having a wavelength in a blue region. In addition, intracellular calcium signals can be inactivated by blocking light having a wavelength of 510 nm or less from the transformant.

  For example, the use of BACCS can regulate intracellular calcium signals in cells expressing Orai1. Moreover, if Orai1-BACCS is used, the intracellular calcium signal not only in the cells expressing Orai1 but also in cells not expressing Orai1 can be regulated.

  It is known that when Orai1 and CAD regions are present in a ratio of 1: 2, Orai1 can be efficiently activated and the responsiveness of intracellular calcium signals is increased (Li et al., 2010). . Based on this, when the ratio of the expression level of BACCS to Orai1 is Orai1: BACCS = 1: 2 or less, it is considered that the responsiveness of intracellular calcium signals decreases. In cells expressing Orai1, in order to make the ratio of BACCS to endogenous Orai1 less likely to be less than or equal to Orai1: BACCS = 1: 2, depending on the amount of endogenous Orai1, BACCS × 2 (only BACCS in tandem) BACCS × 2-2A-Orai1 (Orai1: BACCS = 1: 2), BACCS × 2-IRES-Orai1 (Orai1: BACCS = 1: 4 to 1:10), etc. are preferably used. it can. In addition, IRES (internal ribosome entry site) is a sequence which can start translation from the middle of messenger RNA. IRES-dependent translation has often been reported to be 20-50% more efficient than 5 'cap-dependent translation (Mizoguchi et al., 2000). In other words, for example, in BACCS × 2-IRES-Orai1, IRES-dependent Orai1 translation is predicted to be 20-50% more efficient than 5 ′ cap-dependent BACCS translation. According to the recombinant containing, the expression level ratio of Orai1 and BACCS can be adjusted efficiently. On the other hand, in cells not expressing Orai1, since endogenous Orai1 does not exist, a fusion protein of Orai1 and BACCS × 2 (Orai1-BACCS × 2) can be preferably used.

  Thus, according to the present invention, by controlling the activation of the intracellular calcium channel of the transformant by manipulating the intensity of the irradiated light, 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.) can be regulated.

  The polypeptide of the present invention can be advantageously used in basic medicine or life science research related to calcium signals or 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. Moreover, as a research technique, it can be used in systems such as in vitro, in situ, or in vivo.

  The polypeptide of the present invention can also be used in the development of pharmaceuticals. For example, it can be used for evaluation of drug discovery targets in diseases such as diabetes and hypertension, screening for new drug candidate compounds, and the like.

  The present invention will be described in detail based on the following examples, but the present invention is not limited only to these examples. In this example, the LOV2-Jα region of oat phototropin 1, the human Orai1, and the human STIM1 CAD region were used.

(Example 1: Study on optimization of binding site between LOV2-Jα and CAD)
In order to identify the optimal combination of LOV2-Jα and CAD binding site sequences, a polypeptide in which LOV2-Jα and CAD are bound (hereinafter referred to as “LOV-CAD”) is prepared and subjected to light stimulation. LOV-CAD capable of activating Orai1 was screened. Since the structure of LOV2-Jα is thought to control the region that binds to the C-terminal side of the Jα region, at the time of polypeptide production, the C-terminal side of the base sequence of LOV2-Jα and the base sequence N-terminal of CAD It is necessary to join the side.

(1) Preparation of Recombinant Vector Expressing LOV-CAD An example of preparing a plasmid incorporating the nucleotide sequences of LOV2-Jα (404-538) and CAD (347-448) is shown below. Plasmids expressing other polypeptides were prepared in the same manner. In addition, plasmids of Phototropin 1 LOV2-Jα (ID 22024, PA-Rac1), Orai1-YFP (ID 19756), STIM1-CFP (ID 19755) and NFAT-GFP (ID 11107) were purchased from Addgene.
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 restriction enzymes using BglII and SalI. Next, 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. Furthermore, 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 ( Hereinafter, “ptdTomato-BACCS-WPRE” was prepared.

(2) Examination on N-terminal sequence of CAD region The minimum region necessary for the activation of Orai1 was examined for the N-terminal sequence of the base sequence of the CAD region that binds to the base sequence of LOV2-Jα.
For the study, a mutant that mimics the structure of the LOV2 region under light irradiation conditions (hereinafter referred to as “light mutant”) and a mutant that mimics the structure of the LOV2 region under light-blocking conditions (hereinafter referred to as “dark”). Called "mutants"). For example, it is known that the amino acid sequence of the Jα region becomes a light mutant by mutation of I532E, A536E, I539E, D540R (Harper et al., 2004). In this example, the I539E mutation (I539E) was introduced into the Jα region as a light mutant by recombinant PCR. As a dark mutant, a C450A mutation (C450A) was introduced into the LOV2 region by recombinant PCR. Hereinafter, the light variant LOV2-Jα is referred to as “LOV lit”, and the dark variant LOV2-Jα 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.
First, a protein (hereinafter referred to as “LOV lit-CAD”) in which a CAD region (342-448 to 350-448) lacking various N-terminals was fused to LOV lit (404-546) (hereinafter referred to as “LOV lit-CAD”) was prepared ( 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. Similarly, a recombinant vector “Orai1-YFP” was also prepared so as to express Orai1 in which a yellow fluorescent protein was incorporated on 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. 24-48 hours after gene introduction, cells were fixed with 4% paraformaldehyde in the dark, and the localization of tdTomato fluorescence was observed with a confocal microscope (trade name; Zeiss LSM510, Carl Zeiss).
Since Orai1 is expressed on the cell membrane, LOV lit-CAD bound to Orai1 is localized on the cell membrane (FIG. 4B). Since the 546th amino acid in the Jα region and the 347th amino acid in the CAD region are both “L”, LOV lit (404-546) -CAD (348-448) is LOV lit (404-545) -CAD. Note that it can also be interpreted as (347-448). As a region for activating Orai1, CAD (347-448) was found to be the minimum region with respect to the N-terminus. (FIG. 4C).

(3) Study on C-terminal sequence of LOV2-Jα Polypeptide (hereinafter referred to as “LOV dark-CAD”) by varying the length of the C-terminal of LOV dark so as to include the CAD region (347-448) ) Was produced (FIG. 5A). Since LOV dark-CAD contains sufficient CAD region to activate Orai1, it can bind to Orai1 if there is no steric hindrance due to the protein structure of LOV dark. In this section, we will search for polypeptides that cannot bind to Orai1 due to steric hindrance of LOV dark. LOV dark-CAD that cannot bind to Orai1 was recognized as a polypeptide distributed in the cytoplasm (FIG. 5B).
Regarding the connection to the CAD region on the C-terminal side of LOV2-Jα, the sequence of important hydrophobic amino acids (I532, A536, I539) described in “(2) Examination of the N-terminal sequence of the CAD region” was changed. We devised how to connect amino acid sequences so as not to let them. Even when the hydrophobic amino acid sequence is changed, the amino acid characteristics of the LOV2-Jα region are not changed by the sequence of the CAD region bound to the LOV2-Jα region (that is, the 532nd, 536th, The amino acid sequence was devised so that the 539th amino acid became a hydrophobic amino acid.
The 15 polypeptides shown in FIG. 5C and Orai1 were co-expressed in HEK293 cells, and LOV dark (404-542), LOV dark (404-539), LOV dark (404-538), and LOV dark (404-535) was inhibited from localization to the cell membrane.

(4) Verification using NFAT for the activation of Oral1 by light Whether the polypeptide of the present invention can activate Oral1 by light irradiation is a transcription factor NFAT known to be transferred to the nucleus by a calcium signal Verified using.
First, a protein in which CAD (347-448) is fused to four candidate wild type LOVs (404-542, 404-539, 404-538, 404-535), Orai1, and a nuclear translocation signal are detected. Co-expressed in HEK293 cells with the reporter gene NFAT-GFP.
Specifically, three plasmids, ptdTomato-LOV-CAD-WPRE, Orai1 (produced 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. In addition, 24-48 hours after gene introduction, the medium was changed from MEM + 10% FBS medium to Leibovitz's medium, irradiated with light at 470 nm for 20 minutes, and then fixed with 4% paraformaldehyde. This was used as a sample under bright conditions, and shown as “bright” in FIG.
For each sample, the localization of NFAT was observed when light was blocked and when light of 470 nm was irradiated. FIG. 6A shows the results of NFAT intracellular localization observed with a confocal microscope (trade name: Zeiss LSM510, manufactured by Carl Zeiss).
In cells transfected with LOV (404-542) -CAD (347-448) and LOV (404-539) -CAD (347-448), the efficiency of localization of NFAT to the nucleus even when irradiated with light Was bad. Moreover, in cells into which LOV (404-535) -CAD (347-448) was introduced, although NFAT was efficiently localized to the nucleus by irradiation with light, the NFAT was also introduced into the nucleus even when light was blocked. Localization was seen (Figure 6B).
On the other hand, in cells into which LOV (404-538) -CAD (347-448) was introduced, NFAT was observed in the cytoplasm when light was blocked, and NFAT was observed in the nucleus when irradiated with light. (FIGS. 6A and B).

  Based on the above results, 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). In the amino acid sequence of BACCS, “L” located at the junction of LOV2-Jα and the CAD region seems to play an important role. In order for LOV2-Jα to function as an optical switch, 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 Jα region in a state where light is blocked. On the other hand, this “L” is also important when the CAD region activates Orai1. Therefore, only when the Jα 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.

(5) Verification by calcium imaging In order to directly observe the intracellular calcium signal activated by BACCS, calcium imaging was performed by the following procedure using a calcium sensor.
A recombinant vector of BACCS incorporating a fluorescent protein (ptdTomato-BACCS-WPRE or pmCherry-BACCS-WPRE) and a recombinant vector (ptdTomato-BACCS-2A-Orai1-WPRE) for simultaneously expressing Orai1 with BACCS were prepared. The ptdTomato-BACCS-2A-Orai1-WPRE has a structure in which 22 amino acids of the 2A peptide (Felipe et al., 2006) derived from Thesea assigna virus and Orai1 are fused to the C-terminus of tdTomato-BACCS. When a 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.
Thus, according to the 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.
First, a recombinant vector Orai1-stop codon-SalI (hereinafter referred to as “pOrai1-SalI”) was prepared from the Orai1-YFP plasmid.
In the ptdTomato-BACCS-WPRE prepared in Example 1, the BACCS sandwiched between the BspEI-SalI sites, which are restriction enzyme recognition sites, was replaced with a fragment obtained by treating pOrai1-SalI with restriction enzymes (BspEI and SalI). ptdTomato-BspEI-Orai1-WPRE was produced.
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. By replacing the fragment obtained by treating tdTomato in this plasmid with restriction enzymes (AgeI and BglII) with a fragment obtained by treating the red fluorescent protein reporter vector pmCherry-C1 vector with restriction enzymes (AgeI and BglII), 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 introduction, 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. For each cell, ΔF (amount of fluorescence change) / F0 (fluorescence intensity before light stimulation) is calculated, ΔF is normalized so that the value at the end of observation is 1, plotted on the vertical axis, and plotted on the horizontal axis The result showing time (seconds) is shown in FIG. 7B. As shown in FIG. 7B, in the BACCS-2A-Orai1 expressing cells, an increase in calcium concentration due to light irradiation was observed.
The primers and templates used for preparing each recombinant vector are shown below.

(Example 2: Production of polypeptide of Orai1 and BACCS)
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. Thus, 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. Has been reported (Li et al., 2010). Therefore, a recombinant vector in which two BACCSs were connected in tandem to the C-terminus of Orai1 was prepared by the following procedure.

(1) Preparation of pOrai1-tdTomato-BACCS × 2-WPRE A kozak-like sequence (CCACC) was inserted into the 5 ′ end of the start codon of Orai1-YFP in order to increase translation efficiency. Specifically, the following linker having a restriction enzyme (XhoI) recognition site and a Kozak sequence (hereinafter abbreviated as “KZK”) at the BglII-BspEI site, which is a restriction enzyme recognition site in Orai1-YFP in the plasmid. Inserted.

  In parallel, as in Example 1, 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.
Between Orai1 and tdTomato in the recombinant vector, a linker having an EcoRI-AgeI site, which is a restriction enzyme recognition site, derived from Orai1-YFP is included. For the purpose of removing the restriction enzyme recognition site, 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 by PCR thermal cycler using the following primers and template. The obtained PCR product (BglII-BamHI fragment) was inserted into the BglII site, which is a restriction enzyme recognition site between tdTomato and BACCS in pKKK-Orai1-linker2-tdTomato-BACCS-WPRE, and a recombinant vector (pKKK-Orai1) was obtained. -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).

(2) Preparation of pOrai1-Cherry-BACCS × 2-WPRE, pOrai1-YFP-BACCS × 2-WPRE and pOrai1-BACCS × 2-WPRE pOrai1-tdTomato-BACCS × 2-WPRE with restriction enzymes (AgeI and BglII) The tdTomato part obtained by the treatment was replaced with a fragment obtained by treating the fluorescent protein fusion expression vector pmCherry-C1 with restriction enzymes (AgeI and BglII), and the recombinant vector (pOrai1-Cherry-BACCS × 2-WPRE) was replaced. ) Was produced. Also, the 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. Also, 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.

(3) Preparation of pOrai1-HA-BACCS × 2-WPRE Amplified by a PCR thermal cycler using the following primers and template. The obtained PCR product (3 × HA tag sequence) was inserted into the AgeI site, which is a restriction enzyme recognition site in pOrai1-BACCS × 2-WPRE, to prepare a recombinant vector (pOrai1-HA-BACCS × 2-WPRE).

(4) Preparation of pOrai1-HA-BACCS × 2-igY-WPRE and pOrai1-HA-BACCS × 2-imTm-WPRE In order to visualize pOrai1-HA-BACCS × 2-WPRE-expressing cells with IRES- gapYFP (Imai et al., 2006; hereinafter abbreviated as “igY”) or IRES-membrane-tdTomato-myc (Muzumdar et al., 2007; hereinafter abbreviated as “imTm”) is replaced with pOrai1-HA-BACSS × 2-WPRE 3 'Recombinant vectors (pOrai1-HA-BACCS × 2-igY-WPRE and pOrai1-HA-BACCS × 2-imTm-WPRE, respectively) were prepared by inserting them into the SalI site, which is a restriction enzyme recognition site in the untranslated region. 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.
In any case, the calcium concentration was efficiently increased by irradiation with light (blue light) at 470 nm (FIG. 8B, abbreviations are Che; Orai1-mCherry-BACCS × 2, T; Orai1-tdTomato-BACCS × 2, HA; Orai1-HA-BACCS × 2-imTm is shown). The change in calcium concentration was calculated in the same manner as in Example 1. In addition, calcium imaging results using Fluo-4, a calcium fluorescent probe, after expressing Orai1-mCherry-BACCS × 2 in HEK293 cells and continuously irradiating 470 nm light (blue light) during the test period Is shown in FIG. 8A.
With regard to the length of the linker between Orai1 and BACCS, this experiment confirmed that it works with a minimum of 21 amino acids. Furthermore, it was confirmed that the opening and closing of the calcium channel can be controlled by repeating the irradiation and blocking of light (FIG. 8C).

(Example 3: Examination of the number of BACCS to be bound to Orai1)
There are reports that Orai1 and CAD can be efficiently activated when present in a 1: 2 ratio (Li et al., 2010). Therefore, in order to verify the influence 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 A migration assay was performed.
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.
From the reporter gene NFAT-GFP for detecting the nuclear translocation signal, 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. As a result, the fluorescence of NFAT-YFP (“NFAT-YFP” in FIG. 9A) was observed only in the cytoplasm, not observed in the nucleus, and no nuclear transfer of NFAT was observed. That is, it was shown that it is difficult to activate Orai1 with a polypeptide that binds only one BACCS to Orai1.
As a control experiment, Orai1-tdTomato-BACCS × 2, 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)
The mutation of Orai1 (Orai1 (L273D)) that suppresses the activation of Orai1 by the CAD region is constitutively activated by a construct in which CAD1 is connected in tandem to the C-terminus of Orai1-CAD-CAD. The constitutive calcium influx is not observed (Li et al., 2010).
Therefore, 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 calcium imaging results with Rhod-3, which is 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. 10B, there was no change in the intracellular calcium concentration before and after the light irradiation.
In addition, a polypeptide (Orai1-HA-BACCS (dark) × 2) in which BACCS LOV2-Jα was a dark mutant was prepared. Even when HEK293 cells expressing Orai1-HA-BACCS (dark) × 2 were irradiated with light (blue light) at 470 nm, no increase in intracellular calcium concentration was observed (data not shown).
From the above, it was shown that BACCS acts on Orai1 in response to light and activates Orai1.

(Example 5: Study on Orai1-BACCS including CDI region)
The STIM1 CDI (Calcium dependent inactivation) region (amino acids 470-491) contains the region necessary for rapid inactivation of Orai1 (Mullins et al., 2009). Therefore, by incorporating a CDI region into Orai1-BACCS, a calcium channel that can immediately inactivate Orai1 once activated 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, the cells were irradiated with light of 470 nm (blue light), and calcium imaging using Rhod-3, a red fluorescent calcium sensor, was performed. 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.
As a result, in Orai1-HA-BACCS (347-630) × 2-igY, the concentration of intracellular calcium increased by light irradiation. However, a decrease in calcium concentration (that is, inactivation of Orai1) was not immediately observed unexpectedly, and intracellular calcium concentration did not decrease even when light was blocked (FIG. 11B). , Abbreviations respectively indicate WT; Orai1-BACCS without CDI region, CDI; Orai1-BACCS with CDI region). The change in calcium concentration was calculated in the same manner as in Example 1. This result shows that it is not appropriate to introduce a CDI region into Orai1-BACCS, and that the length of the C-terminal sequence of the CAD region constituting Orai1-BACCS is important in the action of Orai1-BACCS. Suggests.

  It has been reported that a double mutation of G528A mutation and N538E mutation in LOV2-Jα (GVMLIKKTAEN to AVMLIKKTAEE) increases the dynamic range of LOV2 as an optical switch (Strickland et al., 2010). Such 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. As a result, 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)
In order to examine how much BACCS × 2 can activate Orai1 alone without using the Orai1 expression vector, a recombinant vector in which two BACCSs were connected in tandem was prepared by the following procedure. In this example, the effect of BACCS × 2 on endogenous Orai1 expressed in HEK293T was examined.

(1) Preparation of pBACCS × 2-WPRE Amplified by a PCR thermal cycler using the following primers and template. The obtained PCR product (AgeI-BamHI fragment and BamHI-SalI fragment) was converted into pOrai1-HA-BACCS × 2-WPRE modified vector (sequence near the start codon of the insert, NheI-XhoI-KZZK-start codon) -AgeI modified) was inserted into the vector excluding the AgeI-SalI fragment to prepare a recombinant vector (pBACCS × 2-WPRE) (FIG. 12A).
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.

(2) Preparation of pBACCS × 2-igY-WPRE In order to visualize the pBACCS × 2-WPRE-expressing cells obtained above with fluorescence, as in Example 2, IgY was converted to pBACCS × 2-WPRE 3 ′. The recombinant vector (pBACCS × 2-igY-WPRE) was prepared by inserting it into the SalI site, which is a restriction enzyme recognition site in the untranslated region.
Calcium imaging was performed on HEK293T cells expressing BACCS × 2-igY. Rhod-3 (red fluorescent calcium sensor) was used as the calcium fluorescent probe. After 50 seconds from the start of observation, a laser with a wavelength of 442 nm of a confocal microscope was irradiated every 5 seconds to stimulate the cells, and the change in fluorescence intensity was examined for each cell. As fluorescence intensity at each time point, ΔF (fluorescence change amount) / F0 (fluorescence intensity before light stimulation) was calculated. FIG. 12B shows the result of plotting the value of ΔF / F0 on the vertical axis and time (seconds) on the horizontal axis. As a comparative test, calcium imaging was similarly performed on HEK293T cells expressing Orai1-HA-BACCS × 2-igY. In the figure, B × 2 indicates BACCS × 2-igY (analyzed cell number n = 45), and OB × 2 indicates Orai1-HA-BACCS × 2-igY (analyzed cell number n = 35). ΔF / 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.
As shown in FIG. 12B, 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.

(3) Preparation of pYFP-BACCS × 2-WPRE In order to confirm that a fluorescent protein could be fused to the N-terminus of BACCS × 2, amplification was performed by a PCR thermal cycler using the following primers and template. The obtained PCR product (NheI-AgeI fragment) was inserted into the NheI-AgeI site of pBACCS × 2-WPRE to prepare a recombinant vector (pYFP-BACCS × 2-WPRE). Calcium imaging was performed on HEK293T cells expressing YFP-BACCS × 2. In addition, calcium imaging was also performed on HEK293T cells expressing BACCS × 2-igY. Rhod-3 (red fluorescent calcium sensor) was used as the calcium fluorescent probe. 50 seconds after the start of observation, the cells were stimulated by continuously irradiating an LED lamp having a wavelength of 470 nm. For each cell, ΔF / 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. In the figure, B × 2 indicates BACCS × 2-igY (analyzed cell number n = 5), and YB × 2 indicates YFP-BACCS × 2 (analyzed cell number n = 5). ΔF / F0 at each time point is shown as an average value for each cell together with a standard error.
As shown in 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). Each abbreviation in 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.

  Similarly, 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 Jα region is also highly conserved among various plant species (FIG. 3). The abbreviations in FIG. 3 are Os; rice phototropin 1, Zm; corn phototropin 1, At; Arabidopsis phototropin 1 and phototropin 2, Ps; peas phototropin 1, Vf; broad bean phototropin 1, Ac; Phytochrome 3, As; oat phototropin 1 is shown (cited from Harper et al., 2004).

  Furthermore, the LOV2 region is also conserved among many plants as described above. For example, 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%.

  From these facts, there is a possibility that each region can be substituted with various kinds of homologs.

  The plasmid constructs used in this example are shown below.

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Claims (13)

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 Jα 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-Jα, and in which one or more amino acids are substituted in the amino acid sequence of SEQ ID NO: 2 or 3;
It consists of an amino acid sequence that can activate SEQ ID NO: 4 or the calcium channel Orai1 and a CAD 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: 4. A polypeptide comprising:
The LOV2 region, the Jα region and the CAD region are arranged in the order of the LOV2 region, the Jα region and the CAD region from the N-terminus toward the C-terminus,
A polypeptide having no amino acid sequence interposed between the C-terminus of the Jα region and the N-terminus of the CAD region. An Orai1 region that can function as SEQ ID NO: 5 or a calcium channel, and that consists 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: 5,
A first BACCS region comprising the polypeptide of claim 1;
A polypeptide comprising an amino acid sequence comprising a second BACCS region comprising the polypeptide of claim 1,
The Orai1 region, the first BACCS region, and the second BACCS region are arranged in the order of the Orai1 region, the first BACCS region, and the second BACCS region from the N-terminus toward the C-terminus. Polypeptide.   The polypeptide according to claim 2, wherein a first linker is interposed between the C-terminus of the Orai1 region and the N-terminus of the first BACCS region.   The polypeptide according to claim 2 or 3, wherein a second linker is interposed between the C-terminus of the first BACCS region and the N-terminus of the second BACCS region. A first BACCS region comprising the polypeptide of claim 1;
A second BACCS region consisting of the polypeptide of claim 1, and
A polypeptide comprising an amino acid sequence which can function as SEQ ID NO: 5 or a calcium channel and which does not include an 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: 5.   The polypeptide according to claim 5, wherein a linker is interposed between the C-terminus of the first BACCS region and the N-terminus of the second BACCS region.   An isolated nucleic acid encoding the polypeptide of any one of claims 1-6.   A recombinant vector comprising the nucleic acid according to claim 7.   A gene transfer kit comprising the recombinant vector according to claim 8.   A transformant comprising the recombinant vector according to claim 8 and being an isolated cell or a non-human animal.   A method for regulating an intracellular calcium signal, wherein the intracellular calcium signal of the transformant is regulated by manipulating the intensity of light applied to the transformant according to claim 10.   The method for regulating an intracellular calcium signal according to claim 11, wherein the polypeptide is the polypeptide according to any one of claims 1, 5 and 6, and the cell expresses Orai1.   The method for regulating an intracellular calcium signal according to claim 9, wherein the polypeptide is the polypeptide according to any one of claims 2 to 4.