JP5788160B2 - Calcium sensor protein using green fluorescent protein or its homologue substituted with amino acid at specific site - Google Patents

Description

  The present invention relates to a calcium sensor protein using a green fluorescent protein (hereinafter referred to as GFP) substituted with an amino acid at a specific site, or a homologue thereof. More specifically, the present invention relates to a calcium sensor protein having higher sensitivity and reaction amount than a calcium sensor protein using conventional GFP or a homologue thereof, and a calcium sensor gene encoding the calcium sensor protein.

Calcium is a major component of bone that is essential for the maintenance of the structure for the living body, and at the same time as a regulator of various cellular functions such as muscle contraction, nerve excitability, hormone secretion, and changes in enzyme activity. It plays an essential role in maintaining and regulating functions. For this reason, the importance of the calcium sensor used for detecting calcium fluctuation in the living body (extracellular and intracellular) and measuring the calcium concentration is increasing.
Four types of calcium sensors have been developed so far. The outline is shown below.

1) Calcium-sensitive synthetic pigments: These are chemically synthesized pigments sensitive to calcium, and are currently commonly used. When used in a cell, it is necessary to make the cell take in from outside, but it is difficult to make the dye take in only a specific cell, and the dye must be injected into the cell with a glass needle or the like. Have.

2) Aequorin: a protein that emits light in response to calcium, and is directly injected into a cell, or a gene that produces the protein is introduced into a cell and used. In order to function in a cell, it is necessary to supply a coenzyme to the cell, and there is a problem that luminescence is extremely weak.

3) Calcium-sensitive protein using fluorescence resonance energy transfer (FRET): Calcium-sensitive calmodulin (CaM) and a partial sequence of myosin light chain kinase that binds to it, two different colors of GFP or their homologs This protein utilizes the fact that when calcium binds to CaM, its structure changes, and FRET occurs to change the fluorescence intensity emitted by two GFPs or their homologues. The protein is used by directly injecting into a cell or by allowing a gene encoding the protein to be taken into the cell. There is a problem that the fluorescence change in FRET is slight and cannot be measured by a laser microscope equipped with a commonly used argon laser.

4) Calcium-sensitive protein consisting of one GFP: a calcium-sensitive protein in which CaM is bound between positions 144 and 146 of the GFP amino acid sequence (SEQ ID NO: 3). When calcium binds to CaM, the structure of the protein changes. And it utilizes that the fluorescence intensity which GFP emits changes. The protein is also used by directly injecting it into cells or by incorporating other genes into cells. In general, since the sensitivity to calcium is low and the signal / noise ratio is low in an actual cell, there is a problem that measurement is difficult.

  As an application of the above 4), the present inventors have prepared a method for producing a calcium sensor protein capable of controlling the fluorescence characteristics of GFP, a calcium sensor protein produced by the method, and a code for the calcium sensor protein. Provides a calcium sensor gene that can be used (Patent Document 1), has higher sensitivity to calcium than conventional calcium sensors, is easily incorporated into specific cells, and requires special equipment and coenzymes for measurement. Has succeeded in creating a calcium sensor protein (Patent Document 2).

  However, in recent years, there has been an ever-increasing need to detect minute fluctuations of calcium in a living body, and even with the calcium sensor proteins of Patent Document 1 and Patent Document 2, sufficient results have been achieved. The situation is impossible.

Japanese Patent No. 3650815 Japanese Patent Application No. 2009-289789

  In view of the above circumstances, an object of the present invention is to provide a calcium sensor protein that is more excellent in sensitivity and reactivity than a conventional calcium sensor, and a gene encoding the protein.

That is, the present invention relates to the following (1) to (10).
(1) A calcium sensor protein comprising the following amino acid sequences (a) to (i) in order from the N-terminus:
(A) the amino acid sequence represented by SEQ ID NO: 1;
(B) a sequence consisting of three amino acids Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each independently any amino acid) (linker X) (SEQ ID NO: 2);
(C) a myosin light chain kinase protein, or a partial amino acid sequence thereof comprising a calmodulin binding site;
(D) Thr-Ser, Gly-Ser, Leu-Glu, Thr-Tyr, Thr-Asp, Thr-Cys, Thr-Phe linking the sequence of (c) described above and the sequence of (e) described below. Any one amino acid sequence selected from the group consisting of Thr-Met, Thr-Thr, Thr-Glu, Thr-His and Thr-Leu (linker Y);
(E) an amino acid sequence from the Xth to the 239th amino acid sequence of the sequence represented by SEQ ID NO: 3, wherein the 154th and / or 204th and / or 206th amino acids are substituted with other amino acids (where, X is any position from 149 to 151);
(F) A sequence Gly-Gly-Xaa5-Gly-Gly-Xaa6 composed of six amino acid sequences linking the sequence (e) described above and the sequence (g) described later (where Xaa5 and Xaa6 are independent of each other). Any amino acid) (SEQ ID NO: 4);
(G) an amino acid sequence from the first to the Y-th amino acid sequence of the sequence represented by SEQ ID NO: 3, wherein the 106th and / or 125th amino acid is substituted with another amino acid (where Y is 141 to 148 any position);
(H) the amino acid sequence Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg or Thr (linkage) between the sequence of (g) described above and the sequence of (i) described later Linker Z);
(I) An amino acid sequence from the 2nd to 148th amino acid sequence of the sequence represented by SEQ ID NO: 9, wherein the 36th and / or 60th and / or 78th amino acids are substituted with other amino acids.

(2) The number of His present in the 5th to 10th positions of the sequence (a) is 0 to 5 and / or the second Arg of the sequence (a) is deleted, and / or The 154th amino acid of the sequence (e) is substituted with Lys, Arg, His, Thr, Ser, Gly or Ala, and / or the 204th amino acid of the sequence (e) is Val, Leu, Ile, Substituted with Gly or Ala, and / or the 206th amino acid of the sequence of (e) is substituted with Asn, Gln, Thr, Ser, Gly or Ala, and / or the 106th amino acid of the sequence of (g) The amino acid is substituted with Tyr, Phe, Trp, Thr, Ser, Gly or Ala, and / or the 125th amino acid of the sequence (g) is Val, Leu, Ile In the sequence (i), the 36th amino acid of the sequence represented by SEQ ID NO: 9 is substituted with Leu, Ile, Val, Gly or Ala, and / or (i) ), The 60th amino acid of the sequence shown in SEQ ID NO: 9 is substituted with Asp, Glu, Gly or Ala, and / or the 78th sequence of the sequence shown in SEQ ID NO: 9 in the sequence (i) The calcium sensor protein according to (1) above, wherein the amino acid is substituted with Tyr, Phe, Trp, Gly or Ala.

(3) The sequence of (e) is an amino acid sequence from the 150th to 239th of the sequence represented by SEQ ID NO: 3, and the sequence of (g) is the first to 145th of the sequence represented by SEQ ID NO: 3. The calcium sensor protein according to (1) or (2) above, which has an amino acid sequence up to

(4) The sequence of (b) is an amino acid sequence represented by SEQ ID NO: 5 or 6 and / or the sequence of (c) is an amino acid sequence represented by SEQ ID NO: 7, and / or The calcium sensor protein according to any one of (1) to (3) above, wherein the sequence (f) is the amino acid sequence represented by SEQ ID NO: 8.

(5) The sequence of (b) is the amino acid sequence represented by SEQ ID NO: 6, the sequence of (d) is Leu-Glu, and the sequence of (h) is Thr-Arg, The calcium sensor protein according to (4) above.

(6) The sequence of (e) is an amino acid sequence from the 150th to the 239th of the sequence represented by SEQ ID NO: 3 that does not include amino acid substitution, and the sequence of (g) is represented by SEQ ID NO: 3. 1st to 145th, wherein the 106th amino acid is substituted with Tyr, the 125th amino acid is substituted with Val, and the 36th amino acid of the sequence represented by SEQ ID NO: 9 in the sequence of (i) above The calcium sensor protein according to (5) above, wherein is substituted with Leu.

(7) The sequence of (e) is from position 150 to position 239 of the sequence represented by SEQ ID NO: 3, wherein the amino acid at position 154 is substituted with Lys, the amino acid at position 204 is substituted with Val, and (g ) Is the first to 145th amino acid sequence of the sequence represented by SEQ ID NO: 3 that does not include amino acid substitution, and in the sequence of (i), the 60th sequence of the sequence represented by SEQ ID NO: 9 The calcium sensor protein according to (5) above, wherein the amino acid is substituted with Asp and the 78th amino acid is substituted with Tyr.

(8) The number of His present from the fifth to the tenth of the sequence of (a) is 5, and the sequence of (e) is the 150th to 239th of the sequence represented by SEQ ID NO: 3, The 206th amino acid is substituted with Asn, and the sequence of (g) is an amino acid sequence from the 1st to the 145th of the sequence shown in SEQ ID NO: 3 which does not include amino acid substitution, and the sequence of (i) is The calcium sensor protein according to (5) above, which is a sequence from the 2nd to the 148th of the sequence represented by SEQ ID NO: 9 which does not include amino acid substitution.

(9) The number of His present from the 5th to the 10th in the sequence of (a) is 5, and the sequence of (e) is the 150th to 239th of the sequence represented by SEQ ID NO: 3, The amino acid at position 154 is replaced with Lys, the amino acid at position 204 is replaced with Val, the amino acid at position 206 is replaced with Asn, and the sequence (g) is the first to 145th positions of the sequence represented by SEQ ID NO: 3 The amino acid at position 106 is replaced with Tyr, the amino acid at position 125 is replaced with Val, and the amino acid at position 36 in the sequence (i) is replaced with Leu; The calcium sensor protein according to (5) above, wherein the 60th amino acid is substituted with Asp and the 78th amino acid is substituted with Tyr.

(10) A calcium sensor gene encoding the protein according to any one of (1) to (9) above.

According to the present invention, it is possible to provide a calcium sensor protein in which the amount of change in fluorescence intensity is more stable and sensitive than a conventional calcium sensor.

FIG. 1 is an image diagram of a human uterine cancer-derived cell line (HeLa cell) into which DNA of a calcium sensor protein (G-CaMP7) has been introduced and expressed by a transfection method. FIG. 2 is a graph showing how the fluorescence intensity of a calcium sensor protein (G-CaMP7) expressed in HeLa cells changes with respect to ATP. FIG. 3 is a graph comparing the amount of change in fluorescence intensity with respect to ATP treatment on HeLa cells expressing the conventional calcium sensor protein (G-CaMP4) or the calcium sensor protein of the present invention (G-CaMP7). FIG. 4 is a graph showing the relationship between the calcium concentration and the fluorescence amount of purified calcium sensor proteins (G-CaMP4 and G-CaMP7).

The calcium sensor protein of the present invention is a protein characterized by having the following amino acid sequences (a) to (i) in order from the N-terminus;
(A) the amino acid sequence represented by SEQ ID NO: 1;
(B) a sequence consisting of three amino acids Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each independently any amino acid) (linker X) (SEQ ID NO: 2);
(C) a myosin light chain kinase protein, or a partial amino acid sequence thereof comprising a calmodulin binding site;
(D) Thr-Ser, Gly-Ser, Leu-Glu, Thr-Tyr, Thr-Asp, Thr-Cys, Thr-Phe linking the sequence of (c) described above and the sequence of (e) described below. Any one amino acid sequence selected from the group consisting of Thr-Met, Thr-Thr, Thr-Glu, Thr-His and Thr-Leu (linker Y);
(E) an amino acid sequence from the Xth to the 239th amino acid sequence of the sequence represented by SEQ ID NO: 3, wherein the 154th and / or 204th and / or 206th amino acids are substituted with other amino acids (where, X is any position from 149 to 151);
(F) A sequence Gly-Gly-Xaa5-Gly-Gly-Xaa6 composed of six amino acid sequences linking the sequence (e) described above and the sequence (g) described later (where Xaa5 and Xaa6 are independent of each other). Any amino acid) (SEQ ID NO: 4);
(G) an amino acid sequence from the first to the Y-th amino acid sequence of the sequence represented by SEQ ID NO: 3, wherein the 106th and / or 125th amino acid is substituted with another amino acid (where Y is 141 to 148 any position);
(H) the amino acid sequence Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg or Thr (linkage) between the sequence of (g) described above and the sequence of (i) described later Linker Z);
(I) An amino acid sequence from the 2nd to 148th amino acid sequence of the sequence represented by SEQ ID NO: 9, wherein the 36th and / or 60th and / or 78th amino acids are substituted with other amino acids.

Here, the sequence (a) (amino acid sequence represented by SEQ ID NO: 1) is composed of His at the 5th to 10th positions, and the number of His may be any number between 0 and 6. It is preferably 0 to 5, particularly preferably 5. The second Arg may be present or may be deleted.
In addition, the sequences (e), (g) and (i) need only contain the above-mentioned amino acid substitution in at least one sequence ((e), (g) or (i)). Only the 204th and 206th amino acids of (e) are substituted, and the sequence (g) is the first to Yth amino acid sequence of the sequence represented by SEQ ID NO: 3 and does not include amino acid substitution (here Y is an arbitrary position of 141 to 148), and the sequence (i) is an amino acid sequence from the 2nd to the 148th amino acid sequence of the sequence represented by SEQ ID NO: 9 and does not include amino acid substitution Good.

  The calcium sensor protein is mainly composed of modified GFP (e and g), (2) functional molecule (c and i), and (3) linker (d, f and h). Yes.

  The modified GFP is obtained by cleaving the amino acid sequence of GFP in the vicinity of a hot spot amino acid residue that affects the fluorescence properties to modify the structure of the protein, and further substituting the amino acid residue at a specific site.

  The GFP of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 3. The GFP homologue of the present invention is a protein comprising the same or substantially the same amino acid sequence as the amino acid represented by SEQ ID NO: 3. Here, the “protein containing substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, or 82 with the amino acid sequence represented by SEQ ID NO: 3. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Most preferably, it is a protein that contains an amino acid sequence having about 99% amino acid identity and emits fluorescence, and examples thereof include GFP color mutants such as BFP, CFP, GFP, YFP, and RFP.

  Alternatively, as a protein comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3, one or several (preferably about 1 to 30 amino acids, more preferably, the amino acid sequence represented by SEQ ID NO: 3 is more preferable. Is a protein that consists of an amino acid sequence in which about 1 to 10 amino acids, more preferably 1 to 5 amino acids are deleted, substituted, or added, and emits fluorescence.

  In the present invention, the “hot spot amino acid residue” that affects the fluorescence characteristics of GFP is an amino acid residue on GFP that serves as an index of the modified position when creating modified GFP. The desired modified GFP can be created by modifying the structure of GFP in the vicinity of. The hot spot amino acid residue of GFP estimated in the present invention is the 149th amino acid in the amino acid sequence of GFP represented by SEQ ID NO: 3.

  Modifying the structure of GFP preferably means cleaving GFP in the vicinity of a presumed hot spot amino acid residue (preferably, each position within the range of 5 amino acids before and after the hot spot amino acid residue). An appropriate number of amino acids (preferably 1-10 amino acids) are removed from the GFP, and the GFP original N-terminus and the original C-terminus are linked with an appropriate amino acid sequence as necessary.

  The modified GFP substituted with an amino acid residue at a specific site refers to an amino acid residue at a specific site (preferably the 106th and / or 125th and / or 154th and / or the GFP amino acid sequence shown in SEQ ID NO: 3). (Amino acid residue at position 204 and / or 206) is replaced with any amino acid (for example, the amino acid at position 106 is replaced with Tyr, Phe, Trp, Thr, Ser, Gly or Ala, particularly preferably Tyr. And / or the amino acid at position 125 is replaced by Val, Leu, Ile, Gly or Ala, particularly preferably Val, and / or the amino acid at position 154 is Lys, Arg, His, Thr, Ser, Gly or Ala, Particularly preferably, Lys is substituted and / or the 204th amino acid is Val, Leu, Ile, ly or Ala, particularly preferably, using GFP substituted with Val and / or the 206th amino acid substituted with Asn, Gln, Thr, Ser, Gly or Ala, particularly preferably Asn) The GFP whose structure has been modified as described above.

  As a particularly preferred example of the modified GFP, the amino acid sequence from the 150th to the 239th amino acid sequence of SEQ ID NO: 3, wherein the 154th amino acid is replaced with Lys, the 204th amino acid is replaced with Val, The amino acid sequence in which the 206th amino acid is substituted with Asn is the N-terminal side, the first to 145th amino acids of the sequence represented by SEQ ID NO: 3, the 106th amino acid is substituted with Tyr, the 125th amino acid Examples include amino acids in which amino acids are substituted with Val and those linked by an appropriate amino acid sequence with the C-terminal side as the amino acid.

  A functional molecule is a molecule that can cause a change in the three-dimensional structure by the action of a factor acting on the molecule itself, and is linked to the modified GFP. A molecule that can transmit changes to the modified GFP. At this time, the functional molecule functions to change the three-dimensional structure of the modified GFP by changing the three-dimensional structure of the functional molecule to the modified GFP, thereby changing the fluorescence characteristics.

  Therefore, the functional molecule needs to be linked to the modified GFP at a position where it can transmit a change in its three-dimensional structure to the modified GFP and affect the structure of the modified GFP. Therefore, it is preferable that the functional molecule is linked in the vicinity of the part where the original GFP structure is modified. Specifically, it is preferable to be linked via a linker molecule to the position where GFP is cleaved.

  Such a functional molecule may be one molecule or two or more molecules. In the case of two molecules, factors acting on the functional molecule first change the three-dimensional structure of one functional molecule, and then the three-dimensional structure changes the three-dimensional structure of the modified GFP. Examples of the functional molecule in the present invention include a combination of a calmodulin protein and a myosin light chain kinase protein.

  In addition, the functional molecule linked to the modified GFP does not have to have the entire structure as the functional molecule is expressed in the living body, and only has a part where a factor acting on the functional molecule binds. It may be a structure. Preferred examples of the present invention include a calmodulin protein and a part of a myosin light chain kinase having a calmodulin binding function shown in SEQ ID NO: 7 as functional molecules. By appropriately linking such a functional molecule to the modified GFP, the fusion protein can function as a calcium sensor.

  The amino acid sequence from the 1st amino acid to the 148th amino acid sequence of the rat calmodulin protein used as a functional molecule is shown in SEQ ID NO: 9.

  The linker is a cleavage site of the modified GFP, a linking site between the modified GFP and the functional molecule, a protein obtained by linking the modified GFP and the functional molecule, and an amino acid sequence on the N-terminal side (in the present invention). Is a peptide consisting of several residues of amino acids located at the linking site to the amino acid sequence shown in SEQ ID NO: 1. In order to distinguish each linker molecule, the following names are given.

  The amino acid sequence represented by SEQ ID NO: 1 (above sequence (a)) present at the N-terminus of the entire fusion protein composed of modified GFP and a functional molecule, and a partial amino acid sequence including a myosin light chain kinase protein or a calmodulin binding site The linker to be linked is called linker X. The linker X is an arbitrary amino acid sequence including Met, but is preferably an amino acid peptide having 1 to 10 residues, more preferably a sequence Met-Xaa1-Xaa2 consisting of three amino acids (where Xaa1 and Xaa2 are Each is independently an arbitrary amino acid; SEQ ID NO: 2), more preferably Met-Gly-Thr (SEQ ID NO: 5) or Met-Val-Asp (SEQ ID NO: 6).

  The linker that links the N-terminal side of the modified GFP and the functional molecule is referred to as linker Y, and is an arbitrary amino acid sequence, preferably an amino acid peptide of 0 to 10 residues, more preferably composed of two amino acids. The sequence Xaa3-Xaa4 (where Xaa3 is any one amino acid selected from the group consisting of leucine, threonine and glycine, and Xaa4 is any amino acid), more preferably Thr-Ser, Gly-Ser, Any one selected from the group consisting of Leu-Glu, Thr-Tyr, Thr-Asp, Thr-Cys, Thr-Phe, Thr-Met, Thr-Thr, Thr-Glu, Thr-His, and Thr-Leu. Particularly preferred is Leu-Glu.

  The linker that links the C-terminal side of the modified GFP and the functional molecule is referred to as linker Z, and is Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg, or ThrThr-Arg or Thr. And Thr-Arg is particularly preferable.

  The amino acid peptide that links the original N-terminus and C-terminus of GFP is also a linker, preferably a peptide consisting of 2 to 10 amino acids, preferably containing a large amount of Gly, more preferably 6 A sequence consisting of two amino acid sequences Gly-Gly-Xaa5-Gly-Gly-Xaa6 (where Xaa5 and Xaa6 are each independently any amino acid; SEQ ID NO: 3), and particularly preferred examples include Gly-Gly -Thr-Gly-Gly-Ser (SEQ ID NO: 8).

  The calcium sensor protein in the present invention affects the three-dimensional structure of the functional molecule by acting calcium, which is a factor affecting the three-dimensional structure of the functional molecule contained in the protein. This refers to a protein that reversibly changes the fluorescence properties of the modified GFP by affecting the three-dimensional structure of the modified GFP contained in the calcium sensor protein. This change refers to a change that can be detected with a fluorescence microscope or a laser microscope, and preferably a change that can be detected with the naked eye. When the change in fluorescence characteristics is a change in fluorescence intensity, it means that the fluorescence change amount ΔF / F preferably changes by at least 0.1 or more, more preferably changes by 1 or more.

  In the present invention, the fluorescence characteristics refer to fluorescence characteristics such as fluorescence intensity, fluorescence wavelength, fluorescence intensity ratio, absorbance, and absorption wavelength. In the present invention, fluorescence intensity is used as an example of fluorescence characteristics.

  In the present invention, when the fluorescence characteristic is fluorescence intensity, the calcium sensor protein is in a critical state of a state of emitting fluorescence and a state of not emitting fluorescence. In this critical state, the calcium sensor protein may be in a state where it does not emit fluorescence, or may be in a state where fluorescence intensity is low. Or you may exist in a state with high fluorescence intensity. Here, not emitting fluorescence means that fluorescence cannot be confirmed using an optical device. “Low fluorescence intensity” means the above change amount (amount that ΔF / F changes by at least 0.1 or more) due to the presence of calcium and is low enough to change the fluorescence intensity to a high state. Similarly, high fluorescence intensity indicates the amount of change described above (the amount by which ΔF / F changes by at least 0.1) due to the presence of calcium, and is high enough to change the fluorescence intensity to a low state. Say.

  Compared with the conventional calcium sensor protein, the calcium sensor protein in the present invention substitutes the amino acid residues at specific sites of sequence (a), modified GFP and rat calmodulin as described above, and uses the specific linker as described above. Thus, it is characterized in that it causes a greater change in fluorescence characteristics during calcium action. Here, the larger change in fluorescence characteristic means that when the change in fluorescence characteristic is a change in fluorescence intensity, the amount of change ΔF / F in fluorescence is larger than that of the conventional calcium sensor protein, and is preferably enhanced two times or more. That means.

  The fusion protein can be prepared using a known genetic engineering technique. For example, a fragment of a gene encoding each protein part to be fused (that is, a gene encoding a modified GFP accompanied by relaxation of a specific amino acid site and a functional molecule) is prepared by PCR, and these fragments are joined to form a fusion gene. Then, a fusion protein is produced by introducing a plasmid containing the fusion gene into a desired cell and expressing it.

  In the present invention, the calcium sensor gene refers to a gene encoding the calcium sensor protein of the present invention. As described above, the calcium sensor gene is prepared by PCR by generating gene fragments encoding each component constituting the calcium sensor protein to be prepared, and then linking these gene fragments to form a fusion gene. Can be created in shape.

  The calcium sensor protein prepared in the present invention can measure calcium concentration more stably and with high sensitivity inside and outside the cell.

  For example, it is possible to measure the calcium concentration by introducing the calcium sensor gene of the present invention into Escherichia coli or the like and mixing a calcium sensor protein produced in advance with a specimen. It is also possible to measure the intracellular calcium concentration by directly injecting a protein produced using Escherichia coli or the like into the cell whose calcium concentration is to be measured. Alternatively, the intracellular calcium concentration can be measured by introducing the calcium sensor gene of the present invention into a cell whose calcium concentration is to be measured and causing the cell to produce a protein.

  The calcium concentration is measured by applying light of a specific wavelength (for example, excitation light of 488 nm) to the calcium sensor protein and detecting the fluorescence characteristics emitted by the protein with an optical instrument (for example, a laser microscope). . In addition, it is necessary to investigate in advance what fluorescence characteristics the calcium sensor protein used for concentration measurement shows at what calcium concentration. Specifically, it is necessary to measure changes in fluorescence intensity with respect to various calcium concentrations using, for example, a calcium sensor protein produced by Escherichia coli using a fluorescence spectrophotometer (see, for example, FIG. 4). ). By this measurement, results of Kd63nM, Hill coefficient 3.8, and maximum fluorescence change 25.5 were obtained.

  Examples of the present invention are shown below, but these examples are merely illustrative and do not limit the scope of the present invention.

Characteristics of G-CaMP7, a calcium sensor protein of the present invention The calcium sensor protein (G-CaMP7) developed in the present invention introduces a gene encoding the protein into HeLa cells, which are human uterine cancer-derived cell lines. Then, performance evaluation as a calcium sensor was performed (FIG. 1).

  The calcium sensor protein G-CaMP7 of the present invention was expressed in HeLa cells, and then ATP (0.1 mM) was allowed to act as a factor known to increase the intracellular calcium ion concentration. An example of the reaction at that time is shown in FIG. It has been found that the fluorescence intensity of G-CaMP7, which is the calcium sensor protein of the present invention, increases more stably than conventional calcium sensor proteins (G-CaMP4 and the like).

The amount of change in fluorescence intensity for ATP in HeLa cells expressing the calcium sensor protein G-CaMP7 of the present invention was confirmed to be 4.2 times greater than that of the conventional calcium sensor protein G-CaMP4. (FIG. 3). In addition, G-CaMP7 is a calcium sensor protein that exhibits a markedly superior effect over conventional G-CaMP2 because its fluorescence change is about 10 times or more compared to G-CaMP2 from which G-CaMP7 is derived. is there.
Furthermore, as a result of examining the relationship between the calcium concentration of the purified calcium sensor proteins (G-CaMP7 and G-CaMP4) and the amount of fluorescence, G-CaMP7 is approximately twice as sensitive to calcium ions as G-CaMP4. It is high (the Kd value of G-CaMP7 is about twice lower than that of G-CaMP4 and the Hill coefficient of both is about the same), and the amount of change in fluorescence intensity with and without calcium ions is about 4.5 times larger ( When the ratio of the fluorescence intensity with and without calcium ions was determined, it was confirmed that G-CaMP7 was 25.5 times, whereas G-CaMP4 was 5.6 times (FIG. 4).
Thus, it has been proved that the calcium sensor protein of the present invention is more stable and sensitive than conventional ones.

Preparation and measurement method of calcium sensor protein G-CaMP7 (A) Production method of G-CaMP7 (A-1) Plasmid construction for bacterial expression and mammalian expression pRSET _B -GCaMP7 which is a plasmid for bacterial expression of G-CaMP7 and PN1-GCaMP7, which is a plasmid for mammalian expression, is obtained by using pRSET _B- GCaMP2 and pN1-GCaMP2 described in Reference Document 1 (Proc. Natl. Acad. USA, 103, 4753-4758 (2006)). It was constructed by modifying as described below.
That is, first, in the amino acid sequence of GCaMP2 (SEQ ID NO: 10), Met-36 → Leu (the 36th Met in SEQ ID NO: 9) of the calmodulin portion, and Asn-105 → Tyr (the 106th Asn in SEQ ID NO: 3) of the GFP portion. ), Glu-124 → Val (125th Glu in SEQ ID NO: 3) 5'-ATG- encoding Met-36 of the calmodulin moiety in the cDNA sequence (SEQ ID NO: 11) so as to be amino acid substituted. 5 ′ encoding 5′-CTG-3 ′ and 5′-AAC-3 ′ encoding Asn-105 of the GFP moiety 5′-TAC-3 ′ and Glu-124 5 '-GAG-3' was mutated to 5'-GTG-3 'to construct cfGCaMP2.

Next, in the sequence of cfGCaMP2, Met-153 → Lys of the GFP part (154th Met in SEQ ID NO: 3), Thr-203 → Val (204th Thr in SEQ ID NO: 3), Asn-60 → Asp of the calmodulin part It encodes Met-153 of the GFP part in the cDNA sequence so that the amino acid substitution is (Asn at 60th in SEQ ID NO: 9) and Asp-78 → Tyr (Asp at 78th in SEQ ID NO: 9). 5′-ATG-3 ′ is encoded as 5′-AAG-3 ′, 5′-ACC-3 ′ encoding Thr-203 is encoded as 5′-GTT-3 ′, and Asn-60 of the calmodulin moiety is encoded. 5'-AAT-3 'to 5'-GAT-3' and 5'-GAC-3 'encoding Asp-78 to 5'-TAC-3' GCaMP6 was constructed with various mutations.
Furthermore, the tag portion His6 is encoded in the cDNA sequence so that the amino acid substitution is made in the sequence of GCaMP6 from His6 → His5 in the tag portion and Ser-205 → Asn in the GFP portion (206th Ser in SEQ ID NO: 3). 5′-CATCATCATCATCATCAT-3 ′ is mutated to 5′-CATCATCATCATCAT-3 ′, and 5′-TCC-3 ′ encoding Ser-205 of the GFP portion is mutated to 5′-AAT-3 ′. Thus, GCaMP7 was constructed.

More specifically, the following synthetic primer (Operon) using pN1-GCaMP2 and pRSET _B -GCaMP2 as templates first:
5′-ACGGTGCTGCGGTCTCTGGGGCAGA-3 ′ (rCaM-30) (SEQ ID NO: 12)
5′-AGACCGCAGCACCGTCCCCAGCTCCTT-3 ′ (rCaM-31) (SEQ ID NO: 13)
The point mutation was introduced by PCR using the method described later to prepare pN1-GCaMP2 (CaM M36L) and pRSET _B -GCaMP2 (CaM M36L).

Secondly, the following synthetic primer (Operon) using pN1-GCaMP2 (CaM M36L) and pRSET _B -GCaMP2 (CaM M36L) as a template:
5′-GACGGCTACTACAAGACCCGCGCCG-3 ′ (GCaMP-66) (SEQ ID NO: 14)
5′-CTTGTAGTAGCCGTCGTCCTTGAAGAA-3 ′ (GCaMP-67) (SEQ ID NO: 15)
The point mutation was introduced by PCR using the method described later to prepare pN1-GCaMP2 (N105Y / CaM M36L) and pRSET _B -GCaMP2 (N105Y / CaM M36L).

Third, the following synthetic primers (Operon) using pN1-GCaMP2 (N105Y / CaM M36L) and pRSET _B -GCaMP2 (N105Y / CaM M36L) as templates:
5′-CGCATCGTGCTGAAGGGCATCGACT-3 ′ (GCaMP-68) (SEQ ID NO: 16)
5′-CTTCAGCACGATGCGGTTCACCAGGGT-3 ′ (GCaMP-69) (SEQ ID NO: 17)
PN1-cfGCaMP2 and pRSET _B -cfGCaMP2 were prepared by introducing point mutations by PCR using the method described below.

Fourth, the following synthetic primers (Operon) using pN1-cfGCaMP2 and pRSET _B -cfGCaMP2 as templates:
5′-GACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAG-3 ′ (GCaMP-84) (SEQ ID NO: 18)
5′-CTTTCGAAAGTTTGGACTGCACGCTCAGGTAGTGGTTGTC-3 ′ (GCaMP-85) (SEQ ID NO: 19)
The point mutation was introduced by PCR using the method described below to prepare pN1-cfGCaMP2 (T203V) and pRSET _B -cfGCaMP2 (T203V).

Fifth, using pN1-cfGCaMP2 (T203V) and pRSET _B -cfGCaMP2 (T203V) as templates, the following synthetic primers (Operon)
5′-CAAGAAAAATGAAATACACAGACAGTGAAGAAGAAATTAG-3 ′ (GCaMP-86corr) (SEQ ID NO: 20)
5′-CTAATTTCTTCTTCACTGTCTGTGTATTTCATTTTTCTTG-3 ′ (GCaMP-87corr) (SEQ ID NO: 21)
PN1-cfGCaMP3 and pRSET _B -cfGCaMP3 were prepared by introducing point mutations by PCR using the method described below.

Sixth, using pN1-cfGCaMP3 and pRSET _B -cfGCaMP3 as templates, the following synthetic primers (Operon)
5′-GAACGTCTATATCAAGGCCGACAAGCAG-3 ′ (GFP (M153K)) (SEQ ID NO: 22)
5′-GATGCCGACGGTGATGGCACAATCG-3 ′ (CaM (N60D)) (SEQ ID NO: 23)
PN1-GCaMP6 and pRSET _B -GCaMP6 were prepared by performing simultaneous multipoint mutagenesis by PCR using the method described below.

Seventh, using pRSET _B -GCaMP6 as a template, the following synthetic primer (Operon)
5′-TCAGATCTCGCCACCATGCGGGGTTCTCATCATCATCATCAT-3 ′ (N1RSETmod) (SEQ ID NO: 24)
5′-ATATTTCGAAAGTTTNNNCTGNNNGCTCAGGTAGTGGTTGTCGGG-3 ′ (GCaMP-44) (SEQ ID NO: 25)
The mutation was introduced semi-randomly by PCR using the method described below. E. coli KRX was transformed with this PCR product, and a GCaMP6 mutant clone with a bright green fluorescence and a large difference in brightness with and without calcium was screened. As a result, GCaMP6 (His5 / S205N) was discovered. This clone was named GCaMP7 (SEQ ID NO: 26 (amino acid sequence), SEQ ID NO: 27 (nucleic acid sequence)).

Subsequently, the following synthetic primer (Operon) using pRSET _B -GCaMP7 as a template
5′-TCAGATCTCGCCACCATGCGGGGTTCTCATCATCATCATCAT-3 ′ (N1RSETmod) (SEQ ID NO: 28)
5′-GCGCGGCCGCTCACTTCGCTGTCATCATTTGTAC-3 ′ (rCaM-10) (SEQ ID NO: 29)
The full-length cDNA of GCaMP7 was amplified by the PCR method described below, and a 1.37 kb fragment digested with BglII and NotI was ligated with a 3.94 kb fragment digested with BglII and NotI to pN1-GCaMP7. It was created.

  Point mutation introduction by PCR was performed using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) according to the manual. Specifically, 5 ng of the template plasmid, 2.5 μl of 10 × buffer, 0.5 μl of 2.5 mM dNTP, 1 μl of each 10 μM primer, 1 μl of Quik Solution, 0.5 μl of Pfu DNA polymerase, and water were added. Then, the mixed solution with a total volume of 25 μl was subjected to the following conditions.

Step 1
95 degrees Celsius 1 minute step 2
95 degrees Celsius 50 seconds 60 degrees Celsius 50 seconds 68 degrees Celsius 5 minutes 18 cycles step 3
68 degrees Celsius 7 minutes Add 0.5 μl of 20 U / μl DpnI (Agilent) to the above mixed solution, treat at 37 degrees Celsius for 1 hour, and transform from XL-10 Gold to emerge from kanamycin resistant or ampicillin resistant Escherichia coli. It was collected.

  For simultaneous multipoint mutation introduction by PCR, QuikChange Lighting Multi-Site Directed Mutagenesis Kit (Agilent) was used, and the operation was performed according to the manual. Specifically, 1 μl of 100 ng / μl template plasmid, 2.5 μl of 10 × buffer, 1 μl of 2.5 mM dNTP, 0.6 μl of 10 μM each primer, 1 μl of Pfu DNA polymerase, and the total amount by adding water Was mixed under the following conditions.

Step 1
95 degrees Celsius 1 minute step 2
95 degrees Celsius 1 minute 55 degrees Celsius 1 minute 65 degrees Celsius 9 minutes 30 cycles of the above Add 1 μl of 20 U / μl DpnI (Agilent) to the total amount of the above mixture of plasmids containing the multipoint mutation for 1 hour at 37 degrees Celsius After the treatment, the plasmid containing the multipoint mutation was recovered from kanamycin-resistant or ampicillin-resistant Escherichia coli that appeared after transformation into XL-10 Gold as described below.

  Amplification of an arbitrary sequence by the PCR method is a mixed solution in which a template plasmid is 100 ng, 10 μM forward primer is 1 μl, 10 μM reverse primer is 1 μl, PrimeSTAR Max Premix (Takara) is 5 μl, and water is added to make a total volume of 10 μl. Was conducted under the following conditions.

Step 1
98 degrees Celsius 10 seconds step 2
98 degrees Celsius 10 seconds 55 degrees Celsius 10 seconds 72 degrees Celsius 25 seconds
72 degrees Celsius 30 seconds 10 μl of the above mixture was subjected to agarose electrophoresis by the following method to confirm the PCR product.

  Cleavage of DNA with restriction enzymes was performed using NEB, Toyobo, or Takara restriction enzymes, and their attached buffers and attached Bovine Serum Albumin (100 × BSA). The reaction was performed at 37 ° C. for 1 to 3 hours by adding 1 to 2 μg of DNA to the attached buffer (3 μl), attached 100 × BSA (0.3 μl) and each restriction enzyme (10 units) to a total volume of 30 μl. It was.

  Agarose gel (Agarose LE, Nacalai Tesque) was heated with TAE buffer (4.98 g / l Tris base (Nacalai Tesque), 1.142 ml / l glacial acetic acid (Nacalai Tesque), 1 mM EDTA (pH 8) (Dojindo)). Dissolved and prepared to 1% or 2%. Using λHindIII digest (Toyobo) or 100 bp DNA Ladder (Toyobo) as a DNA size marker, the DNA sample is a SYBR Green diluted 100-fold with DMSO (Sigma) in 1/10 volume of 10 × sample buffer attached to the restriction enzyme. What added 1/10 amount of I (Invitrogen) was electrophoresed at 100V using TAE buffer, and detected using Safe Imager (Invitrogen).

  MagExtractor (Toyobo) was used for DNA recovery from the gel, and the operation was performed according to the manual. Specifically, first, after agarose gel electrophoresis, the target band was cut out with a scalpel so as to be as small as possible on the Safe Imager, and 400 μl of the adsorbed solution was added and left at room temperature to completely dissolve the gel. Next, 30 μl of magnetic beads were added and left at room temperature for 2 minutes with occasional stirring. The magnetic beads adsorbed with DNA were adsorbed using a magnet stand, and the supernatant was discarded. 600 μl of the washing solution was added to the collected magnetic beads and stirred for 10 seconds with a vortex mixer, and the magnetic beads adsorbed with DNA using a magnetic stand were collected by the same method. 1 ml of 75% ethanol was added thereto and stirred for 10 seconds with a vortex mixer, and the magnetic beads adsorbed with DNA were collected using a magnet stand. This was spun down, and the supernatant was completely discarded. After drying at 55 ° C. for 2 minutes, 25 to 100 μl of water or TE was added and allowed to stand at room temperature for 2 minutes with occasional stirring. The magnetic beads after the DNA was dissociated were separated using a magnet stand, and the supernatant containing the DNA was collected.

  In the Ligation reaction, DNA Ligation Kit Ver. 2 (Takara) was used and the operation was performed according to the manual. Specifically, an equal volume of Ligation Mix was added to and mixed with a mixed solution of about 25 fmol of plasmid vector and about 25 to 250 fmol of insert DNA, and then reacted at 16 ° C. for 30 minutes.

  Transformation is performed in E. coli. E. coli competent cells DH5a (Takara), XL-10 Gold (Agilent), or KRX (Takara) were used. Specifically, 100 μl of competent cells were dissolved on ice, 1 μl of DNA solution or 1 μl of ligation reaction solution was added and left on ice for 30 minutes, and then heated at 42 degrees Celsius for 45 seconds. Thereafter, the mixture is further left on ice for 5 minutes, added with 500 μl of LB medium, cultured at 37 ° C. for 1 hour, and then planted in a selective medium (LB medium) containing 100 μg / ml ampicillin or 50 μg / ml kanamycin (Wako Chemicals). Incubate overnight at 37 degrees Celsius. The next day, the colonies were inoculated into 1 to 5 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin or 50 μg / ml kanamycin and cultured at 37 degrees Celsius for 16 hours.

  Miniprep Kit (Qiagen) was used to recover the plasmid from E. coli, and the operation was performed according to the manual. Specifically, 5 ml of the E. coli culture solution was first centrifuged at about 2000 × g for 10 minutes, and the supernatant was removed by decantation or pipetting to obtain an E. coli precipitate. To this precipitate, 250 μl of ice-cooled RNase-containing P1 buffer was added and suspended, 250 μl of P2 buffer was added and left at room temperature for 5 minutes, and the cells were disrupted with alkaline SDS. Thereafter, 350 μl of N3 buffer was added for neutralization. The cell disruption solution was transferred to a spin column and centrifuged at about 13200 × g for 30 seconds to 1 minute to adsorb the plasmid to the column. The column passage liquid was removed by decantation. Next, 750 μl of PE buffer was added to the column, and the column was washed by centrifugation at about 13200 × g for 30 seconds to 1 minute. The column passage liquid was removed by decantation. Further, the liquid droplets remaining on the column were completely removed by centrifugation at about 13200 × g for 30 seconds to 1 minute without adding any buffer. The column was attached to a new collection microtube, 50 μl of EB buffer was added to the column, and centrifuged at about 13200 × g for 30 seconds to 1 minute to elute and collect the plasmid from the column.

LB liquid medium 10 g / l Bacto-tryptone (Nacalai Tesque), 5 g / l Bacto-yeast extract (Nacalai Tesque), 5 g / l NaCl (Nacalai Tesque), 1 g / l glucose (Wako Chemicals). Sterilized in an autoclave.

LB agar medium 10 g / l Bacto-tryptone (Nacalai Tesque), 5 g / l Bacto-yeast extract (Nacalai Tesque), 5 g / l NaCl (Nacalai Tesque), 1 g / l glucose (Wako Chemicals), 15 g / l Agar Tesque). After sterilization in an autoclave, when the temperature drops to about 45 degrees, antibiotics (100 μg / ml ampicillin or 50 μg / ml kanamycin (Wako Chemicals)) are added and poured into a plastic dish.

  TE (pH 8) (10 mM Tris-HCl, 1 mM EDTA) (Wako Chemicals)

(A-2) Purification of protein G-CaMP7 protein is purified using Ni-NTA agarose (Qiagen), which specifically binds to the His tag, using the fact that these proteins have His tags. The operation was performed according to the manual. Specifically, pRSET _B -GCaMP7 E. coli competent cells KRX were transformed into LB selection medium containing 100 μg / ml ampicillin and cultured overnight at 37 degrees Celsius. The colony was inoculated into 10 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin and cultured at 37 degrees Celsius for 16 hours. 10 ml of the culture solution is further planted in 200 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin and cultured at 37 degrees Celsius until the absorbance OD600 is 0.5 to 1, after which the final concentration becomes 1%. Rhamnose (Promega) was added and cultured at 18-225 degrees Celsius for 4-5 hours.

After centrifugation at 3000 rpm for 15 minutes (6200 centrifuge, Kubota), E. coli was recovered. Escherichia coli was suspended in 1 ml of LB medium. After freezing at -20 degrees Celsius for 30 minutes, it was thawed at room temperature for 30 minutes. Freezing and thawing were repeated once more. 40 ml suspension buffer (25 mM Tris-HCl (pH 8) (Sigma), 1 mM 2-mercaptoethanol (Nacalai Tesque)), protease inhibitor (0.1 mM PMSF, 5 μg / ml leupeptin (Wako Chemicals)) chilled on ice Suspended Escherichia coli at 4 degrees Celsius and centrifuged at 100,000 xg for 15 minutes to obtain a supernatant, 5M NaCl was added to a final concentration of 0.3M, and 2ml of 50ml was added. % Ni-NTA agarose (Qiagen; protein binding ability 5 to 10 mg / ml resin) was further added, and the mixture was gently mixed at room temperature for 1 hour to allow the reaction to proceed, and the reaction solution was added to an empty column (Econo column; column size ˜20 ml (Bio- Rad)) and the excess liquid is not dripped from the column. After washing twice with 10 ml of washing solution (50 mM NaH ₂ PO ₄ (pH 8) (Nacalai Tesque), 0.3 M NaCl, 20 mM imidazole (Nacalai Tesque)), 3 to 4 ml of recovery solution (50 mM The His-tagged protein was recovered from the column by elution with NaH ₂ PO ₄ (pH 8) (Nacalai Tesque), 0.3 M NaCl, 250 mM imidazole (Nacalai Tesque). In sanko junyaku), it was dialyzed with 125 ml or more of KM buffer (0.1 M KCl (Nacalai Tesque), 20 mM MOPS-Tris (pH 7.5) (Dojindo)) at 4 degrees Celsius. After changing every hour, and after changing liquids 3 times or more It was recovered solution of the protein from the dialysis tube.

  The protein concentration was measured using a protein assay kit (Bio-Rad), and the operation was measured by the Bradford method (Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.) According to the manual. First, 1 ml of Bradford reagent was added to 50 μl of a protein solution diluted with water to 10 to 200 μg / ml, and the absorbance at 595 nm was measured 30 minutes later. The reference concentration of the protein was determined by measuring using bovine serum albumin as the reference protein. The measurement was performed at room temperature.

(B) Measurement method using G-CaMP7 (B-1) Measurement of calcium binding ability The calcium binding ability of G-CaMP7 protein is obtained by measuring the fluorescence intensity in various calcium concentration solutions. Calculations were based on intensity volume response curves. The G-CaMP7 protein purified as described above was diluted with KM buffer to a final concentration of 0.3 μM. For fluorescence intensity measurement, a fluorescence spectrophotometer F-2500 (Hitachi) was used to excite at 470 nm and record fluorescence at 510 nm. First, 20 mM BAPTA ((Dojindo)) was added to the measurement sample and measurement was performed in the absence of calcium, and then CaCl ₂ was successively added at various concentrations and measured. The measurement was performed at room temperature.

(B-2) HeLa cell culture and plasmid introduction Using a carbon dioxide incubator, HeLa cells were cultured in a medium (DMEM (Gibco), 10% Fetal Bovine Serum (Gibco), 1x penicillin streptomycin (Gibco)). After culturing at 37 degrees Celsius, the plasmid pN1-GCaMP7 was introduced into the cultured cells using Lipofectamine 2000 (Invitrogen). The introduction operation was performed according to the reagent manual. First, 0.8 μg of plasmid was diluted with 50 μl of DMEM without serum. Next, 2 μl of Lipofectamine 2000 was added to 50 μl of serum-free DMEM and left at room temperature for 5 minutes. Thereafter, both dilutions were mixed and allowed to stand at room temperature for 20 minutes. The entire amount of this mixed solution was administered to HeLa cells in a 24-well culture dish to introduce the plasmid. After introducing the plasmid, the cells were cultured at 37 degrees Celsius for 1-3 days.

(B-3) Fluorescence measurement with HeLa cells An inverted fluorescence microscope (IX70 (Olympus)) equipped with a CCD camera (ORCA-ER, Hamamatsu Photonics) controlled by a computer (Aquacosmos (Hamamatsu Photonics)) for fluorescence measurement , NIBA filter set (Olympus), objective lens 20x or 40x (Olympus)). Cells into which the plasmid was introduced were set in a microscope, and HBS buffer (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO ₄ (Nacalai Tesque), 2 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ (Nacalai Tesque)), 11.5 mM glucose , 20 mM HEPES (Dojindo) (pH 7.4)) was refluxed as an extracellular solution, and 100 μM ATP (Sigma) was administered extracellularly to stimulate the cells. Detected as. The measurement was performed at room temperature.

  The present invention provides a calcium concentration that plays an essential role in the maintenance and regulation of biological functions as a regulator of various cellular functions such as muscle contraction, nerve excitability, hormone secretion, and changes in enzyme activity. Provides a calcium sensor protein that can measure the fluctuations of the protein with extremely high sensitivity compared to the conventional one, and its use value is high, and it greatly contributes to the field of elucidation of biological mechanisms, medicine and drug discovery.

Claims (2)

Calcium sensor protein having the following amino acid sequences (a) to (i) in order from the N-terminus:
(A) an amino acid sequence in which the number of His present at positions 5 to 10 in the amino acid sequence represented by SEQ ID NO: 1 is 5;
(B) the amino acid sequence represented by SEQ ID NO: 6;
(C) the amino acid sequence represented by SEQ ID NO: 7;
(D) the amino acid sequence of Leu-Glu (linker Y) linking the sequence of (c) described above and the sequence of (e) described below;
(E) 150th to 239th of the sequence shown in SEQ ID NO: 3, wherein the 154th amino acid is replaced with Lys, the 204th amino acid is replaced with Val, and the 206th amino acid is replaced with Asn. Amino acid sequence;
(F) the amino acid sequence represented by SEQ ID NO: 8;
(G) an amino acid sequence from the first to the 145th amino acid sequence of the sequence represented by SEQ ID NO: 3, wherein the 106th amino acid is substituted with Tyr and the 125th amino acid is substituted with Val;
(H) the amino acid sequence of Thr-Arg (linker Z) linking the sequence of (g) described above and the sequence of (i) described below;
(I) The amino acid sequence from the 2nd to the 148th of the sequence shown in SEQ ID NO: 9, wherein the 36th amino acid is replaced with Leu, the 60th amino acid is replaced with Asp, and the 78th amino acid is Tyr An amino acid sequence substituted with A calcium sensor gene encoding the protein according to claim 1 .