Protein scintillator

Abstract

The present invention addresses the problem of providing: a protein that acts as a scintillator; and a method for designing a protein that acts as a scintillator. According to the present invention, a use of a β-barrel-type fluorescent protein as a scintillator is provided, wherein the β-barrel-type fluorescent protein has a fluorescence peak at 500 nm or less. According to the present invention, a method for designing a protein that acts as a scintillator is also provided, the method comprising (A) a step for identifying a specific amino acid residue in an amino acid sequence for a β-barrel-type fluorescent protein being mutated and (B) a step for constructing, on the basis of the amino acid sequence for the protein being mutated, an amino acid sequence having a structure such that the amino acid residue identified in the step (A) is substituted by another specific amino acid residue.

Description

Protein Scintillators REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2023-105343 (filing date: June 27, 2023), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a protein scintillator. The present invention also relates to a method for designing and producing a protein that functions as a scintillator, and a method for photosynthesis using radiation.

[0003] Fluorescent proteins have been widely used as a method for in vivo molecular imaging. Since its discovery, Aequorea victoria green fluorescent protein (avGFP) has been the fluorescent protein that has been most modified and improved, and has been used in a variety of applications. By introducing various mutations into avGFP, fluorescent proteins with enhanced functions, such as wavelength, brightness, pH sensitivity, and maturation rate, have been obtained. Regarding wavelength, fluorescent proteins that emit a wide range of visible light from blue to red have been obtained, and even those that emit fluorescence at shorter wavelengths in the blue region have been obtained (Non-Patent Documents 1 and 2).

[0004] However, there have been no reports of proteins that emit fluorescence when excited by radiation (α-rays, γ-rays, β-rays, X-rays, electron beams, etc.).

[0005] Sugiura K. and Nagai T., Communications Biology 5, 1172 (2022)Tomosugi W. et al., Nature Methods 6(5), 351-353 (2009)

[0006] The present inventors have now found that β-barrel fluorescent proteins that have a fluorescence peak at a short wavelength function as scintillators, and the present invention is based on this finding.

[0007] An object of the present invention is to provide a protein that functions as a scintillator. Another object of the present invention is to provide a method for designing and producing a protein that functions as a scintillator, as well as a method for applying and utilizing the protein to the photoresponse of an organism when it is exposed to radiation.

[0008] The present invention provides the following: [1] Use of a β-barrel fluorescent protein as a scintillator, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less. [2] The use according to [1] above, wherein the β-barrel fluorescent protein is a protein selected from the group consisting of the following (a), (b), and (c): (a) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4, (b) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 4 and emitting light when exposed to β-rays or an electron beam, and (c) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4 in which one or more amino acids have been deleted, substituted, inserted, and / or added, and emitting light when exposed to β-rays or an electron beam. [3] The use according to [1] or [2] above, wherein the β-barrel fluorescent protein is selected from the group consisting of Sumire, Sirius, moxBFP, mTurquoise2, mTurquoise, SCFP1, SCFP2, SCFP3A, mTFP1 and oxBFP.[4] A method for designing a protein that functions as a scintillator, comprising the steps of: (A) and (B) (A) specifying one or more amino acids selected from the group consisting of the following (1) to (3) in the amino acid sequence of a β-barrel fluorescent protein to be mutated: (1) an amino acid corresponding to the amino acid at position 65 in the amino acid sequence of any of SEQ ID NOs: 1 to 4; (2) an amino acid corresponding to the amino acid at position 66 in the amino acid sequence of any of SEQ ID NOs: 1 to 4; (3) an amino acid corresponding to the amino acid at position 203 in the amino acid sequence of any of SEQ ID NOs: 1 to 4 (B) constructing an amino acid sequence based on the amino acid sequence of a protein to be mutated, in which one or more amino acids specified in step (A) are substituted with the following amino acids: (1) an amino acid corresponding to the amino acid at position 65 in the amino acid sequence of any of SEQ ID NOs: 1 to 4: serine (S), glycine (G), or glutamine (Q); (2) an amino acid corresponding to the amino acid at position 66 in the amino acid sequence of any of SEQ ID NOs: 1 to 4: tryptophan (W), histidine (H), tyrosine (Y), or phenylalanine (F); (3) A design method comprising an amino acid corresponding to the amino acid at position 203 in the amino acid sequence of any one of SEQ ID NOs: 1 to 4: threonine (T) or valine (V). [5] A method for preparing a protein that functions as a scintillator, comprising a step of expressing in a host a polynucleotide encoding the amino acid sequence constructed by the design method described in [5] above. [6] A method for producing a fluorescent protein by radiation, in which a β-barrel fluorescent protein is caused to emit light by β-rays or an electron beam, wherein the β-barrel fluorescent protein has a fluorescence peak at 500 nm or less. [7] The method for producing a β-barrel fluorescent protein by radiation, in which the β-barrel fluorescent protein is present in the body of a non-human animal. [8] A method for photosynthesis by radiation, in which a β-barrel fluorescent protein is caused to emit light by β-rays or an electron beam, and photosynthesis is driven by the fluorescence emitted by the protein, wherein the β-barrel fluorescent protein has a fluorescence peak at 500 nm or less.[9] A photosynthetic apparatus comprising a β-barrel fluorescent protein and a photosynthetic organism whose photosynthesis is driven by fluorescence emitted by the protein, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

[10] The photosynthetic apparatus according to [9] above, further comprising a β-ray or electron beam emission source.

[11] A photosynthetic organism which possesses a β-barrel fluorescent protein in an expressible manner, wherein photosynthesis is driven by fluorescence emitted by the protein and the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

[12] The photosynthetic organism according to

[11] above, wherein the photosynthetic organism is a cyanobacterium.

[0009] The present invention provides a protein scintillator that has not been reported before. Because protein scintillators can be expressed by genetic manipulation, the present invention will contribute to the elucidation of biological functions through optogenetics and the development of new therapeutic methods, as well as to radiation-assisted photosynthesis technology.

[0010] Figure 1 shows the amino acid sequences of Sumire, Sirius, moxBFP, and mTurquoise2. Figure 2 shows the amino acid sequences of each fluorescent protein. ３５Figure 3 shows the emission of light from radiation derived from S. The values ​​obtained by subtracting the background (count value in a solution without protein) from the fluorescence count value per minute for each protein are plotted. Lowercase letters indicate significant differences between different signs (p<0.05) (Tukey's test). Figure 3 shows the relationship between the intensity of radiation irradiated to each fluorescent protein and the resulting fluorescence count. The values ​​obtained by subtracting the background (count value in a solution without protein) from the count value for each protein are plotted. Figure 4 shows (A) the excitation wavelength spectrum and (B) the fluorescence wavelength spectrum of mTurquoise2 and mTurquoise2 in which the threonine (T) at position 203 in the amino acid sequence has been mutated to tyrosine (Y) (hereinafter referred to as the T203Y mutant). Figure 5 shows the three-dimensional structures and predicted three-dimensional structures of mTurquoise2 and the T203Y mutant, respectively. (A) shows the three-dimensional structure of mTurquoise2 (PDB ID: 3ZTF). The numbers indicate the distance between the 203rd amino acid residue and the chromophore. (B) shows the predicted three-dimensional structure of the T203Y mutant. The numbers indicate the distance between the 203rd amino acid residue and the chromophore. Specific Description of the Invention

[0011] <β-barrel fluorescent protein> In the present invention, a β-barrel fluorescent protein can be used as a scintillator. A β-barrel fluorescent protein is a protein that has a barrel-shaped three-dimensional structure called a β-barrel and emits fluorescence of a longer wavelength when it absorbs light of a specific wavelength.

[0012] Fluorescent proteins with a β-barrel structure are well known to those skilled in the art and include the green fluorescent protein from Aequorea victoria (avGFP) or derivatives thereof that have the same folding conformation as avGFP.

[0013] In the present invention, the β-barrel fluorescent protein includes a fluorescent protein having a fluorescence peak at 500 nm or less, preferably a fluorescent protein having a fluorescence peak at 480 nm or less, 450 nm or less, or 420 nm or less. In the present invention, the term "fluorescence peak" refers to the peak of the fluorescence wavelength.

[0014] Among the fluorescent proteins having a β-barrel structure derived from avGFP, examples of fluorescent proteins having a fluorescence peak at 500 nm or less include purple fluorescent protein, ultramarine fluorescent protein, blue fluorescent protein (BFP), and cyan fluorescent protein (CFP).

[0015] Fluorescent proteins that can be used as protein scintillators in the present invention include proteins selected from the group consisting of the following (a), (b), and (c): (a) proteins consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4, (b) proteins consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 4 and that emit light when exposed to β rays or an electron beam, and (c) proteins consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4 and that emit light when exposed to β rays or an electron beam. The amino acid sequences of SEQ ID NOs: 1 to 4 are as shown in Figure 1.

[0016] The protein consisting of the amino acid sequence of SEQ ID NO: 1 in (a) is called Sumire and has a fluorescence peak at 414 nm (Non-Patent Document 1). This fluorescent protein is a purple fluorescent protein derived from avGFP.

[0017] The protein consisting of the amino acid sequence of SEQ ID NO: 2 in (a) is Sirius, which has a fluorescence peak at 424 nm (Non-Patent Document 2). This fluorescent protein is an ultramarine fluorescent protein derived from avGFP.

[0018] The protein consisting of the amino acid sequence of SEQ ID NO: 3 in (a) is moxBFP, which has a fluorescence peak at 445 nm (Heim R. & Tsien RY, Current Biology, 6(2), pp. 178-182 (1996)). This fluorescent protein is a blue fluorescent protein (BFP) derived from avGFP.

[0019] The protein (a) consisting of the amino acid sequence of SEQ ID NO: 4 is mTurquoise2 (sometimes referred to as "mTQ2" in this specification and drawings), which has a fluorescence peak at 474 nm (J. Goedhart et al., Nature Communications Vol. 3, Article number: 751 (2012)). This fluorescent protein is a cyan fluorescent protein (CFP) derived from avGFP.

[0020] In the present invention, the amino acid positions in the amino acid sequences of SEQ ID NOs: 1 to 4 are represented without counting the second V (valine). This is a representation commonly used in the art, following the amino acid position numbering of wild-type GFP. For example, the amino acids at positions 65, 66, and 203 of Sumire (the amino acid sequence of SEQ ID NO: 1) are G (glycine), Y (tyrosine), and V (valine), respectively, which correspond to the amino acids at positions 66, 67, and 204 in the actual amino acid sequence of SEQ ID NO: 1 (counting the second V).

[0021] In the present invention, "emitting light when exposed to β rays or electron beams" means that a transition from a ground state to an excited state occurs when exposed to β rays or electron beams, and fluorescence is emitted when returning to the ground state.

[0022] In (b) above, "identity" is used in a sense that includes "homology." Here, "identity" refers to, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the percentage of exact amino acid matches between the sequences. When calculating identity, for example, the presence of gaps in the sequences and the properties of the amino acids are taken into consideration (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The alignment can be performed, for example, using any algorithm. Specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated, for example, using a publicly available homology search program such as the above. For example, identity can be calculated using the default parameters of the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0023] The identity in (b) is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more.

[0024] The protein (c) may have one or more modifications selected from the group consisting of deletions, substitutions, insertions, and additions in the amino acid sequence of any of SEQ ID NOS: 1 to 4. The number of modified amino acids is, for example, 1 to 47, preferably 1 to 35, more preferably 1 to 23, even more preferably 1 to 21, 1 to 19, 1 to 16, 1 to 14, 1 to 11, 1 to 9, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, and particularly preferably 1 to several, 1 to 3, 1 to 2, or 1. The number of modified amino acids may also be the number of mutations occurring by known methods such as site-directed mutagenesis, or the number of naturally occurring mutations. Therefore, the term "modification" is used to include mutations. The modifications may occur consecutively or discontinuously in the amino acid sequence. The modifications may also be multiple homogeneous modifications (eg, multiple substitutions) or multiple heterogeneous modifications (eg, a combination of one or more deletions and one or more substitutions).

[0025] The insertion of amino acids in (c) above can be, for example, insertion into the interior of the amino acid sequence. Furthermore, the addition of amino acids can be, for example, addition to either the N-terminus or the C-terminus of the amino acid sequence, or addition to both the N-terminus and the C-terminus.

[0026] The amino acid modification may be a conservative modification (e.g., a conservative mutation). "Conservative modification" or "conservative mutation" means modifying or mutating one or more amino acids so as not to substantially alter the function of the protein. The amino acid substitution may also be a conservative substitution. "Conservative substitution" means substituting one or more amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. In conservative substitution, the substituted amino acid and the substituted amino acid preferably have similar properties and / or functions, for example. Specifically, chemical properties such as hydrophobicity and hydrophilicity index, polarity, and charge, or physical properties such as secondary structure, are preferably similar. As such, amino acids or amino acid derivatives with similar properties and / or functions are known in the art. For example, nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Examples of polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, lysine, etc. Examples of negatively charged amino acids (acidic amino acids) include aspartic acid, glutamic acid, etc.

[0027] In the present invention, amino acids can be identified by the following three-letter or one-letter symbols well known in the art: Alanine: Ala A Arginine: Arg R Asparagine: Asn N Aspartic acid: Asp D Cysteine: Cys C Glutamine: Gln Q Glutamic acid: Glu E Glycine: Gly G Histidine: His H Isoleucine: Ile I Leucine: Leu L Lysine: Lys K Methionine: Met M Phenylalanine: Phe F Proline: Pro P Serine: Ser S Threonine: Thr T Tryptophan: Trp W Tyrosine: Tyr Y Valine: Val V

[0028] In the amino acid sequences of (b) and (c), it is preferable that the amino acids at positions 65, 66, and / or 203 (particularly the amino acid at position 203) in each of the amino acid sequences of SEQ ID NOs: 1 to 4 remain in their original sequence (are not modified).

[0029] In (b) and (c) above, whether or not the test protein is a "protein that emits light when exposed to β rays or an electron beam" can be evaluated using the fluorescent properties of the test protein as an indicator, for example, by irradiating the test protein with β rays or an electron beam and measuring the scintillation light using a scintillation counter or the like.

[0030] In addition to mTurquoise2, CFPs that can be used as protein scintillators include the following proteins derived from avGFP: mTurquoise (sometimes referred to as "mTQ" in the present specification and drawings) (J. Goedhart et al., Nature Methods Vol. 7, 137-139 (2010)) SCFP3A (GJ Kremers et al., Biochemistry, Vol. 45(21), 6570-6580 (2006)) SCFP2 (GJ Kremers et al., Biochemistry, Vol. 45(21), 6570-6580 (2006)) SCFP1 (GJ Kremers et al., Biochemistry, Vol. 45(21), 6570-6580 (2006)) mTFP1 (Hui-wang Ai et al., Biochem J. 400(3):531-540(2006))

[0031] In addition to moxBFP, other BFPs that can be used as protein scintillators include the following proteins derived from avGFP: oxBFP (Costantini Lm et al., Nature Communications, 6(1), 7670 (2015))

[0032] <Method for Designing a Radioactive Scintillator Protein> The present invention provides a method for designing a protein scintillator. The design method of the present invention comprises the steps of: (A) identifying one or more amino acids selected from the group consisting of (1) to (3) below in the amino acid sequence of a β-barrel fluorescent protein to be mutated; and (1) an amino acid corresponding to the amino acid at position 65 in the amino acid sequence of any of SEQ ID NOs: 1 to 4; (2) an amino acid corresponding to the amino acid at position 66 in the amino acid sequence of any of SEQ ID NOs: 1 to 4; or (3) an amino acid corresponding to the amino acid at position 203 in the amino acid sequence of any of SEQ ID NOs: 1 to 4. (B) constructing an amino acid sequence in which one or more amino acids identified in step (A) are substituted with specific amino acids, based on the amino acid sequence of the protein to be mutated.

[0033] Step (A) can be carried out by aligning the β-barrel fluorescent protein to be mutated with any of the amino acid sequences of SEQ ID NOs: 1 to 4, and identifying, in the β-barrel fluorescent protein to be mutated, an amino acid corresponding to an amino acid at a specific position in any of the amino acid sequences of SEQ ID NOs: 1 to 4. The amino acid sequence of SEQ ID NOs: 1 to 4 to be aligned with the β-barrel fluorescent protein to be mutated can be selected to have the highest identity with the mutation target.

[0034] In step (B), the amino acid after substitution can be any of the following amino acids: (1) an amino acid corresponding to the amino acid at position 65 of the amino acid sequence of any of SEQ ID NOs: 1 to 4: serine (S), glycine (G), or glutamine (Q); (2) an amino acid corresponding to the amino acid at position 66 of the amino acid sequence of any of SEQ ID NOs: 1 to 4: tryptophan (W), histidine (H), tyrosine (Y), or phenylalanine (F); (3) an amino acid corresponding to the amino acid at position 203 of the amino acid sequence of any of SEQ ID NOs: 1 to 4: threonine (T) or valine (V).

[0035] In (1) above, when the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 3 or 4, the amino acid at position 65 after substitution can be serine (S). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 1, the amino acid at position 65 after substitution can be glycine (G). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 2, the amino acid at position 65 after substitution can be glutamine (Q).

[0036] In (2) above, when the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 4, the amino acid at position 66 after substitution can be tryptophan (W). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 3, the amino acid at position 66 after substitution can be histidine (H). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 1, the amino acid at position 66 after substitution can be tyrosine (Y). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 2, the amino acid at position 66 after substitution can be phenylalanine (F).

[0037] In (3) above, when the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 3 or 4, the amino acid at position 203 after substitution can be threonine (T). When the amino acid sequence aligned with the β-barrel fluorescent protein to be mutated is the amino acid sequence of SEQ ID NO: 1 or 2, the amino acid at position 203 after substitution can be valine (V).

[0038] If any of the amino acids at the specific positions (1) to (3) of the β-barrel fluorescent protein to be mutated is already one of the above amino acids, there is no need to substitute the amino acid in that sequence.

[0039] Among the amino acids (1) to (3) above, the amino acid (3) is associated with the inner surface of the β-barrel structure. By selecting the amino acid (3) as a specific amino acid as described above, it is possible to ensure the function as a protein scintillator.

[0040] Among the amino acids (1) to (3) above, the amino acids (1) and (2) are related to the luminophore. By making the amino acids (1) and (2) specific amino acids as described above, it is possible to maintain the function as a protein scintillator.

[0041] <Method for Preparing a Protein Scintillator> The present invention provides a method for preparing a protein scintillator. The preparation method of the present invention enables the preparation of a protein scintillator based on an amino acid sequence constructed by the design method of the present invention, and specifically comprises the step of (P) expressing in a host a polynucleotide encoding the amino acid sequence constructed by the design method of the present invention.

[0042] Step (P) can be carried out by culturing a host transformed with an expression vector to which a polynucleotide is operably linked. The preparation method of the present invention may further comprise, after the culturing step, a step of collecting an expression product from the host and / or culture obtained by culturing, and, in some cases, isolating or purifying the protein to be prepared.

[0043] In the present invention, the polynucleotide encoding the amino acid sequence of the protein to be prepared is not particularly limited as long as it is expressible in the host used and is composed of codons that encode a protein that functions as a protein scintillator, and codon optimization may be performed to enable expression in the host used or to increase the expression level. Codon optimization can be performed by known methods commonly used in this field.

[0044] In one embodiment of the preparation method of the present invention, an expression vector is prepared to which a polynucleotide encoding the amino acid sequence of the protein to be prepared is operably linked, and a host transformed with this vector can be cultured.

[0045] In the preparation method of the present invention, the expression vector into which the polynucleotide can be inserted can be selected depending on the host, and is not particularly limited as long as it is an expression vector commonly used in this field. For example, a vector capable of autonomous replication in the host or a vector that can be integrated into the host chromosome can be used as the expression vector. When a host microorganism such as Escherichia coli or Bacillus subtilis is used as the host, for example, pET, pBAD, or pUC can be used as the expression vector.

[0046] In the present invention, the promoter that can be linked to the expression vector can be selected depending on the host, and is not particularly limited as long as it is a promoter that is commonly used in this field. For example, in E. coli, promoters derived from E. coli or phages, such as the trp promoter, lac promoter, tac promoter, and PL promoter, or modified versions thereof, can be used as promoters.

[0047] The host to be transformed in the preparation method of the present invention is not particularly limited as long as it is a host commonly used in this field. For example, microorganisms or cells such as bacteria (such as Escherichia coli and Bacillus subtilis), yeast, filamentous fungi, insect cells, and eukaryotic cells can be used as hosts.

[0048] The host transformed in the preparation method of the present invention can be cultured in a medium suitable for culturing the host. The medium to be used is not particularly limited as long as it is a nutrient medium in which the host can grow and the target protein of the present invention can be produced, and may be either a synthetic medium or a natural medium. Furthermore, culture conditions such as time and temperature can be appropriately selected to suit the host to be cultured.

[0049] <Use as a protein scintillator> Luminescence method According to the present invention, there is provided a method for making a β-barrel fluorescent protein emit light by irradiation with β-rays or an electron beam. The fluorescent protein used in the luminescence method of the present invention can be the above-mentioned β-barrel fluorescent protein.

[0050] In the present invention, "causing luminescence by β-rays or electron beams" means that β-barrel fluorescent proteins are transitioned from the ground state to an excited state by β-rays or electron beams, and then emit fluorescence when returning to the ground state.

[0051] In the luminescence method of the present invention, a β-ray emission source or an electron beam emission source can be used to cause the β-barrel fluorescent protein to emit light. Examples of the β-ray emission source include radioactive substances that emit β-rays. The radioactive substances that emit β-rays are not particularly limited, and include: ３ H. １４ C. ３２ P. ３５ S. ９０ Sr, １３１ I, １３７ Examples of the electron beam emission source include radioactive materials such as Cs. Examples of the electron beam emission source include a device that emits an electron beam. The device that emits an electron beam (electron beam emitter) is not particularly limited, but includes, for example, a linear accelerator.

[0052] In the luminescence method of the present invention, the arrangement of the β-barrel fluorescent protein and the β-ray emission source or the electron beam emission source can be determined taking into consideration the flight distance of β-rays or electron beams in air or liquid and the penetration distance within a body. That is, the luminescence method of the present invention includes a step of arranging the β-ray emission source or the electron beam emission source and the β-barrel fluorescent protein so that the β-barrel fluorescent protein is excited by β-rays or electron beams.

[0053] The light-emitting method of the present invention can be used in, for example, optogenetics. The light-emitting method of the present invention can also be used in photosynthesis. Each of these will be explained below.

[0054] Use in Optogenetics In the light-emitting method of the present invention, a β-barrel fluorescent protein can be present in a cell or tissue in vitro. That is, in this embodiment, β-rays or an electron beam are irradiated from the outside, and the β-barrel fluorescent protein present in the cell or tissue can be made to emit light by the β-rays or electron beam that have penetrated the cell or tissue. In the light-emitting method of the present invention, a β-barrel fluorescent protein can also be present inside a human or non-human organism. That is, in this embodiment, a human or organism can be irradiated from the outside with β-rays or an electron beam, and the β-barrel fluorescent protein present in the body can be made to emit light by the β-rays or electron beam that have passed through the organism. The non-human organism is not particularly limited, and non-limiting examples of non-human organisms include non-human mammals such as mice, rats, monkeys, and rabbits, as well as insects, plants, and microorganisms.

[0055] In the field of optogenetics, a technique is known in which a photosensitive protein (e.g., opsin) is present in a cell, tissue, or living organism, and cellular function is manipulated by light irradiation. In the light-emitting method of the present invention, a β-barrel fluorescent protein is present in a living organism, which is poorly penetrated by visible light, and the protein is made to emit light by β-rays or an electron beam. The resulting fluorescence stimulates the photosensitive protein, allowing for remote manipulation of the function of cells or proteins in the living organism. The light-emitting method of the present invention, in which a β-barrel fluorescent protein is present in a living organism, is advantageous in that it does not require surgery to implant an optical fiber into the living organism and can express the β-barrel fluorescent protein at a target site. In such an embodiment, the β-barrel fluorescent protein can be present near the site where the photosensitive protein is present, and the β-barrel fluorescent protein can be expressed in a target site-specific manner, for example, by tissue-specific expression technology.

[0056] In the light-emitting method of the present invention, light can be emitted by β-rays or an electron beam, and photosynthesis can be driven by the fluorescence of the β-barrel fluorescent protein. That is, according to the present invention, there is provided a radiation-induced photosynthesis method in which a β-barrel fluorescent protein is emitted by β-rays or an electron beam, and photosynthesis is driven by the fluorescence of the β-barrel fluorescent protein.

[0057] In the present invention, by driving photosynthesis with fluorescence, light energy can be converted into storable chemical energy (e.g., carbohydrates), and therefore the present invention is advantageous in that it can convert radiation energy into industrially usable energy.

[0058] The light-emitting method of the present invention can, for example, drive photosynthesis in photosynthetic organisms. Utilizing the photosynthetic pathway of photosynthetic organisms, it is possible to assimilate carbon dioxide using light energy and synthesize organic matter. Usable photosynthetic organisms include: cyanobacteria (blue-green algae) such as Spirulina; eukaryotic algae such as green algae (Chlorella, Lumbricus, Chlamydomonas, etc.) and Euglena (Euglena, etc.); and plants such as mosses and higher plants.

[0059] In the photosynthesis method of the present invention, the combination and arrangement of a β-barrel fluorescent protein and a photosynthetic organism can be determined taking into consideration the driving of photosynthesis by fluorescence emitted by the β-barrel fluorescent protein. The combination of a β-barrel fluorescent protein and a photosynthetic organism can be selected so that the fluorescent wavelength emitted by the β-barrel fluorescent protein at least overlaps with the light wavelength received by the photosynthetic organism. The arrangement of the β-barrel fluorescent protein and the photosynthetic organism can be determined so that the fluorescence emitted by the β-barrel fluorescent protein reaches a photoreceptor (e.g., a light-harvesting pigment protein) of the photosynthetic organism (e.g., a photosynthetic bacterium) without attenuation; specifically, the distance between the β-barrel fluorescent protein and the photosynthetic organism can be determined.

[0060] In the photosynthesis method of the present invention, the positions of the β-barrel fluorescent protein and the β-ray emission source or the electron beam emission source can be determined taking into consideration the flight distance and transmittance of the β-ray or electron beam. As long as the β-barrel fluorescent protein is excited by the β-ray or the electron beam and emits fluorescence sufficient to drive photosynthesis, there are no limitations on the positional relationship or distance between the β-barrel fluorescent protein and the β-ray emission source or the electron beam emission source.

[0061] The present invention provides a photosynthetic apparatus comprising a β-barrel fluorescent protein and a photosynthetic organism in which photosynthesis is driven by the fluorescence of the protein. The photosynthetic apparatus of the present invention can be implemented according to the luminescence method of the present invention and the photosynthesis method of the present invention.

[0062] The photosynthetic apparatus of the present invention may not be equipped with a beta-ray emission source or an electron beam emission source, and the apparatus of the present invention may be installed and used near a beta-ray emission source or an electron beam emission source. In such an embodiment, a beta-ray emission source that is difficult to move may be used. The photosynthetic apparatus of the present invention may also further include a beta-ray emission source or an electron beam emission source. Such an embodiment includes, for example, a photosynthetic apparatus of the present invention that further includes spent nuclear fuel prepared in advance for use in photosynthesis. These embodiments are advantageous in that radioactive waste that is difficult to reuse can be recycled through photosynthesis, and further contribute to a low-carbon society through carbon dioxide fixation. Furthermore, they are advantageous in that they are less susceptible to environmental fluctuations such as temperature, do not occupy aboveground space, can be installed underground, and can be stacked.

[0063] In the photosynthesis method of the present invention, a β-barrel fluorescent protein can also be present in a photosynthetic organism (particularly a photosynthetic microorganism). In this embodiment, the β-barrel fluorescent protein can be present in the vicinity of a photoreceptor in the photosynthetic organism; for example, the β-barrel fluorescent protein can be expressed at a desired site using genetic engineering techniques. The presence of a β-barrel fluorescent protein in the vicinity of a photoreceptor in a photosynthetic organism is advantageous in that the fluorescent energy of the β-barrel fluorescent protein can be efficiently transferred to the photoreceptor. That is, according to another aspect of the present invention, there is provided a photosynthetic organism that possesses an expressible β-barrel fluorescent protein, and in which photosynthesis is driven by the fluorescence of the protein. The photosynthetic organism of the present invention can be produced according to the luminescence method and photosynthesis method of the present invention. Considering the application of genetic engineering techniques, the photosynthetic organism is preferably a photosynthetic microorganism, more preferably a cyanobacterium.

[0064] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0065] Example 1: Identification of proteins that emit fluorescence upon excitation by radiation (1) Method Each of the fluorescent proteins, Sumire, Sirius, moxBFP, mTurquoise2, mTFP1, EGFP, Venus, Achilles, mScarlet, and mRaspberry, was highly expressed in Escherichia coli using the pET system. Specifically, a nucleotide sequence encoding a fusion protein containing a 6xHis tag at the C-terminus of each fluorescent protein was inserted immediately after the T7 promoter to create the plasmid pRSETB (Thermo Fisher Scientific). Escherichia coli JM109(DE3) (Promega) was then transformed with this plasmid, and the E. coli was cultured in 200 ml of LB medium at 22°C for 3 days. After cultivation, the fluorescent proteins were purified using Ni-NTA Agarose (QIAGEN). The DNA sequences encoding the fluorescent proteins used for transformation of E. coli were as follows: Sumire: SEQ ID NO: 5, Sirius: SEQ ID NO: 6, moxBFP: SEQ ID NO: 7, mTurquoise2: SEQ ID NO: 8, mTFP1: SEQ ID NO: 9, EGFP: SEQ ID NO: 10, Venus: SEQ ID NO: 11, Achilles: SEQ ID NO: 12, mScarlet: SEQ ID NO: 13, mRaspberry: SEQ ID NO: 14.

[0066] Each purified fluorescent protein solution was dispensed into a 96-well plate, and radioisotopes were added to the wells. ３５ S was added and the content was measured using a liquid scintillation counter MicroBeta 2 (PerkinElmer Japan). ２ Measurement was carried out for 1 minute using a microplate RI glow luminescence measurement system.

[0067] In addition to the above, a fluorescent protein (hereinafter referred to as T203Y) was created by introducing a mutation into mTurquoise2 (mTQ2) in which T (threonine) at position 203 was replaced with Y (tyrosine). After high expression of the protein using the same method as above, the expressed protein was purified and subjected to a luminescence test. The excitation and emission spectra of mTQ2 and T203Y were measured. The DNA sequence encoding T203Y used to transform E. coli was a DNA sequence in which bases ACC at positions 610 to 612 in the DNA sequence of mTurquoise2 (SEQ ID NO: 8) were replaced with TAT.

[0068] (2) Results and Discussion Each purified fluorescent protein was incubated at 10 μM, ３５ When measured using a liquid scintillation counter under conditions of S 800 Bq / μl and a final solution volume of 100 μl, strong fluorescence counts were obtained with Sumire, Sirius, moxBFP, and mTurquoise2, but very weak fluorescence counts were observed with mTFP1, EGFP, Venus, Achilles, mScarlet, and mRaspberry, and no fluorescence was observed with mTQ2 mutated with T203Y (Figure 2). The radiation intensity dependence of mTQ2 mutated with T203Y and EGFP was examined. The counts of mTQ2 increased linearly with the radiation dose, whereas the counts of T203Y and EGFP did not increase even with increasing radiation dose (Figure 3). These results demonstrate that β-barrel fluorescent proteins with a fluorescence peak at 500 nm or less emit light when exposed to radiation, and that Sumire, Sirius, moxBFP, and mTurquoise2 in particular emit strong fluorescence when exposed to radiation.

[0069] While mTQ2 emitted light upon irradiation, the mutated T203Y did not. However, T203Y, like mTQ2, remained a fluorescent protein. Analysis of the T203Y fluorescence properties revealed that the excitation and emission peak wavelengths were shifted to longer wavelengths compared to mTQ2 (Figure 4). Specifically, the three peaks in the excitation spectrum (233 nm, 281 nm, and 438 nm) of mTQ2 were shifted to 256 nm, 284 nm, and 455 nm, respectively, in T203Y. Furthermore, the emission spectrum peak was 479 nm for mTQ2 and 515 nm for T203Y. In other words, when the T203Y mutation was added to the CFP mTurquoise2, a shift in the fluorescence wavelength was observed. Although not bound by the following theory, it is believed that the shift in the wavelength of the fluorescence peak due to the T203Y mutation is one of the causes of the reduced scintillation ability of mTurquoise 2. From the above, the observation results for the T203Y mutation in mTurquoise 2, together with the observation results for Sumire, Sirius, moxBFP, and mTurquoise 2, indicate that the fact that β-barrel fluorescent proteins have a fluorescence peak at 500 nm or less is correlated with the phenomenon of luminescence due to radiation.

[0070] It has been reported that the introduction of the T203Y mutation into EGFP, a β-barrel fluorescent protein similar to mTurquoise2, results in a shift in the absorption wavelength peak from 434 nm to 463 nm and in the fluorescence wavelength peak from 476 nm to 506 nm (Sawano A and Miyawaki A, Nucleic Acids Res. 2000 Aug 15;28(16):E78). This result is nearly consistent with the results observed in this example when the T203Y mutation was introduced into mTurquoise2. It has also been reported that the fluorescence quantum yield of EGFP with the T203Y mutation, which was 0.4, was significantly reduced to 0.14 in the T203Y mutant of EGFP (Sawano A and Miyawaki A, supra). It is expected that the decrease in fluorescence quantum yield observed when the T203Y mutation was introduced into EGFP may also occur with mTurquoise2. Therefore, the results observed when the T203Y mutation was introduced into mTurquoise2 are thought to be due in part to the fact that the T203Y mutation lengthened the absorption wavelength, making it less susceptible to excitation by radiation, and also to the fact that the amount of light emitted by radiation was significantly reduced due to a decrease in fluorescence quantum yield.

Claims

1. Use of a β-barrel fluorescent protein as a scintillator, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

2. The use according to claim 1, wherein the β-barrel fluorescent protein is a protein selected from the group consisting of the following (a), (b) and (c): (a) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 4; (b) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 4, and which emits light when exposed to β-rays or an electron beam; and (c) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 4, and which emits light when exposed to β-rays or an electron beam.

3. The use according to claim 1 or 2, wherein the β-barrel fluorescent protein is selected from the group consisting of Sumire, Sirius, moxBFP, mTurquoise2, mTurquoise, SCFP1, SCFP2, SCFP3A, mTFP1 and oxBFP.

4. A method for designing a protein that functions as a scintillator, comprising the steps of (A) and (B) below: (A) identifying one or more amino acids selected from the group consisting of (1) to (3) below in the amino acid sequence of a β-barrel fluorescent protein to be mutated: (1) an amino acid corresponding to the amino acid at position 65 of the amino acid sequence of any of SEQ ID NOs: 1 to 4; (2) an amino acid corresponding to the amino acid at position 66 of the amino acid sequence of any of SEQ ID NOs: 1 to 4; (3) an amino acid corresponding to the amino acid at position 203 of the amino acid sequence of any of SEQ ID NOs: 1 to 4 (B) constructing an amino acid sequence based on the amino acid sequence of a protein to be mutated, in which one or more amino acids identified in step (A) are replaced with the following amino acids: (1) an amino acid corresponding to the amino acid at position 65 of the amino acid sequence of any of SEQ ID NOs: 1 to 4: serine (S), glycine (G) or glutamine (Q); (2) an amino acid corresponding to the amino acid at position 66 of the amino acid sequence of any one of SEQ ID NOs: 1 to 4: tryptophan (W), histidine (H), tyrosine (Y) or phenylalanine (F); (3) an amino acid corresponding to the amino acid at position 203 of the amino acid sequence of any one of SEQ ID NOs: 1 to 4: threonine (T) or valine (V).

5. A method for preparing a protein that functions as a scintillator, comprising the step of expressing in a host a polynucleotide that codes for an amino acid sequence constructed by the design method described in claim 4.

6. A method for making a fluorescent protein emit light by radiation, comprising the step of causing a β-barrel fluorescent protein to emit light by β-rays or an electron beam, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

7. The method for luminescence according to claim 6, wherein the β-barrel fluorescent protein is present in the body of a non-human animal.

8. A radiation-based photosynthesis method in which a β-barrel fluorescent protein is made to emit light by β-rays or an electron beam, and photosynthesis is driven by the fluorescence emitted by the protein, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

9. A photosynthetic apparatus comprising a β-barrel fluorescent protein and a photosynthetic organism in which photosynthesis is driven by the fluorescence emitted by the protein, wherein the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

10. The photosynthetic device of claim 9, further comprising a beta ray or electron beam emitting source.

11. A photosynthetic organism having an expressible β-barrel fluorescent protein, wherein photosynthesis is driven by the fluorescence emitted by the protein, and the β-barrel fluorescent protein is a fluorescent protein having a fluorescence peak at 500 nm or less.

12. The photosynthetic organism of claim 11, wherein the photosynthetic organism is a cyanobacterium.

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