Protein set that induces temperature-dependent interactions with biological materials

Abstract

To provide a combination of proteins that contains a TlpA mutant and can induce substance interaction by heating.SOLUTION: A protein set for inducing substance interaction in a temperature-dependent manner comprises: a protein set comprising a protein having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein a, the following protein b, and the binding-inducing protein; or a protein set comprising a protein having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein b, the following protein a, and the binding-inducing protein; a: a protein comprising the whole or a part of a coiled-coil region of a protein having a specific amino acid sequence, and having mutations indicated by E180R and E250R; and b: a protein comprising the whole or a part of a coiled-coil region of a protein having the specific amino acid sequence and having mutations indicated by R179E and R251E.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a protein set capable of controlling the binding and dissociation of biological substances (such as proteins) in a temperature-dependent manner, and a method for controlling the interaction of substances using the protein set. [Background technology]

[0002] Proteins, which are biological substances, perform a variety of functions in living organisms. For example, protein-protein interactions are involved in the control of systems fundamental to life, such as intracellular signal transduction and material transport. Tools for controlling protein-protein interactions in living organisms using chemical compounds or light and for genetic manipulation have been reported. One tool that applies optogenetics is photoswitch proteins, which are protein pairs whose binding and dissociation can be controlled by turning light on and off (Patent Document 1). Photoswitch proteins are expected to be a tool that can control intracellular signal transduction, genome editing, gene expression, and other processes through the binding and dissociation of protein pairs using light manipulation (Non-Patent Document 1 and Non-Patent Document 2). When using tools that are activated by light manipulation, blue light (up to 470 nm) or near-infrared light is used. However, blue light is absorbed by hemoglobin and other substances, making it less permeable to biological tissue. Therefore, photoswitch proteins that can be controlled with blue light have not been sufficiently effective for use in in vivo light manipulation. Furthermore, there are concerns that near-infrared light may be difficult to deliver to tissues deeper than the subcutaneous tissue.

[0003] Compared to the tissue penetration ability of light, ultrasound and radio waves have a higher tissue penetration ability, allowing them to reach deep into biological tissue and heat the area they reach. Therefore, by utilizing temperature-sensitive proteins that can control binding and dissociation by temperature changes, it is thought that thermogenetic tools will be available that can control the interactions of substances in vivo, especially in deep tissues, by heating using ultrasound or radio waves (Non-Patent Documents 3 and 4). Salmonella ( Salmonella typhimurium TlpA, a transcription factor derived from T. tumefaciens, is a protein consisting of an N-terminal DNA-binding domain and a domain that forms an α-helix dimer called a coiled-coil. TlpA forms a homodimer at around 37°C and reversibly dissociates into a monomer when the temperature rises above 37°C (Patent Document 2, Non-Patent Document 5). By binding two different protein fragments to be interacted with each other to two TlpA monomers and then changing the temperature, it is thought that the dissociation of the TlpA monomers can be controlled, thereby controlling the binding and dissociation of the two protein fragments. However, because wild-type TlpA forms homodimers, it is possible for the same protein fragments to bind to each other. In light of this situation, a TlpA mutant that forms a heterodimer rather than a homodimer was created (Non-Patent Document 6).

[0004] When using thermogenetic tools in vivo, it is desirable to have tools that induce protein interactions at desired times and locations when temperatures different from the body temperature, for example, above 37°C, occur, thereby inducing various functions such as genome editing. However, the TlpA heterodimer described above binds at around 37°C and dissociates when the temperature rises above 37°C. Therefore, it is not suitable as a tool for inducing interactions in vivo based on the principle that protein fragments bind together as TlpA dimers are formed by heating. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2015-165776 [Patent Document 2] WO2007 / 142208 issue [Non-patent literature]

[0006] [Non-Patent Document 1] Kawano et al., Nature Chemical Biology 12:1059-1064 2016. [Non-patent document 2] Kawano et al., Nature Communications 6:6256 2015. [Non-patent document 3] Piraner et al., Nature Chemical Biology 13:75-80 2017. [Non-patent document 4] Paulides et al., Advanced Drug Delivery Reviews 163-164:3-18 2020. [Non-Patent Document 5] Hurme et al., Cell 90:55-64 1997. [Non-patent document 6] Piraner et al., ACS Synth. Biol. 8: 2256-2262 2019. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above circumstances, an objective of the present invention is to provide a protein combination (protein set) that contains the above TlpA mutant and is capable of inducing substance interactions upon heating. A further objective of the present invention is to provide a method for controlling interactions of substances in vivo using the protein set.

[0008] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that by using a protein set consisting of three proteins: a TlpA mutant protein (TlpA mutant 1), another TlpA mutant (TlpA mutant 2) that forms a dimer with this mutant and a protein in which an SsrA peptide is bound (TlpA mutant 2-SsrA), and Stringent starvation protein B (SspB) that binds to SsrA, it is possible to induce the binding and dissociation of two interacting proteins in a temperature-dependent manner. An example of the mechanism of action of the protein set of the present invention is shown schematically in Figure 1. At temperatures around 37°C, TlpA mutant 1 and TlpA mutant 2-SsrA (a protein in which the SsrA peptide is bound to TlpA mutant 2) form a coiled-coil structure with the TlpA mutants themselves. As a result, the SsrA peptide is covered by the TlpA mutant dimer, inhibiting its interaction with SspB. When heated to around 40°C, TlpA mutant 1 and TlpA mutant 2 dissociate, exposing the SsrA peptide and inducing binding between the SsrA peptide and SspB. Of the proteins in this set, protein A was bound to TlpA mutant 2-SsrA and protein B was bound to SspB, and the temperature was changed. At around 37°C, the binding between the SsrA peptide and SspB was inhibited, preventing protein A and protein B from coming close to each other and suppressing their interaction. However, when the temperature was raised to around 40°C, the binding between the SsrA peptide and SspB occurred, bringing protein A and protein B close to each other, thereby inducing their interaction. The present invention was completed based on the properties of the above three proteins.

[0009] That is, the present invention provides the following (1) to (10). (1) A protein set for inducing a temperature-dependent interaction of substances, A protein set including the following protein a having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein a, the following protein b, and the binding-inducing protein, or a protein set including a protein having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein b, the following protein a, and the following binding-inducing protein; a: a protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having mutations represented by E180R and E250R; b: A protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having the mutations represented by R179E and R251E. (2) The protein a is a protein having mutations represented by E180R and E250R in the amino acid sequence represented by SEQ ID NO: 1, the protein b is a protein having mutations represented by R179E and R251E in the amino acid sequence represented by SEQ ID NO: 1; the binding-inducing protein is Stringent starvation protein B (SspB); The binding-inducing peptide is an SsrA peptide, The protein set described in (1) above, characterized in that the SsrA peptide is bound to the C-terminus of protein a or protein b, which has Z amino acids deleted from the C-terminus (where Z is an integer between 0 and 7). (3) The set of proteins according to (2) above, wherein the protein a further has a mutation represented by Δ1-X, where X is 2 or more and 93 or less, and the protein b further has a mutation represented by Δ1-Y, where Y is 2 or more and 93 or less. (4) The set of proteins according to (3) above, wherein X and Y are 65. (5) A protein set according to any one of (1) to (4) above, wherein, among the proteins constituting the protein set, a protein having a binding-inducing peptide bound to protein a or a protein having a binding-inducing peptide bound to protein b, and the binding-inducing protein are each bound to or fused with another protein, and the other proteins interact with each other. (6) The set of proteins according to (5) above, wherein the other protein is a site-specific recombinase or a fragment of Cas9 nuclease. (7) A method for controlling the distance between two proteins, comprising: A step of binding or fusing a protein having a binding-inducing peptide bound to protein a or a protein having a binding-inducing peptide bound to protein b, which are included in the protein set described in any one of (1) to (3) above, to one of the two proteins; A step of binding or fusing a binding-inducing protein included in the set of proteins described in any one of (1) to (3) above to the other of the two proteins; and heating the protein set. (8) The method according to (7) above, wherein the other protein is a site-specific recombinase or a fragment of Cas9 nuclease. (9) The method according to (7) above, characterized in that the protein set is heated by irradiating it with ultrasound or radio waves. (10) A kit for controlling the interaction of substances within a cell, the kit comprising nucleic acids encoding each protein included in the protein set described in any one of (1) to (4) above. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0010] By using the protein set according to the present invention, it becomes possible to efficiently control the interactions between substances in vivo, particularly in the deep tissues of living organisms. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of the mechanism of action of the protein set according to the present invention. [Figure 2] Figure 2 shows the results of evaluating the function of heterodimers formed by various TlpA mutants. Panel A is a graph showing the percentage of molecules forming heterodimers at 37°C and 40°C for various TlpA mutant combinations. Panel B shows the results of examining the change in the percentage of molecules forming heterodimers when the heating temperature is changed for the #8 mutant combination shown in Panel A. Panel C shows the calculated percentage of molecules forming heterodimers when the #8 mutant was heated at 40°C for 5 minutes and then incubated at 37°C for the specified time. [Figure 3] Figure 3 shows the results of Western blotting performed to evaluate the function of the protein set according to the present invention. After crosslinking the protein set solution, Western blotting was performed. The results for the TlpAa-SsrA molecule, in which SsrA (AANDENYF; SEQ ID NO: 7) was fused to each of the amino acids 357 to 371 of TlpAa, correspond to the lanes indicated by the amino acid numbers in the figure. Heat-: No heating (37°C), +: Heating (40°C), [Figure 4] Figure 4 shows the results of evaluating the DNA recombination activity of Cre recombinase induced by the protein set of the present invention. Panel A shows the results of fusing split fragments of the DNA recombinase Cre to TlpAa-SsrA and SspB, respectively, and evaluating the DNA recombination efficiency upon heating using the reporter gene expression level (assessed as luciferase luminescence value) as an index. Panel B shows the fold increase in reporter gene expression level under 40°C culture conditions relative to 37°C culture conditions. The numbers on the horizontal axis of each graph identify various combinations of TlpAa-SsrA and SspB. Cre: Wild-type Cre [Figure 5]Figure 5 shows the results of confirming the functionality of ThrmoCre1.0. Panel A shows the DNA recombination efficiency when a protein set consisting of ThermoCreN, ThermoCreC, and TlpAb was heated, evaluated using reporter gene expression (assessed as luciferase luminescence). Panel B shows the fold increase in reporter gene expression at 40°C compared to 37°C. In addition to the combination of ThermoCreN, ThermoCreC, and TlpAb, the DNA recombination efficiency was also evaluated using the following controls: ThermoCreN and TlpAb combination, ThermoCreC and TlpAb combination, TlpAb alone, ThermoCreN and ThermoCreC combination, and wild-type Cre alone. [Figure 6] Figure 6 shows the results of confirming that DNA recombination by ThermoCre1.0 can be induced by ultrasound irradiation. When cells into which ThermoCre was introduced were heated for a short period of time by ultrasound irradiation, an increase in protein expression efficiency of approximately 9% was confirmed after 30 minutes of heating. [Figure 7] Figure 7 shows the results of confirming the safety of ThermoCre 1.0 for cells. Relative cell viability was calculated by setting the value for cells transfected with a control vector (pcDNA3.1 / myc-His ver. B) at 100. [Figure 8] Figure 8 shows the results of confirming the reversibility of the ThermoCre system. "37°C → 37°C" indicates that a specific gene was introduced into cells and cultured at 37°C for 24 hours, after which a reporter gene was introduced into the cells and cultured for another 24 hours at 37°C, and luminescence levels were measured. The same applies to "37°C → 40°C," "40°C → 37°C," and "40°C → 40°C." [Figure 9]Figure 9 shows the results of evaluating the DNA recombination efficiency of the ThermoCre system. ThermoCre and a reporter gene (LSL-GFP) were introduced into cells, which were then cultured at 37°C or 40°C and observed under a fluorescence microscope. ThermoCre represents a fluorescent image of TlpAb-mCherry-positive cells, and LSL-GFP represents a fluorescent image of cells in which DNA recombination had occurred. Nucleus represents an image of nuclei stained with Hoechst33342. [Figure 10] FIG. 10 is a diagram showing a schematic diagram of the molecular structures that constitute ThermoCre1.0, ThermoCre1.1, and ThermoCre1.2. [Figure 11] Figure 11 shows the results of comparing the DNA recombination activity of ThermoCre1.0, ThermoCre1.1, and ThermoCre1.2. Panel A shows the DNA recombination efficiency when ThermoCre1.0, ThermoCre1.1, and ThermoCre1.2 were heated, evaluated using reporter gene expression levels (assessed as luciferase luminescence values). The inset shows an enlarged view of the graph for all genes except Cre. Panel B shows the fold increase in reporter gene expression levels when cultured at 40°C compared to 37°C. [Figure 12] FIG. 12 is a diagram showing a schematic diagram of the molecular structure that constitutes ThermoCre1.3. [Figure 13] FIG. 13 is a diagram showing a schematic diagram of the structure of an integrated plasmid for expressing the molecules constituting ThermoCre1.3 shown in FIG. [Figure 14] Figure 14 shows the results of comparing the DNA recombination activity of ThermoCre expressed using each vector shown in Figure 13. Panel A shows the results of evaluating the DNA recombination efficiency when each ThermoCre was heated, using the reporter gene expression level (assessed by luciferase luminescence value) as an index. Panel B shows an enlarged view of the graph for all but Cre. Panel C shows the fold increase in reporter gene expression level under 40°C culture conditions compared to 37°C culture conditions. ThermoCre1.3* shows the results of an experiment in which the molecules that make up ThermoCre1.3 shown in Figure 12 were separately expressed. [Figure 15] Figure 15 shows the time course of recombination kinetics using ThermoCre1.3. The upper panel shows the experimental schedule. Panel A shows the time course of reporter gene expression (assessed by luciferase luminescence) under each condition: Nluc (report gene) alone, loxP-NLuc (loxP-NLuc;LLNlucLL) + ThermoCre1.3, loxP-NLuc (loxP-NLuc;LLNlucLL) + Cre, and loxP-NLuc (loxP-NLuc;LLNlucLL) alone. Panel B shows the time course of the fold increase in reporter gene expression under 40°C culture conditions relative to 37°C culture conditions. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described. Note that, unless otherwise specified, the term "the present embodiment" refers to all or any of the embodiments described in this specification. The first embodiment is a protein set for inducing temperature-dependent substance interactions (hereinafter also referred to as the "protein set according to this embodiment"), which includes a TlpA mutant (hereinafter also referred to as "TlpA mutant 1") or a TlpA mutant that forms a heterodimer with the mutant (hereinafter also referred to as "TlpA mutant 2"), a protein in which a binding-inducing peptide is bound to TlpA mutant 1 or TlpA mutant 2 (hereinafter also referred to as "TlpA mutant 1-peptide" or "TlpA mutant 2-peptide"), and a binding-inducing protein. More specifically, the following protein sets: "A protein set comprising TlpA mutant 1, TlpA mutant 2 peptide and binding-inducing protein" or "A protein set comprising TlpA mutant 2, TlpA mutant 1 peptide and binding-inducing protein".

[0013] In this embodiment, wild-type TlpA is a protein consisting of amino acids represented by SEQ ID NO: 1 shown below (the nucleic acid sequence is listed in the sequence listing: SEQ ID NO: 2), or a protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. Here, "a protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 1" refers to a protein consisting of an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity, and which forms a homodimer. Alternatively, a "protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 1" refers to a protein consisting of an amino acid sequence in which one or several (preferably about 1 to 30, more preferably about 1 to 10, and even more preferably 1 to 5) amino acids have been deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, and which forms a homodimer.

[0014] SEQ ID NO:1; MRPATYEPEQIIEAGLALQAEGRNITGFALRNQVGGGNPTRLRQIWDEYQASQSTVVTEPVAELPVEVAEEVKAVSAALSERITQLATELNDKAVRAAERRVAEVTRAAGEQTAQAERELADAAQTVDDLEEKLDELQDRYDSLTLALESERSLRQQHDVEMAQLKERLAAAEENTRQREERYQE QKTVLQDALNAEQAQHKNTREDLQKRLEQISAANARTEELKSERDKVNTLLTRLESQENALASERQQHLATRETLQQRLEQAIADTQARAGEIALERDRVSSLTARLESQEKASSEQLVRMGSEIASLTERCTQLENQRDDARLETMGEKETVAALRGEAEALKRQNQSLMAALSGNKQTGGQNA

[0015] In this embodiment, a "binding-inducing peptide" refers to a peptide that binds to a specific protein, and a "binding-inducing protein" refers to a protein that binds to the "binding-inducing peptide." Examples of combinations of "binding-inducing peptides" and "binding-inducing proteins" include the combination of "SsrA peptide" and "SspB," the combination of "ARVCF peptide" and "Erbin PDZ" (Hung et al., J. Mol. Biol. 392:1221-1231 2009), the combination of "SSADTWV peptide" and "engineered PDZ (ePDZ)" (Strickland et al., Nature Methods 9:379-384 2012), as well as combinations of a peptide of about 1 to 20 amino acids and a single-chain antibody that specifically binds to the peptide, such as the combination of "gp41 peptide" and its single-chain antibody (Boersma et al., Cell 178:458-472 2019) and the combination of "GCN4 peptide" and its single-chain antibody (Tanenbaum et al., Cell. 2014 October 23; 159:635-646 2014). Here, "SspB" refers to a protein consisting of amino acids represented by SEQ ID NO: 3 (the nucleic acid sequence is listed in the sequence listing: SEQ ID NO: 4) (hereinafter also referred to as "SspBnano"), or a protein consisting of amino acids represented by SEQ ID NO: 5 (the nucleic acid sequence is listed in the sequence listing: SEQ ID NO: 6) (hereinafter also referred to as "SspBmicro"), or a protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.

[0016] Here, "a protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 (or SEQ ID NO: 5)" refers to a protein consisting of an amino acid sequence that has 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 3 (or SEQ ID NO: 5), more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity, and which binds to the "SsrA peptide" described below. Alternatively, a "protein substantially identical to a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 (or SEQ ID NO: 5)" refers to a protein consisting of an amino acid sequence in which one or several (preferably about 1 to 30, more preferably about 1 to 10, and even more preferably 1 to 5) amino acids have been deleted, substituted, inserted, or added to the amino acid sequence represented by SEQ ID NO: 3 (or SEQ ID NO: 5), and which binds to the "SsrA peptide" described below.

[0017] SEQ ID NO:3 (SspBnano); SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQIVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEIYDELNIG SEQ ID NO:5 (SspBmicro); SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQIVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEIYDELNIG

[0018] In this embodiment, the term "SsrA peptide" refers to a peptide consisting of approximately 6 to 15 amino acids, including at least the amino acid sequence shown in SEQ ID NO: 7. Those skilled in the art can easily identify such peptides, for example, peptides consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 11 (see, for example, Levchenko et al., Science 289:2354-2356, 2000). Alternatively, the term refers to a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, inserted, or added, and which binds to the aforementioned "SspB protein." Examples of SsrA peptides that can be used in this embodiment include a peptide consisting of seven amino acids, X1-X2-X3-X4-X5-X6-X7, where X1 is A, G, P, S, or V, X2 is A, S, or V, X3 is N or H, X4 is D, C, or E, X5 is any amino acid, X6 is any amino acid, and X7 is F, W, or Y. For details, see Flynn et al., Proc. Natl. Acad. Sci., 98:10584-10589 2001, for example. SEQ ID NO:7; AANDEN SEQ ID NO:8; AANDENYF SEQ ID NO:9; AANDENYALAA SEQ ID NO:10; AANDENYALDD SEQ ID NO:11; AANDENYA

[0019] As used herein, when specifying the position of the amino acid sequence of a TlpA mutant, "the amino acid corresponding to the Zth amino acid in the amino acid sequence represented by SEQ ID NO: 1" refers to the amino acid in the amino acid sequence of the TlpA mutant that corresponds to the Zth amino acid in the amino acid sequence represented by SEQ ID NO: 1 when the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence of the TlpA mutant are aligned to maximize the degree of identity. Furthermore, when a specific amino acid in an amino acid sequence is indicated herein, it is described in the order of "specific amino acid (single-letter notation)" and "amino acid position number" in the amino acid sequence. For example, glutamic acid at position 180 in the amino acid sequence of wild-type TlpA (SEQ ID NO: 1) is described as E180. Furthermore, in this embodiment, point mutations are described in the order of "amino acid," "amino acid position number," and "substituted amino acid" in the wild-type sequence. For example, a mutation in which the 180th amino acid in wild-type TlpA is substituted from E (glutamic acid) to R (arginine) is described as E180R, and a TlpA mutant having this mutation is described as a TlpA mutant having an E180R point mutation. Furthermore, for deletion mutants, the range of deleted amino acids is indicated after Δ. For example, a deletion of amino acids 1 through 65 in the amino acid sequence of wild-type TlpA is described as Δ1-65.

[0020] As used herein, the term "amino acid" is used in the broadest sense and includes not only natural amino acids but also their derivatives and artificial amino acids. As used herein, amino acids include, for example, natural amino acids as well as unnatural amino acids. Furthermore, "amino acids" also include amino acids whose main chain structure differs from that of natural amino acids, amino acids whose side chain structure differs from that of natural amino acids, amino acids having an extra methylene in the side chain, and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group.

[0021] Because the above-described TlpA mutant 1 and TlpA mutant 2 interact via the coiled-coil region, the mutants must contain at least all or a portion of the coiled-coil region. The coiled-coil region of TlpA has been reported to be approximately the region from amino acid 69 to amino acid 359 (see Non-Patent Document 4, figure legend of Supplementary Figure S2). Examples of TlpA mutants containing all or a portion of the coiled-coil region in this embodiment include, but are not limited to, mutants lacking all or a portion of the DNA-binding region near the N-terminus, such as mutants lacking all or a portion of the amino acids 1 to 93 (Δ1-65, Δ1-93, etc.), and mutants lacking a portion of the C-terminal region (Δ258-371).

[0022] Furthermore, TlpA variant 1 and TlpA variant 2, which form a heterodimer, may each have at least the following point mutations, where the amino acid numbers shown below are those in the amino acid sequence represented by SEQ ID NO: 1. TlpA mutant 1: E180R, E250R TlpA mutant 2: R179E, R251E Furthermore, in some cases, TlpA1 and TlpA2 may have point mutations D135V, A217V, and / or L236F. More specifically, the following combinations of mutations can be exemplified as combinations of TlpA mutants of this embodiment that have only point mutations or that have both the deletion mutation and point mutations. Combination 1 TlpA mutant 1: Δ1-65, E180R, E250R TlpA mutant 2: Δ1-65, R179E, R251E Combination 2 TlpA mutant 1: Δ1-65, D135V, E180R, A217V, L236F, E250R TlpA mutant 2: Δ1-65, D135V, R179E, A217V, L236F, R251E Combination 3 TlpA mutant 1: Δ1-93, D135V, E180R, A217V, L236F, E250R TlpA mutant 2: Δ1-93, D135V, R179E, A217V, L236F, R251E Combination 4 TlpA mutant 1: Δ258-371, D135V, E180R, A217V, L236F, E250R TlpA mutant 2: Δ258-371, D135V, R179E, A217V, L236F, R251E Combination 5 TlpA mutant 1: E180R, E250R TlpA mutant 2: R179E, R251E Combination 6 TlpA mutant 1: D135V, E180R, A217V, L236F, E250R TlpA mutant 2: D135V, R179E, A217V, L236F, R251E

[0023] The binding-inducing peptide, such as the SsrA peptide, may be attached to any part of the TlpA mutant, but must be attached at a position such that when the TlpA mutant forms a heterodimer, the binding-inducing peptide is covered by the dimer and not exposed on the surface. For example, the N-terminus or C-terminus of the TlpA mutant, particularly the C-terminus, is preferred. The TlpA mutant and the binding-inducing peptide, such as the SsrA peptide, may be directly attached or linked via a flexible linker consisting of several amino acids (approximately 2 to 10 amino acids).

[0024] More specifically, examples of the protein set according to the first embodiment include the following protein sets: That is, a protein set including a protein having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein a, the following protein b, and a binding-inducing protein, or A protein set including the following protein b having a binding-inducing peptide bound to the N-terminus or C-terminus thereof, the following protein a, and the following binding-inducing protein; a: a protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having mutations represented by E180R and E250R; b: A protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having the mutations represented by R179E and R251E.

[0025] Furthermore, as the protein set according to the first embodiment, in the above protein set, Protein a is a protein having mutations represented by Δ1-X, E180R, and E250R in the amino acid sequence represented by SEQ ID NO: 1, wherein X is 2 or more and 93 or less; Protein b is a protein having mutations represented by Δ1-Y, R179E, and R251E in the amino acid sequence represented by SEQ ID NO: 1, wherein Y is 2 or more and 93 or less; the binding-inducing protein is Stringent starvation protein B (SspB), and An example of a protein set is one in which the binding-inducing peptide is an SsrA peptide, and the SsrA peptide is bound to the C-terminus of protein a or protein b, which has Z amino acids deleted from the C-terminus (where Z is an integer between 0 and 7). Here, X and / or Y may be 65.

[0026] More specifically, the protein set for inducing substance interaction in a temperature-dependent manner according to this embodiment includes: A protein set including the following protein a having an SsrA peptide bound to its C-terminus, the following protein b, and an SspB protein, or A protein set including the following protein b having an SsrA peptide bound to its C-terminus, the following protein a, and an SspB protein: Protein a or protein b to which SsrA binds may be missing Z amino acids from the C-terminus (where Z is an integer between 0 and 7); a: a protein having mutations represented by Δ1-65, E180R, and E250R in the amino acid sequence represented by SEQ ID NO: 1; b: A protein having the mutations Δ1-65, R179E, and R251E in the amino acid sequence shown in SEQ ID NO:1.

[0027] Each protein included in the protein set of this embodiment may be bound to various signal peptides (e.g., nuclear transport (localization) signal peptide, mitochondrial transport (localization) signal peptide, chloroplast transport (localization) signal peptide, etc.), various tag sequences (FLAG, MYC, His, V5, etc.), fluorescent proteins, etc.

[0028] Among the proteins included in the protein set of this embodiment, the protein in which a binding-inducing peptide (e.g., SsrA peptide) is bound to protein a, the protein in which a binding-inducing peptide is bound to protein b, and the binding-inducing protein may each be bound to or fused with "another substance" (e.g., various biological substances such as proteins, lipids, sugars, and nucleic acids). It is desirable that the "other substances" are substances that interact with each other. For example, suppose the protein set according to this embodiment includes the TlpA mutant 1-SsrA peptide, TlpA mutant 2, and SspB protein. TlpA mutant 1 and TlpA mutant 2 form a dimer at temperatures below approximately 37°C, with SsrA coated inside the dimer and not exposed on the surface. When this protein set is heated to approximately 40°C by irradiation with ultrasound or the like, the dimer of TlpA mutant 1 and TlpA mutant 2 dissociates, exposing the SsrA peptide and making it possible for the SspA peptide to bind to the SspB protein. When protein 1 is bound to the TlpA mutant 1-SsrA peptide and protein 2 is bound to the SspB protein, if protein 1 and protein 2 interact, at temperatures below 37°C, the SsrA peptide is coated within the dimer of TlpA mutant 1 and TlpA mutant 2, suppressing binding to the SspB protein and thus suppressing the interaction between protein 1 and protein 2. When heated to approximately 40°C, the dimer of TlpA mutant 1 and TlpA mutant 2 dissociates, exposing the SsrA peptide and making it available to bind to the SspB protein, bringing protein 1 and protein 2 into close proximity and enabling them to interact (see Figure 1). As described above, it is possible to regulate the interactions of various substances by binding one interacting substance to the TlpA mutant 1-binding-inducing peptide (such as the SsrA peptide) and then subjecting the protein set in which the other substance is bound to the binding-inducing protein to a temperature change (mutual change between approximately 37°C and approximately 40°C).

[0029] In this embodiment, when the "another substance" is a protein, the "another protein" may be any protein, preferably an interacting protein. The other protein used in this embodiment may be, for example, a divided fragment of a protein, which may exhibit its original function when the divided fragments bind. Alternatively, the other protein may be a protein that is originally a separate protein and exhibits its function by forming a dimer, such as a protein that exhibits the activity required for genome editing through dimerization, for example, a site-specific recombinase (Cre recombinase, Flp recombinase, VCre recombinase, Dre recombinase, etc.), a TALE protein, Cas9 nuclease, dCas9, Cpf1 nuclease, etc. Examples of protein fragments include, but are not limited to, fragments of Cre recombinase (such as a fragment consisting of amino acids 19 to 59 and a fragment consisting of amino acids 60 to 343 in SEQ ID NO: 12), as well as fragments of Flp recombinase, VCre recombinase, and Cas9 nuclease. The TlpA mutant-binding-inducing peptide or binding-inducing protein and the "other protein" may be fused together or linked via a flexible linker (e.g., a peptide linker consisting of approximately 10 to 50 amino acids). SEQ ID NO: 12 (Cre); MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTL LRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD

[0030] The second embodiment is a nucleic acid (DNA or RNA) that encodes each protein included in the protein set according to this embodiment (hereinafter also referred to as "nucleic acid according to this embodiment"). The nucleic acid according to this embodiment can be prepared by a method known in the art or a method obtained by appropriately modifying the method.

[0031] Each protein included in the protein set according to this embodiment can be prepared by incorporating nucleic acids encoding the protein into an appropriate expression vector, transforming or transfecting appropriate host cells with the expression vector, culturing the cells in an appropriate medium, and purifying the expressed proteins. The expression vectors for each protein may be separate vectors or a single vector, and a vector suitable for the intended purpose can be used.

[0032] Host cells for protein expression include, for example, bacterial cells (e.g., Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans , Pseudomonas putida ), mold (e.g. Penicillium camembertii , Acremonium chrysogenum), animal cells, plant cells, baculovirus / insect cells or yeast cells (e.g. Saccharomyces cerevisiae and Pichia pastoris etc.) may also be used.

[0033] Protein expression vectors suitable for various host cells can be used. Examples of expression vectors that can be used include pBR322, pBR325, pUC118, and pET (E. coli hosts), pEGFP-C and pEGFP-N (animal host cells), pVL1392 and pVL1393 (insect host cells, baculovirus vectors), and pG-1, Yep13, and pPICZ (yeast host cells). These expression vectors contain a replication origin, selection marker, and promoter appropriate for each vector, and may also contain an enhancer, transcription termination sequence (terminator), ribosome binding site, and polyadenylation signal, as needed. Furthermore, to facilitate purification of the expressed polypeptide, the expression vector may contain a nucleotide sequence for fusion expression of a FLAG tag, His tag, HA tag, GST tag, or other tag. Furthermore, in order to express multiple proteins using one vector, the vector may contain a base sequence encoding an IRES sequence, a 2A (P2A, T2A) peptide sequence, or the like. The expression vector can be prepared by a method known to those skilled in the art, and can also be prepared using a commercially available kit, if appropriate.

[0034] When extracting expressed proteins from cultured bacteria or cells, the bacteria or cultured cells are collected after cultivation using known methods, suspended in an appropriate buffer, and disrupted by ultrasound, lysozyme, and / or freeze-thawing, followed by centrifugation or filtration to obtain a soluble extract. In particular, when cultured cells are used as hosts, it is preferable to obtain the protein expressed in the culture supernatant by recovering the supernatant. The target protein can be obtained from the resulting extract or culture supernatant by appropriately combining known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that mainly utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography (for example, a method that uses a resin bound to glutathione when a polypeptide is expressed with a GST tag, a Ni-NTA resin or a Co-based resin when a polypeptide is expressed with a His tag, an anti-HA antibody resin when a polypeptide is expressed with an HA tag, or an anti-FLAG antibody-bound resin when a polypeptide is expressed with a FLAG tag); methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0035] The third embodiment is a kit for controlling the interaction of substances within cells, comprising three nucleic acids (i.e., "nucleic acids according to this embodiment") encoding the three proteins included in the protein set according to this embodiment: the TlpA mutant, the TlpA mutant-binding-inducing peptide, and the binding-inducing protein. Furthermore, each of the TlpA mutant-binding-inducing peptide and the binding-inducing protein may be bound to or fused with one of two interacting proteins, and nucleic acids encoding these proteins may be included in the kit according to this embodiment. The nucleic acids according to this embodiment may be provided in a state where they are inserted into various expression vectors so that they can be expressed in the desired cells. An example of a kit according to this embodiment is a kit containing at least a nucleic acid encoding TlpA mutant 1 having mutations Δ1-65, E180R, and E250R in the amino acid sequence represented by SEQ ID NO: 1, a nucleic acid encoding a fusion protein of TlpA mutant 2 having mutations Δ1-65, R179E, and R251E in the amino acid sequence represented by SEQ ID NO: 1 and an SsrA peptide, and a nucleic acid encoding SspB.

[0036] A fourth embodiment is a method for controlling the distance between two proteins, comprising: a step of binding or fusing a protein having a binding-inducing peptide bound to the protein a or a protein having a binding-inducing peptide bound to the protein b, both of which are included in the protein set according to this embodiment, to one of the two proteins; A step of binding or fusing a binding-inducing protein included in the protein set according to this embodiment to the other of the two proteins; and heating the protein set. Here, the "two proteins" correspond to the "another protein" in the first embodiment and may be proteins that interact with each other, or the two proteins may be two fragments generated by splitting one protein, and the two fragments may be split in such a way that the activity of the original protein is restored when recombined. The protein included in this embodiment and each of the "two proteins" may be bonded via a linker or the like, or may be directly bonded (fused).

[0037] The fourth embodiment can also be carried out intracellularly. Here, "cells" includes both in vitro and in vivo cells. Those skilled in the art can appropriately select a method for introducing into cells a conjugate of a protein included in the protein set according to this embodiment and one of the "two proteins." For example, a vector expressing the protein complex may be introduced into the cell to express the protein complex within the cell, or the protein complex may be directly introduced into the cell using a cell membrane-permeable peptide or the like. When the protein complex is to be translocated to the nucleus or intracellular organelles, the protein complex may be fused to a translocation signal peptide (e.g., a nuclear localization signal peptide, a mitochondrial localization signal peptide, etc.) and then introduced into the cell. The protein set according to this embodiment can be easily heated by a method that can be appropriately selected by those skilled in the art. Examples include irradiating the protein set with ultrasound or radio waves (see, for details, Non-Patent Documents 3 and 4). Furthermore, it is desirable to heat the protein set according to this embodiment so that its temperature reaches approximately 39°C to 43°C. In this case, the optimal frequency when using ultrasound or radio waves for heating varies depending on the location of the site to be heated, such as the depth of the site in the living body, and the temperature to which the site is heated. Those skilled in the art can select an appropriate frequency. While not particularly limited, for example, when using ultrasound, the frequency may be approximately 0.1 MHz to 4 MHz or 0.5 MHz to 4 MHz. When using radio waves, the frequency may be approximately 3 Hz to 300 GHz, 1 MHz to 1 GHz, 30 MHz to 500 MHz, or 100 MHz to 300 MHz.

[0038] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include the plural as well as the singular, unless the context clearly indicates otherwise. Also, in this specification, "about" or "to the extent" means a numerical range of ±10%. The present invention will be further explained below by showing examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Example]

[0039] 1. Evaluation of heterodimers formed in TlpA mutants 1-1. Experimental method Three milliliters of cell culture medium (DMEM [Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque], 10% heat-inactivated fetal bovine serum (FBS; SIGMA-ALDRICH)) was added to a 60-mm dish (Corning). HEK293T cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). Two micrograms of plasmid DNA encoding the TlpA mutant (containing a FLAG tag), 6 μL of PEI MAX (Polysciences), and 600 μL of Opti-MEM I Reduced Serum Media (Thermo Fischer Scientific) were mixed and incubated for 30 minutes. Then, the mixture was added to the HEK293T cell culture medium and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After 24 hours of culture, the medium was removed and the cells were washed with PBS. TM Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with an EDTA-free protease inhibitor cocktail (Roche), and the expressed protein was collected. Protein yield was assessed by measuring absorbance at 660 nm using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific) on an iMark microplate reader (BIO-RAD).

[0040] The solution containing the TlpA mutant was premixed and 95 μL was transferred to a PCR tube (STAR). After heating for 5 minutes at a predetermined temperature (37°C or 40°C) in a thermal cycler ProFlex PCR System (Thermo Fisher Scientific), bis(sulfosuccinimidyl)suberate (BS3) (Thermo Fisher Scientific) was added to a final concentration of 3 mM and heated for an additional 30 minutes to crosslink interacting proteins. To terminate the crosslinking reaction, a final concentration of 50 mM Tris·HCl (pH 7.5) solution was added and heated for an additional 15 minutes. After crosslinking, the sample was added to 5x sample buffer (50 mM Tris-HCl, 2% sodium dodecyl sulfate, 5% glycerol, 1% 2-mercaptoethanol, 0.004% bromophenol blue) and heated at 95°C for 5 minutes to denature the proteins and prepare samples for Western blotting. Samples were separated by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (GE Healthcare). After transfer, the membrane was washed with TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.05% (v / v) Tween-20) and shaken in 1% skim milk for 1 hour. It was then shaken overnight at 4°C in a primary antibody solution diluted with 1% skim milk. The primary antibody used was Anti-FLAG M2 Monoclonal Antibody (MilliporeSigma) diluted 1:5000. After washing the membrane with TBS-T, it was shaken in a secondary antibody solution diluted with 1% skim milk for 1 hour. The secondary antibody used was ECL Anti-mouse IgG, Horseradish Peroxidase-linked whole antibody (GE Healthcare) diluted 1:10000. The membrane was washed with TBS-T, and signals were detected using Western Lightning ECL Pro (Perkin Elmer) as a detection reagent with an LAS4000mini (GE Healthcare).

[0041] 1-2.Results The percentage of molecules forming multimers was calculated from the Western blotting data and expressed as the mean ± standard deviation (n = 3) (Figure 2A). For the combination of TlpAa (Δ1-65, E180R, E250R) and TlpAb (Δ1-65, R179E, R251E) molecules (#8 in Figure 2A), the percentage of molecules forming multimers was calculated when the heating temperature was varied and expressed as the mean ± standard deviation (n = 3) (Figure 2B). Additionally, the percentage of molecules forming multimers was calculated from the experimental results when the mixture was heated at 40°C for 5 minutes before the addition of the cross-linking agent, followed by incubation at 37°C for the indicated time and expressed as the mean ± standard deviation (n = 3). The horizontal axis of Figure 2A indicates the number of each TlpA mutant combination. Among these mutant combinations, the combinations with a 2.5-fold or higher increase in monomerization upon heating from 37°C to 40°C (the ratio of molecules forming multimers at 37°C divided by the ratio of molecules forming multimers at 40°C) were #1, #2, #4, #7, #8, #9, and #10. The mutant combinations and their corresponding increases are shown below. #1 (approximately 2.5 times); Mutations including D135V, E180R, A217V, L236F and E250R Mutations including D135V, R179E, A217V, L236F and R251E #2 (approximately 2.9 times); Mutants including Δ1-65, D135V, E180R, A217V, L236F and E250R Mutants including Δ1-65, D135V, R179E, A217V, L236F and R251E #4 (approximately 3.1 times); Mutants including Δ258-371, D135V, E180R, A217V, L236F and E250R Mutants including Δ258-371, D135V, R179E, A217V, L236F and R251E #7 (approximately 3.1 times); Mutants including E180R and E250R Mutants including R179E and R251E #8 (approximately 10.1 times) Mutants including Δ1-65, E180R and E250R Mutants containing Δ1-65, R179E, and R251E #9 (about 4.5 times) Mutants including Δ1-93, D135V, E180R, A217V, L236F and E250R Mutants including Δ1-93, D135V, R179E, A217V, L236F and R251E #10 (about 3.5 times); Mutants including Δ258-371, E180R and E250R Mutants containing Δ258-371, R179E and R251E

[0042] TlpA normally forms a homodimer, but when a molecule in which the amino acids corresponding to positions 180 and 250 are substituted with arginine is mixed with a molecule in which the amino acids corresponding to positions 179 and 251 are substituted with glutamic acid, a heterodimer is formed, which is known to dissociate into monomers upon heating to 42°C. We then examined the monomerization upon heating from 37°C to 40°C, and confirmed a 2.5-fold increase (#1). Similar experiments were performed with candidate molecules incorporating other mutations and deletions of the N-terminal region. The combination of a TlpA mutant (TlpAa) in which the amino acids corresponding to positions 180 and 250 in the TlpA sequence were replaced with arginine and positions 1 to 65 were deleted (also referred to as "TlpAa") and a TlpA mutant (TlpAb) in which the amino acids corresponding to positions 179 and 251 in the TlpA sequence were replaced with glutamic acid and positions 1 to 65 were deleted (also referred to as "TlpAb") (#8) showed a 10.1-fold change in the monomerization activity upon heating from 37 to 40°C. The TlpAa / TlpAb combination showed a significant change in monomerization activity over the temperature range from 37 to 39°C (Figure 2B). Furthermore, dimerization occurred within 1 minute after heating, confirming that the heat-induced monomerization was a reversible reaction (Figure 2C).

[0043] 2. Functional evaluation of the protein set according to the present invention 2-1. Experimental method Plasmid DNAs expressing TlpAa-SsrA, TlpAb, and SspB were prepared using Escherichia coli TaKaRa Competent Cells BL21 (TaKaRa). For TlpAa-SsrA, SsrA was attached to the C-terminus of TlpAa (amino acid 371). Fourteen expression plasmids were prepared: one in which SsrA was attached to the C-terminus of TlpAa, and the other in which one amino acid was deleted from the C-terminus to the 358th amino acid. E. coli was transformed with each expression plasmid DNA, and transformed colonies were cultured at 37°C with shaking in liquid LB medium supplemented with ampicillin (Wako) to a final concentration of 100 μg mL-1. OD 600 When the pH reached 0.4-0.5, the mixture was cooled to 15°C. After 30 minutes, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and the mixture was cultured with shaking at 15°C for 24 hours. After the culture, the E. coli solution was centrifuged, and the E. coli pellet was subjected to complete culture. TMPBS containing EDTA-free protease inhibitor cocktail (Roche) was added, followed by sonication and centrifugation. The supernatant was collected. The yield of recovered protein was assessed using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific) and measuring absorbance at 660 nm on an iMark microplate reader (BIO-RAD). The target molecule solution was premixed and 95 μL was transferred to a PCR tube (STAR). After heating at the designated temperature for 5 minutes in a ProFlex PCR System (Thermo Fisher Scientific), BS3 (bis(sulfosuccinimidyl) suberate) (Thermo Fisher Scientific) was added to a final concentration of 3 mM and heated for an additional 30 minutes to crosslink interacting proteins. To terminate the crosslinking reaction, Tris·HCl (pH 7.5) solution was added to a final concentration of 50 mM and heated for an additional 15 minutes. After crosslinking, the samples were added to 5x sample buffer (50 mM Tris-HCl, 2% sodium dodecyl sulfate, 5% glycerol, 1% 2-mercaptoethanol, 0.004% bromophenol blue) and heated at 95°C for 5 minutes to denature the proteins. The samples were then separated by SDS-PAGE and transferred to a Nitrocellulose Blotting Membrane (GE Healthcare). After transfer, the membrane was washed with TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.05% (v / v) Tween-20) and shaken in 1% skim milk for 1 hour. It was then shaken overnight at 4°C in a primary antibody solution diluted with 1% skim milk. The primary antibody used was Anti-FLAG M2 Monoclonal Antibody (MilliporeSigma) (Thermo Fisher Scientific) diluted 1:5000. After washing the membrane with TBS-T, it was shaken for 1 hour in a secondary antibody solution diluted with 1% skim milk.The secondary antibody used was ECL™ Anti-mouse IgG, Horseradish Peroxidase-linked whole antibody (GE Healthcare) diluted 1:10,000. After washing with TBS-T, Western Lightning ECL Pro (Perkin Elmer) was used as the detection reagent, and signals were detected using an LAS4000mini (GE Healthcare).

[0044] 2-2.Results The results of Western blotting are shown in Figure 3. The results for the TlpAa-SsrA molecule, in which SsrA (AANDENYF; SEQ ID NO: 8) was fused to each of the amino acids 357 to 371 of TlpAa, correspond to the lanes indicated by the numbers in the figure. The following conditions were examined: TlpAb and SspB were added to TlpAa-SsrA; TlpAb alone was added; SspB alone was added; TlpAb alone without TlpAa-SsrA; or SspB alone was added. SsrA and SspB are known to interact with each other. Protein solutions of TlpA-SsrA, TlpAb, and SspB were mixed and cross-linking experiments were performed. For TlpAa-SsrA, in which TlpAa was fused to SsrA at specific amino acid positions (e.g., positions 371 to 364), the interaction between SsrA and SspB increased upon heating from 37°C to 40°C, resulting in the formation of molecules in which TlpAa-SsrA and SspB were cross-linked (indicated by arrowheads in the figure). These results demonstrate that the protein set containing TlpAa-SsrA, TlpAb, and SspB can induce interactions between proteins, and between TlpAa-SsrA and SspB, upon heating (at approximately 40°C).

[0045] 3. Evaluation of the function of controlling the activity of split Cre recombinase using the protein set of the present invention 3-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). Plasmid DNA of the following genes (a gene expressing a molecule in which TlpAa-SsrA is bound to split Cre (containing a nucleic acid sequence encoding a nuclear localization signal (NLS)), a gene expressing a molecule in which SspB is bound to split Cre (containing a nucleic acid sequence encoding an NLS), and a gene expressing TlpAb (containing a nucleic acid sequence encoding an NLS)) and the reporter gene loxP-NLuc were mixed in an amount of 0.25 μg each, and then mixed with 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fischer Scientific). The mixture was left to stand for 30 minutes, then added to COS-7 cell culture medium, and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After 24 hours of incubation, the medium was removed and the cells were washed with PBS. 500 μL of Nluc assay solution (a 5-fold dilution of Nano-Glo Luciferase Assay Buffer (Promega) in HBSS, calcium, magnesium, and no phenol red (Thermo Fisher Scientific) followed by a 1:10,000 volume of Nano-Glo Luciferase Assay Substrate (Promega)) was added and incubated for 3 minutes at room temperature. 100 μL of the solution was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (greiner bio-one)), and the luminescence intensity was measured for 0.2 seconds per well using a TriStar LB941 (BERTHOLD) luminescence analyzer.

[0046] 3-2.Results TlpAa-SsrA and SspB were fused to the Cre recombinase fragments, respectively, and various combinations of fragment sites, fusion methods, and SspB mutants (SspBnano or SspBmicro) were expressed in cultured cells. The efficiency of DNA recombination upon heating was evaluated by measuring the expression level of the luciferase (Fig. 4). Luminescence values ​​at 37°C or 40°C are shown as mean ± standard deviation (n = 3) (Fig. 4A). The Ford increase of expression (the fold increase in expression at 40°C relative to 37°C) was calculated by dividing the luminescence value at 40°C by the luminescence value at 37°C and is shown as mean ± 95% CI (n = 3) (Fig. 4B). The numbers on the horizontal axis of each graph identify the various TlpAa-SsrA and SspB combinations.

[0047] The combinations that resulted in an increase in luminescence intensity of 2-fold or more are shown below. #1 (Increase in light output: approx. 3.3 times) TlpAa(371)-SsrA-CreC(60-343) + SspBmicro-CreN(19-59) (These are the arrangements from the N-terminus to the C-terminus. The same applies below.) *The above "TlpAa(371)-SsrA-CreC(60-343)" refers to a molecule in which SsrA is linked to the 371st amino acid of TlpAa, and CreC(60-343) (a fragment from the 60th to the 343rd amino acids in the amino acid sequence of the Cre recombinase represented by SEQ ID NO: 8) is further linked. "SspBmicro-CreN(19-59)" refers to a molecule in which CreN(19-59) (a fragment from the 19th to the 59th amino acids in the amino acid sequence of the Cre recombinase represented by SEQ ID NO: 8) is linked to the C-terminus of SspBmicro. The TlpAa-SsrA and Cre fragment, and the SspB and Cre fragment, are linked by a peptide linker (LEASPSNPGASNGSGT: SEQ ID NO: 13). The same applies to the following combinations.

[0048] #5 (increase in luminescence intensity: approximately 3.3 times) TlpAa(371)-SsrA-CreC(60-343) + SspBnano-CreN(19-59) #9 (increase in luminescence intensity: approximately 5.5 times) CreC(60-343)-TlpAa(371)-SsrA + SspBmicro-CreN(19-59) #11 (increase in luminescence intensity: approximately 2.7 times) CreN(19-59)-TlpAa(371)-SsrA + SspBmicro-CreC(60-343) #13 (increase in luminescence intensity: approximately 5.5 times) CreC(60-343)-TlpAa(371)-SsrA + SspBnano-CreN(19-59) #15 (increase in luminescence intensity: approximately 2.7 times) CreN(19-59)-TlpAa(371)-SsrA + SspBnano-CreC(60-343) #17 (increase in luminescence intensity: approximately 4.5 times) TlpAa(371)-SsrA-CreC(60-343) + CreN(19-59)-SspBmicro #21 (increase in luminescence intensity: approximately 4.1 times) TlpAa(371)-SsrA-CreC(60-343) + CreN(19-59)-SspBnano #23 (increase in luminescence intensity: approximately 2.1 times) TlpAa(371)-SsrA-CreN(19-59) + CreC(60-343)-SspBnano #25 (increase in luminescence intensity: approx. 4.1 times) CreC(60-343)-TlpAa(371)-SsrA + CreN(19-59)-SspBmicro #27 (increase in luminescence intensity: approximately 2.1 times) CreN(19-59)-TlpAa(371)-SsrA + CreC(60-343)-SspBmicro #29 (increase in light output: approximately 6.5 times) CreC(60-343)-TlpAa(371)-SsrA + CreN(19-59)-SspBnano #31 (increase in luminescence intensity: approximately 2.4 times) CreN(19-59)-TlpAa(371)-SsrA + CreC(60-343)-SspBnano #33 (increase in luminescence intensity: approximately 3.6 times) TlpAa(364)-SsrA-CreC(60-343) + SspBmicro-CreN(19-59) #37 (increase in luminescence intensity: approximately 2.4 times) TlpAa(364)-SsrA-CreC(60-343) + SspBnano-CreN(19-59) #41 (increase in luminescence intensity: approximately 8.9 times) CreC(60-343)-TlpAa(364)-SsrA + SspBmicro-CreN(19-59) #43 (increase in luminescence intensity: approximately 2.2 times) CreN(19-59)-TlpAa(364)-SsrA + SspBmicro-CreC(60-343) #45 (increase in luminescence intensity: approximately 22.5 times) CreC(60-343)-TlpAa(364)-SsrA + SspBnano-CreN(19-59) #47 (increase in luminescence intensity: approximately 3.4 times) CreN(19-59)-TlpAa(364)-SsrA + SspBnano-CreC(60-343) #49 (increase in luminescence intensity: approx. 5.3 times) TlpAa(364)-SsrA-CreC(60-343) + CreN(19-59)-SspBmicro #53 (increase in luminescence intensity: approximately 4.8 times) TlpAa(364)-SsrA-CreC(60-343) + CreN(19-59)-SspBnano #57 (increase in luminescence intensity: approx. 7.9 times) CreC(60-343)-TlpAa(364)-SsrA + CreN(19-59)-SspBmicro #59 (increase in luminescence intensity: approximately 2.3 times) CreN(19-59)-TlpAa(364)-SsrA + CreC(60-343)-SspBmicro #61 (increase in luminescence intensity: approximately 8.4 times) CreC(60-343)-TlpAa(364)-SsrA + CreN(19-59)-SspBnano #63 (increase in luminescence intensity: approximately 2.3 times) CreN(19-59)-TlpAa(364)-SsrA + CreC(60-343)-SspBnano

[0049] Among the above combinations, a molecule in which CreN (19-59) was fused to the C-terminus of SspBnano (hereinafter also referred to as "ThermoCreN") and a molecule in which CreC (60-343) was fused to the N-terminus of TlpAa-SsrA (hereinafter also referred to as "ThermoCreC") were combined, and an approximately 22.5-fold increase in the expression level of the photoprotein was confirmed (#45). Hereinafter, the protein set #45 will be referred to as "ThermoCre1.0," and the DNA recombination induction system using ThrmoCre will be referred to as the "ThermoCre system." The above results demonstrate that when Cre fragments are bound to each of TlpAa-SsrA and SspB from the protein set of the present invention and the temperature is raised to approximately 40°C, interactions between the Cre fragments are induced, making it possible to induce DNA recombination by Cre recombinase.

[0050] 4. Functionality of ThrmoCre1.0 4-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). Plasmid DNA of the desired genes (ThermoCreN (SspBnano-CreN(19-59)) expressing gene, ThermoCreC (CreC(60-343)-TlpAa(364)-SsrA) expressing gene, TlpAb expressing gene, Cre expressing gene) and the reporter gene loxP-NLuc (0.25 μg each) were added to the cells. If necessary, empty vector pcDNA3.1 / myc-His ver.B (Thermo Fisher Scientific) was added to the cells to make a total of 1 μg. Then, 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) were added. The cells were incubated for 30 minutes, then added to COS-7 cell culture medium, and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After 24 hours of culture, the medium was removed and the cells were washed with PBS. 500 μL of Nluc assay solution (a 5-fold dilution of Nano-Glo Luciferase Assay Buffer (Promega) with HBSS, calcium, magnesium, and no phenol red (Thermo Fisher Scientific) followed by the addition of 1:10,000 of Nano-Glo Luciferase Assay Substrate (Promega)) was added, and the cells were left to stand at room temperature for 3 minutes.100 μL was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96 Well, F-Bottom, Chimney Well, White (greiner bio-one)), and the luminescence intensity was measured for 0.2 seconds per well using a luminescence measuring device TriStar LB941 (BERTHOLD).

[0051] 4-2.Results The results are shown in Figure 5. No luminescence was detected when ThermoCreN and TlpAb, or ThermoCreC and TlpAb, were expressed together with a reporter gene, demonstrating that these molecules alone do not possess DNA recombination activity. Furthermore, when ThermoCreN and ThermoCreC were expressed together with a reporter gene, DNA recombination activity was confirmed regardless of whether heating was applied, and heating increased DNA recombination efficiency by approximately two-fold. On the other hand, when ThermoCreN, ThermoCreC, and TlpAb were co-expressed, the DNA recombination efficiency before heating was lower than when ThermoCreN and ThermoCreC were expressed alone, demonstrating that the rate of change in DNA recombination activity due to heating was significantly improved (Figure 5B). Therefore, it was revealed that TlpAb suppresses the spontaneous reconstitution of ThermoCreN and ThermoCreC before heating, and that heating releases the TlpAb-mediated reconstitution, allowing ThermoCreN and ThermoCreC to interact, reconstituting Cre and restoring DNA recombination ability.

[0052] 5. Confirmation that ThermoCre1.0 is activated by ultrasonic heating 5-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). ThermoCre1.0 consisted of 0.25 μg of plasmid DNA each for TlpAb, ThermoCreN, ThermoCreC, and the reporter gene loxP-NLuc, 3 μL of PEI MAX (Polysciences), and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fischer Scientific). After 30 minutes of incubation, the mixture was added to COS-7 cell culture medium and cultured in a CO2 incubator (37°C, 5% CO2). After 4 hours of incubation, transfected cells were harvested with 0.5 g / L trypsin / 0.53 mmol / L EDTA solution (Nacalai tesque). The supernatant was removed by centrifugation, and the cell pellet was resuspended in 1 mL of cell culture medium. 200 μL of the cell solution was transferred to a PCR tube (STAR). A 25-mm-thick agarose gel was placed on the transducer of a HIFU irradiation device (Sonitron GTS base station, HIFU transducer module 3.5 MHz, 20 mm depth, 20 mm diameter; Nepagene). A hole approximately 5 mm deep was drilled in the center of the gel, ultrasound diagnostic gel LOGIQLEAN (GE HealthCare) was poured into it, and a PCR tube containing the cell solution was placed in the hole. The cell solution was heated to 40 ± 0.5°C for 10 or 30 minutes while measuring the temperature with a temperature sensor (HDS-120E, SFS-E-100-ASP; Anritsu Meter Co., Ltd.). One mL of cell culture medium was added to a 35-mm dish (Corning), and the entire volume of the heated cell solution was then added. The dish was then cultured in a CO2 incubator (37°C, 5% CO2).After 24 hours of incubation, the medium was removed and the cells were washed with PBS. 500 μL of Nluc assay solution (a 5-fold dilution of Nano-Glo Luciferase Assay Buffer (Promega) in HBSS, calcium, magnesium, and no phenol red (Thermo Fisher Scientific) followed by a 1:10,000 volume of Nano-Glo Luciferase Assay Substrate (Promega)) was added and incubated for 3 minutes at room temperature. 100 μL of the solution was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (Greiner Bio-One)), and luminescence intensity was measured for 0.2 seconds per well using a TriStar LB941 (Berthold) luminescence analyzer.

[0053] 5-2.Results The results are shown in Figure 6. Values ​​were calculated by dividing the luminescence value under 40°C culture conditions by the luminescence value under 37°C culture conditions, and are expressed as the mean ± standard deviation (n = 3). When cells expressing ThermoCre1.0 were heated for a short time by ultrasonic irradiation, an increase in protein expression efficiency of approximately 9% was observed after 30 minutes of heating. Therefore, it was demonstrated that the Cre-mediated DNA recombination system using ThermoCre1.0 (hereinafter referred to as the "ThermoCre" system) can be driven by ultrasonic irradiation.

[0054] 6. Confirmation of the safety of ThermoCre 1.0 6-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). 1 μg of the desired gene (ThermoCreN, ThermoCreC, TlpAb, pcDNA3.1 / myc-His ver.B (Thermo Fisher Scientific)), 3 μL of PEI MAX (Polysciences), and 200 μL of Opti-MEM (Opti-MEM I Reduced Serum Media, Thermo Fisher Scientific) were mixed and left to stand for 30 minutes, then added to COS-7 cell culture medium and cultured in a CO2 incubator (37°C, 5% CO2). After 24 hours of culture, the transfected cells were harvested with 0.5 g / L trypsin / 0.53 mmol / L EDTA solution (Nacalai tesque) and cultured at 3.0 × 10 4 The cells were transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (Greiner Bio-One)) and incubated with 100 μL of cell culture medium in a CO2 incubator (37°C, 5% CO2). After 24 hours, alamarBlue Cell Viability Reagent (Thermo Fisher Scientific) was added according to the manufacturer's protocol to assess cytotoxicity. Absorbance at 570 nm and 595 nm was measured using an iMark microplate reader (BIO-RAD).

[0055] 6-2.Results Figure 7 shows the results of comparing the cell viability of cells transfected with ThermoCreN, ThermoCreC, or TlpAb with cells not transfected with these genes. Relative cell viability was calculated using the value of cells transfected with a control vector (pcDNA3.1 / myc-His ver. B) as 100, and is expressed as the mean ± standard deviation (n = 3). No significant difference was observed in the cell viability of cells transfected with ThermoCreN, ThermoCreC, or TlpAb compared with cells transfected with the control vector. This indicates that ThermoCreN, ThermoCreC, and TlpAb are not cytotoxic.

[0056] 7. Reversibility of the ThermoCre System 7-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). 0.25 μg of each of the designated genes (ThermoCreN, ThermoCreC, TlpAb, Cre) and, if necessary, the empty vector pcDNA3.1 / myc-His ver.B (Thermo Fisher Scientific) were added to a total of 1 μg, and then 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM (Opti-MEM I Reduced Serum Media, Thermo Fisher Scientific) were added. The mixture was left to stand for 30 minutes, then added to COS-7 cell culture medium and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After removing the medium and washing with PBS, 500 μL of Nluc assay solution (a 5-fold dilution of Nano-Glo Luciferase Assay Buffer (Promega) in HBSS, calcium, magnesium, and no phenol red (Thermo Fisher Scientific) followed by a 1:10,000 volume of Nano-Glo Luciferase Assay Substrate (Promega)) was added and incubated for 3 minutes at room temperature. 100 μL of the solution was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (Greiner Bio-One)), and the luminescence intensity was measured for 0.2 seconds per well using a TriStar LB941 (Berthold) luminescence analyzer.

[0057] 7-2.Results The results are shown in Figure 8. Cells transfected with ThremoCreN, ThremoCreC, and TlpAb (ThremoCre), the empty vector pcDNA3.1 / myc-His ver. B (pcDNA), or Cre alone were cultured at 37°C or 40°C for 24 hours, then a reporter gene was transfected and cultured at 37°C or 40°C for 24 hours, after which luminescence levels were measured. The results are shown in Figure 8. Luminescence levels at 37°C and 40°C are expressed as mean ± standard deviation (n = 3). Relative fold increase of expression was calculated by dividing the luminescence level at 40°C by the luminescence level at 37°C, normalized to the value under the 37°C → 37°C condition (24 hours of culture at 37°C followed by another 24 hours of culture at 37°C), and expressed as mean ± 95% CI (n = 3). DNA recombination was confirmed in cells cultured at 40°C, then transfected with a reporter gene and incubated at 40°C, whereas DNA recombination was not confirmed in cells cultured at 40°C, then transfected with a reporter gene and cultured at 37°C. This indicates that Cre, which had previously been reconstituted by heating at 40°C through the interaction between ThermoCreN and ThermoCreC and had restored DNA recombination ability, lost DNA recombination ability again upon subsequent culture at 37°C, demonstrating that the ThermoCre system is reversible to heat stimulation.

[0058] 8. Recombination efficiency by the ThermoCre system 8-1. Experimental method COS-7 cells (ATCC) were cultured in a 35 mm glass-bottom dish (IWAKI) with 1 mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) in a CO2 incubator (37°C, 5% CO2). 0.25 μg each of the plasmid DNAs of TlpAb-mCherry, ThermoCreN, and ThermoCreC, which make up the ThermoCre system, and the reporter gene loxP-STOP-loxP-EGFP, were mixed with 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fischer Scientific) and left to stand for 30 minutes. After this, the mixture was added to COS-7 cell culture medium and the cells were cultured in a CO2 incubator (37°C, 5% CO2). After 24 hours of culture, nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific), and the medium was replaced with microscopy medium (HBSS, calcium, magnesium, no phenol red (Thermo Fisher Scientific), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)). The cells were then observed using a confocal fluorescence microscope IX-81 FV-1000D (Olympus). The objective lens magnification was 40x. Images were analyzed using ImageJ (NIH).

[0059] 8-2.Results Figure 9 shows the results of inducing recombination with the ThermoCre system at 37°C or 40°C. When recombination occurs within the cells, they become GFP-positive. The DNA recombination efficiency was calculated by dividing the number of LSL (lox-stop-lox)-GFP-positive cells by the number of ThermoCre (TlpAb-mCherry)-positive cells. In the cells expressing the ThermoCre system, the DNA recombination efficiency was 12.8% in the cells heated at 37°C, while it was 81.6% in the cells heated at 40°C. Therefore, it was revealed that the proportion of cells that had undergone DNA recombination increased in cells into which the ThermoCre system was introduced upon heat stimulation.

[0060] 9.Improvements to ThermoCre 1.0 9-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). Plasmid DNA of the designated genes (ThermoCre1.0, ThermoCre 1.1, ThermoCre 1.2, Cre) and the reporter gene loxP-NLuc was added in an amount of 0.25 μg each, and, if necessary, the empty vector pcDNA3.1 / myc-His ver.B (Thermo Fisher Scientific) was added to a total of 1 μg. Then, 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) were added, and the mixture was left to stand for 30 minutes. After this, the mixture was added to COS-7 cell culture medium and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After 24 hours of incubation, the medium was removed and the cells were washed with PBS. 500 μL of Nluc assay solution (a 5-fold dilution of Nano-Glo Luciferase Assay Buffer (Promega) with HBSS, calcium, magnesium, and no phenol red (Thermo Fisher Scientific) followed by a 1:10,000 volume of Nano-Glo Luciferase Assay Substrate (Promega)) was added and incubated for 3 minutes at room temperature. 100 μL of the solution was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (Greiner Bio-One)), and the luminescence intensity was measured for 0.2 seconds per well using a TriStar LB941 (Berthold) luminescence analyzer.

[0061] 9-2.Results The molecular structures of ThermoCre1.0, ThermoCre1.1, and ThermoCre1.2 are shown in Figure 10. ThermoCre1.1 was created by changing the ThermoCreN linker site in ThermoCre1.0 to LEGGGGSGGGGSGGGGSGT (SEQ ID NO: 14). ThermoCre1.2 was created by changing the ThermoCreN linker site to LEGGGGSGGGGSGGGGSGT (SEQ ID NO: 14), shifting the NLS fusion site to the C-terminus, and shifting the NLS fusion site to the C-terminus of ThermoCreC. Figure 11 shows a comparison of the recombination efficiencies of ThermoCre1.0, ThermoCre1.1, and ThermoCre1.2. Luminescence values ​​measured when each ThermoCre was cultured at 37°C and 40°C are expressed as the mean ± standard deviation (n = 3) (Figure 11A). The fold increase of expression was calculated by dividing the luminescence value at 40°C by the luminescence value at 37°C, and is expressed as the mean ± 95% CI (n = 3) (Fig. 11B). As a result, a 5.0-fold increase in DNA recombination was observed in ThermoCre1.0, and a 50.1-fold increase in DNA recombination in ThermoCre1.2. In other words, by changing the linker site and the NLS fusion position, a 10-fold or greater improvement in the ability to induce DNA recombination was observed.

[0062] 10. Development of ThermoCre 1.3 10-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). Plasmid DNA of the desired genes (0.25 μg each of the three plasmids constituting ThermoCre1.3, 0.75 μg each of IRES-P2A, T2A-P2A, T2A-T2A, P2A-T2A, or P2A-P2A shown in Figure 13, and 0.5 μg of Cre) and the reporter gene loxP-NLuc were added to the cells. If necessary, empty vector pcDNA4 V5_His ver. B (Thermo Fisher Scientific) was added to the cells to bring the total to 1 μg. The cells were then mixed with 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific). The mixture was left to stand for 30 minutes, then added to COS-7 cell culture medium, and cultured in a CO2 incubator (37°C or 40°C, 5% CO2). After 24 hours of incubation, the medium was removed and the cells were washed with PBS. 500 μL of Nluc assay solution (Nano-Glo Luciferase Assay Buffer (Promega) diluted 4-fold with cell culture medium, followed by the addition of 1:50,000 of Nano-Glo Luciferase Assay Substrate (Promega)) was added and the cells were incubated at room temperature for 3 minutes. 100 μL of the solution was transferred to a 96-well plate (PS Microplate, CELLSTAR, 96-well, F-Bottom, Chimney Well, White (greiner bio-one)), and the luminescence intensity was measured for 0.2 seconds per well using a TriStar LB941 (BERTHOLD) luminescence analyzer.

[0063] 10-2.Results The structures of the three molecules that make up ThermoCre1.3 are shown in Figure 12. ThermoCre1.3-N was created by changing the linker site of ThermoCre1.1N to LEGGGGSGGGGSGGGGSGTGGGGSGGGGSGGGGS (SEQ ID NO: 15), and the NLS was located at the N-terminus, the same as in ThermoCre1.1N. ThermoCre1.3-C used the same linker sequence (SEQ ID NO: 15) as ThermoCre1.1C, but the NLS sequence was moved to the C-terminus. ThermoCre1.3-suppressor is the same as ThermoCre1.1-suppressor. All integrated ThermoCre expression vectors were arranged in the following order from the N-terminus: ThermoCre1.3-Suppressor, ThermoCre1.3-C, and ThermoCre1.3-N. The molecules were fused via IRES, P2A peptide, or T2A peptide in the combinations shown in Figure 13. Figure 14 shows a comparison of the recombination efficiency of integrated ThermoCre vectors. Luminescence values ​​measured when each ThermoCre vector was cultured at 37°C and 40°C are expressed as the mean ± standard deviation (n = 3). Fold change upon heating was calculated by dividing the luminescence value at 40°C by the luminescence value at 37°C and expressed as the mean ± 95% CI (n = 3). As a result, 18-fold DNA recombination was confirmed with IRES-P2A and T2A-P2A. That is, by appropriately selecting the type and position of the IRES and 2A peptide, we were able to produce a ThermoCre set with a good S / N ratio at 37°C and 40°C, and obtained a thermoresponsive DNA recombination ability comparable to that of ThermoCre1.3 expressed without integration ("ThermoCre1.3*" in Figure 14). Here, the T2A-P2A with good thermoresponsiveness was named ThermoCre1.3 and used in the following experiments.

[0064] 11. Time course of recombination kinetics using ThermoCre1.3 11-1. Experimental method One mL of cell culture medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH)) was added to a 35 mm dish (Corning), and COS-7 cells (ATCC) were cultured in the culture medium in a CO2 incubator (37°C, 5% CO2). After 23 hours of culture, the medium was removed and replaced with 1.5 mL of luminescence measurement medium (DMEM (Dulbecco's Modified Eagle Medium (High Glucose) with L-Glutamine and Phenol-Red; Nacalai tesque), 10% heat-inactivated FBS (Fetal Bovine Serum; SIGMA-ALDRICH), 2.5% HEPES (1 M) (Thermo Fisher Scientific), 0.5% Endurazine (Promega)). Thirty minutes after medium change, 0.75 μg of ThermoCre1.3 or 0.5 μg of Cre were added to the cultures, along with 0.25 μg of the reporter gene (loxP-NLuc or Nluc) and, if necessary, the empty vector pcDNA4 V5_His ver. B (Thermo Fisher Scientific) to a total volume of 1 μg. The cultures were then mixed with 3 μL of PEI MAX (Polysciences) and 200 μL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific), and incubated for 30 minutes before being added to the COS-7 cell culture medium. Immediately after addition, luminescence intensity measurements were initiated using an AB-2550 Kronos Dio (ATTO) luminescence analyzer. Measurements were repeated at 5-minute intervals, with 1-second readings per dish, at 37°C or 40°C, without CO2, over two days.

[0065] 11-2.Results The time course of luminescence intensity when cells transfected with each gene combination were cultured at 37°C and 40°C is shown (Fig. 15A), and the time course of fold change calculated by dividing the luminescence intensity at 40°C by the luminescence intensity at 37°C is shown (Fig. 15B). As a result, with the integrated ThermoCre1.3, the fold change peaked after 14 hours and then fluctuated between 7-17 fold, indicating that the heat responsiveness of DNA recombination ability is sustained over a long period of time. [Industrial Applicability]

[0066] The present invention provides a protein set that allows precise control of the binding and dissociation of substances (such as proteins) depending on temperature changes. The protein set of the present invention, once introduced into a living body, can be heated from outside the body using ultrasound or other methods to control its function, making it suitable for use in the treatment of diseases and other conditions, and is expected to be used in the medical field and other fields. Furthermore, the protein set can be used as a research and development resource for genetically modified animals, and because temperature changes are used to control the protein set, it can also be used industrially in plants and microorganisms, which have previously been difficult to apply optogenetics to.

Claims

1. A protein set for inducing a temperature-dependent interaction of a substance, comprising: A protein set including the following protein a having a binding-inducing peptide bound to the N-terminus or C-terminus of the following protein a, the following protein b, and a binding-inducing protein, or a protein set including the following protein b having a binding-inducing peptide bound to the N-terminus or C-terminus thereof, the following protein a, and the following binding-inducing protein; a: a protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having mutations represented by E180R and E250R; b: A protein comprising all or part of the coiled-coil region of a protein having the amino acid sequence represented by SEQ ID NO: 1 and having the mutations represented by R179E and R251E.

2. the protein a is a protein having mutations represented by E180R and E250R in the amino acid sequence represented by SEQ ID NO: 1; the protein b is a protein having mutations represented by R179E and R251E in the amino acid sequence represented by SEQ ID NO: 1; the binding-inducing protein is Stringent starvation protein B (SspB), The binding-inducing peptide is an SsrA peptide, The protein set according to claim 1, characterized in that the SsrA peptide is bound to the C-terminus of protein a or protein b, which has Z amino acids deleted from its C-terminus (where Z is an integer between 0 and 7).

3. The set of proteins according to claim 2, wherein the protein a further has a mutation represented by Δ1-X, where X is 2 or more and 93 or less, and the protein b further has a mutation represented by Δ1-Y, where Y is 2 or more and 93 or less.

4. The set of proteins according to claim 3, wherein X and Y are 65.

5. The protein set according to any one of claims 1 to 4, wherein, among the proteins constituting the protein set, a protein having a binding-inducing peptide bound to protein a or a protein having a binding-inducing peptide bound to protein b, and the binding-inducing protein are each bound to or fused with another protein, and the other proteins interact with each other.

6. The set of proteins according to claim 5 , wherein the additional protein is a site-specific recombinase or a fragment of Cas9 nuclease.

7. A method for controlling the distance between two proteins, comprising: A step of binding or fusing a protein having a binding-inducing peptide bound to protein a or a protein having a binding-inducing peptide bound to protein b, which are included in the protein set according to any one of claims 1 to 3, to one of the two proteins; A step of binding or fusing a binding-inducing protein included in the set of proteins according to any one of claims 1 to 3 to the other of the two proteins; and heating the protein set.

8. 8. The method of claim 7, wherein the additional protein is a site-specific recombinase or a split fragment of a Cas9 nuclease.

9. The method according to claim 7, wherein the protein set is heated by irradiating it with ultrasound or radio waves.

10. A kit for controlling the interaction of substances within a cell, the kit comprising nucleic acids encoding each protein included in the protein set described in any one of claims 1 to 4.

Images