Photoswitch protein functioning under irradiation of long-wavelength light

Abstract

The present invention addresses the problem of providing an improved RpBphP1 mutant and an improved RpPpsR2 mutant in such a manner that the binding and dissociation can be reliably controlled by light irradiation and shielding of near-infrared light. More specifically, the present invention is a proteome comprising two types of proteins that bind to each other under irradiation of near-infrared light, the proteome being a group of RpBphP1 mutants and wild-type RpPpsR2, a group of wild-type RpBphP1 mutants and RpPpsR2 mutants, or a group of RpBphP1 mutants and RpPpsR2 mutants, the binding of the proteome being greater than that of the wild-type RpBphP1 mutants and that of the wild-type RpPpsR2, and the binding of the proteome being less than that of the wild-type RpBphP1 mutants and that of the wild-type RpPpsR2. The binding in a dark place is weak and / or the binding under the irradiation of near-infrared light is strong.

Description

Technical Field

[0001] The present invention relates to a protein group whose binding and dissociation can be controlled under light irradiation. Background Art

[0002] Proteins, as one of the biological substances, play various functions in living organisms. For example, proteins interact with each other to participate in the control of systems that form the basis of life, such as signal transmission and material transport within cells. In recent years, with the development of optogenetics, technologies have been developed to control the interaction between proteins in living organisms through light manipulation, such as photoswitchable proteins, which are now attracting much attention.

[0003] Photoswitch proteins refer to protein pairs whose binding and dissociation can be controlled by turning on / off light irradiation (Patent Document 1). Various phenomena within cells, such as signal transduction, genome editing, gene expression, etc., can be regulated by light manipulation through the binding / dissociation of photoswitch proteins (Non-Patent Document 1 and Non-Patent Document 2). In addition, photoswitch proteins that can be controlled by blue light (below 470nm) are currently widely used in photomanipulation. However, since blue light can be absorbed by hemoglobin and the like, the transmittance of biological tissues is low, and photoswitch proteins that can be manipulated by blue light cannot fully exert their effects in the use of photomanipulation in organisms. Therefore, it is desirable to use photoswitch proteins that are not absorbed by hemoglobin, have high transmittance to biological tissues, and can be manipulated by near-infrared light. At this time, a research team reported that Rhodopseudomonas palustris ( Rhodopseudomonas palustris ) can be used as a photoswitch protein by irradiating with near-infrared light (Non-patent Documents 3 and 4).

[0004] Since the binding and dissociation of RpBphP1 and RpPpsR2 can be controlled by near-infrared light with high transmittance to biological tissues, and biliverdin, a cofactor required for RpBphP1 to receive light, is widely present in the cells of various animals including mammals, it is expected to be used in the medical field. However, since the wild-type RpBphP1 and RpPpsR2 protein pair can bind even in the dark, that is, when not irradiated with light, there is a problem that it is difficult to reliably control the binding and dissociation by light irradiation.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2015-165776

[0008] Non-patent literature

[0009] Non-patent literature 1: Kawano et al., Nature Chemical Biology 12: 1059-1064 2016.

[0010] Non-patent literature 2: Kawano et al., Nature Communications 6:6256 2015.

[0011] Non-patent literature 3: Kaberniuk et al., Nature Methods 13: 591-597 2016.

[0012] Non-patent document 4: Redchuk et al., Nature Chemical Biology 13:633-639 2017. Summary of the invention

[0013] In view of the above circumstances, an object of the present invention is to provide RpBphP1 mutants and RpPpsR2 mutants improved so that binding and dissociation can be reliably controlled by irradiation and shielding of near-infrared light.

[0014] The inventors prepared various mutants of RpBphP1 and RpPpsR2 and succeeded in finding a combination of a mutant and a wild type, or a combination of mutants, that can reliably control binding and dissociation by turning on / off near-infrared light irradiation.

[0015] That is, the present invention is the following (1) to (13).

[0016] (1) A proteome consisting of two proteins that bind under near-infrared light irradiation,

[0017] The protein group is a group of RpBphP1 mutant and wild-type RpPpsR2, a group of wild-type RpBphP1 and RpPpsR2 mutant, or a group of RpBphP1 mutant and RpPpsR2 mutant. Compared with the binding of wild-type RpBphP1 and wild-type RpPpsR2, the binding of the protein group is weaker in the dark and / or stronger under near-infrared light irradiation.

[0018] (2) The protein group according to (1) above, wherein the wild-type RpBphP1 is a protein composed of the amino acid sequence represented by sequence number 1 or sequence number 2, or a protein having a sequence homology of more than 90% with the amino acid sequence represented by sequence number 1 or sequence number 2, and the wild-type RpPpsR2 is a protein composed of the amino acid sequence represented by sequence number 3, or a protein having a sequence homology of more than 90% with the amino acid sequence represented by sequence number 3.

[0019] (3) The proteome according to (2) above, wherein the proteome is a combination of wild-type RpPpsR2 and RpBphP1 mutant,

[0020] The RpBphP1 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2,

[0021] The point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, It is composed of E513P, E516A, F518A, S525A, D607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, and Q521A.

[0022] (4) The proteome according to (2) above, wherein the proteome is a group of wild-type RpBphP1 and RpPpsR2 mutant,

[0023] The RpPpsR2 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 3,

[0024] The point mutation group consists of Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, and V146A.

[0025] (5) The proteome according to (2) above, wherein the proteome is a group of wild-type RpBphP1 and RpPpsR2 mutant,

[0026] The RpPpsR2 mutant is modified with the amino acid sequence represented by SEQ ID NO: 3 to be D1-D1-D2, D3-D1-D2, D1-D2 (del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2 (L137E), D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2 (del N 88), D1-D2 (del N 89), D1-D2 (del N 90), D1-D2 (del N 91), 91), D1-D2(del N 92), D1-D2(del N 93), D1-D2(del N94), D1-D2(del N 95), D1-D2(del N 96), D1-D2(del N 97), D1-D2(del C 5), D1-D2(del C 15), D1-D2(del N 93)(del C 5), D1-D2(del N 93)(del C 6), D1-D2(del N 93)(del C 11), D1-D2(del N 93)(del C12), D1-D2-D3(del C 25), D1-D2-D3(del C 41), D1-D2-D3(del C 49), D1-D2-D3(del C 70), D1-D2-D3(del C 87), D1-D2-D3(del C 96), D1-D2-D3(del C 104), D1-D2(del N 64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15), D1-D2(del N 33), D1-D2 (del N 47) or D1-D2-D3 (del C 9).

[0027] (6) The proteome according to (2) above, wherein the proteome is a group of RpBphP1 mutant and RpPpsR2 mutant,

[0028] The RpBphP1 mutant is any one of mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C , Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D 607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D60 7Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A;

[0029] The RpPpsR2 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 3, wherein the point mutation group consists of Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A , L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, and V146A.

[0030] (7) The proteome according to (2) above, wherein the proteome is a group of RpBphP1 mutant and RpPpsR2 mutant,

[0031] The RpBphP1 mutant is any one of mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C , Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D 607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D60 7Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A;

[0032] For RpPpsR2, the same sequence as D1-D1-D2 and D3-D1-D2(from D1-D2) 77)、D1-D2、D2-D1、D2-D2、D3-D2、D3-D2(L137E)、D4-D2、D1-D2-D1、D1-D2-D2、D1-D2-D4-D1、D2、D2 -D2、D2-D3-D4、D4-D1-D2、D2-D3-D4-D1、D2-D3-D4-D2、D2-D3-D4-D3、D2-D3-D4-D5、D1-D2(del N 88)、D1-D2(of N 89)、D1-D2(of N 90)、D1-D2(of N 91)、D1-D2(of N 92)、D1-D2(of N 93)、D1-D2(of N91-D)、 95)、D1-D2(of N 96)、D1-D2(of N 97)、D1-D2(of C 5)、D1-D2(of C 15)、D1-D2(of N 93)(of C 5)、D1-D2)(of N 93)、of N 93)(from C 11)、D1-D2(from N 93)(from C12)、D1-D2-D3(from C 25)、D1-D2-D3(from C 41)、D1-D2-D3(from C 49)、D10(D7)-D2-D3 87)、D1-D2-D3(of C 96)、D1-D2-D3(of C 104)、D1-D2(of N 64)、D2-D3、D1-D2-D3、D2-D2-D3、D2-D3-D1、D2-D3-D3、D3 -D2-D3、D4-D2-D3、D1-D1-D2-D3、D1-D2-D3-D1、D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D3-4D2、D3-D2-D2 N 15)、D1-D2 (of N 33)、D1-D2 (of N 47)or D1-D2-D3 (of C 9)

[0033] (8) An RpBphP1 mutant, comprising an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutations are Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P , F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D607A, D60 7G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D6 07F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E 693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A.

[0034] (9) An RpPpsR2 mutant, comprising an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 3, wherein the point mutations are Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115 A. Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R1 34A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, V146A.

[0035] (10) A kind of RpPpsR2 mutant, the amino acid sequence shown by sequence number 3 is modified to D1-D1-D2, D3-D1-D2, D1-D2 (del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2(L137E), D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1 -D2、D2-D3-D4、D4-D1-D2、D2-D3-D4-D1、D2-D3-D4-D2、D2-D3-D4-D3、D2-D3-D4-D5、D1-D2 N 88), D1-D2(del N 89), D1-D2(del N 90), D1-D2(del N91), D1-D2(del N 92), D1-D2(del N93), D1-D2(del N 94), D1-D2(del N 95), D1-D2(delN 96), D1-D2(del N 97), D1-D2(del C 5), D1-D2(del C 15), D1-D2(del N 93)(del C5), D1-D2(del N 93)(del C 6), D1-D2(del N 93)(del C 11), D1-D2(del N 93)(del C12), D1-D2-D3(del C 25), D1-D2-D3(del C 41), D1-D2-D3 (del C 49), D1-D2-D3(del C70), D1-D2-D3(del C 87), D1-D2-D3(del C 96), D1-D2-D3(del C 104), D1-D2(del N64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2 N 15), D1-D2(del N 33), D1-D2(del N47), D1-D2-D3(del C 9) A mutant consisting of an amino acid sequence of any of the above.

[0036] (11) A nucleic acid encoding the RpBphP1 mutant described in (8) above.

[0037] (12) A nucleic acid encoding any one of the RpPpsR2 mutants described in (9) above.

[0038] (13) A nucleic acid encoding any one of the RpPpsR2 mutants described in (10) above.

[0039] In addition, the symbol "to" in this specification represents a numerical range including the values ​​on both sides thereof.

[0040] The photoswitch protein of the present invention (i.e., a combination (group) of RpBphP1 mutant and wild-type RpPpsR2, a combination (group) of wild-type RpBphP1 and RpPpsR2 mutant, or a combination (group) of RpBphP1 mutant and RpPpsR2 mutant) can reliably control its binding and dissociation by irradiation and shielding of near-infrared light. Therefore, the photoswitch protein of the present invention can also reliably control its binding and dissociation by near-infrared light in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the confirmation of the function of the photoswitch protein composed of wild-type RpBphP1 and wild-type RpPpsR2. (a) is a schematic diagram of CPTS (CRISPR-Cas9-based light-activated transcription system). (b) is the result of confirming the binding state of wild-type RpBphP1 and wild-type RpPpsR2 in the dark and under near-infrared light irradiation.

[0042] Figure 2 Functional analysis of photoswitch proteins composed of RpBphP1 mutants and wild-type RpPpsR2 (Part 1). Results of analysis of the binding states of RpBphP1 into which point mutations were introduced and wild-type RpPpsR2 in the dark and under near-infrared light irradiation are shown (a to c).

[0043] Figure 3 Functional analysis of photoswitch proteins composed of RpBphP1 mutants and wild-type RpPpsR2 (Part 2). Results of analysis of the binding states of RpBphP1 into which point mutations were introduced and wild-type RpPpsR2 in the dark and under near-infrared light irradiation (a and b) are shown.

[0044] Figure 4 Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 mutants (Part 1). Results of analysis of the binding state between RpPpsR2 into which point mutations were introduced and wild-type RpBphP1 in the dark and under near-infrared light irradiation are shown (a and b).

[0045] Figure 5Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 mutants (Part 2) The results of analyzing the binding states of RpPpsR2 into which point mutations were introduced and wild-type RpBphP1 in the dark and under near-infrared light irradiation are shown (a to c).

[0046] Figure 6 Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 domain variants (Part 1). Results of analysis of the binding states of wild-type RpBphP1 and RpPpsR2 domain variants in the dark and under near-infrared light irradiation are shown (a and b).

[0047] Figure 7 Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 domain variants (Part 2) The results of analyzing the binding states of wild-type RpBphP1 and RpPpsR2 domain variants in the dark and under near-infrared light irradiation are shown.

[0048] Figure 8 Functional analysis of a photoswitch protein composed of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain (part 1). The amino acid sequence shown in a (sequence number 6) is the amino acid sequence of the D1-D2 domain of RpPpsR2. D1-D2 (del N 77), D1-D2 (del N 93) and D1-D2 (del C 15) represent the amino acid sequences of D1-D2 variants having 77 amino acid deletions on the N-terminal side, 93 amino acid deletions on the N-terminal side and 15 amino acid deletions on the C-terminal side, respectively. b shows the results of analyzing the binding state of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain under dark conditions and near-infrared light irradiation.

[0049] Fig. 9 Functional analysis of a photoswitch protein composed of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain (part 2). The amino acid sequence shown in a (sequence number 6) is the amino acid sequence of the D1-D2 domain of RpPpsR2. D1-D2 (del N 47) and D1-D2 (del N 64) represent the amino acid sequences of D1-D2 variants having 47 amino acid deletions on the N-terminal side and 64 amino acid deletions on the N-terminal side, respectively. b shows the results of analyzing the binding state of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain under dark conditions and near-infrared light irradiation.

[0050] Fig.10Functional analysis of a photoswitch protein composed of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain (part 3). The amino acid sequence shown in a (sequence number 6) is the amino acid sequence of the D1-D2 domain of RpPpsR2. D1-D2 (del N 88), D1-D2 (del N 93) and D1-D2 (del N 97) represent the amino acid sequences of D1-D2 variants having 88 amino acid deletions on the N-terminal side, 93 amino acid deletions on the N-terminal side and 97 amino acid deletions on the N-terminal side, respectively. b shows the results of analyzing the binding state of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain under dark conditions and near-infrared light irradiation.

[0051] Fig.11 Functional analysis of a photoswitch protein composed of wild-type RpBphP1 and a RpPpsR2 domain variant containing a deletion in the domain (part 4). The amino acid sequence shown in a (sequence number 6) is the amino acid sequence of the D1-D2 domain of RpPpsR2. D1-D2 (del N 93), D1-D2 (del N 93) (del C 5) and D1-D2 (del N 93) (del C 12) respectively represent the amino acid sequences of D1-D2 variants having 93 amino acid deletions on the N-terminal side, 93 amino acid deletions on the N-terminal side and 5 amino acid deletions on the C-terminal side, and 93 amino acid deletions on the N-terminal side and 12 amino acid deletions on the C-terminal side. b shows the results of analyzing the binding state of wild-type RpBphP1 and RpPpsR2 domain variants containing a deletion in the domain under dark and near-infrared light irradiation.

[0052] Fig.12 This is a combination in which multiple mutations and modifications (mutations and modifications that reduce dark leak signals and mutations and modifications that increase NIR signals) having beneficial effects in RpBphP1 and RpPpsR2 are accumulated to improve the functions. A total of 12 patterns are shown.

[0053] Fig.13 The photo-switch protein is composed of a RpBphP1 mutant with multiple beneficial point mutations and wild-type RpPpsR2, and the photo-switch protein is composed of a RpPpsR2 mutant with multiple beneficial point mutations and wild-type RpBphP1. (a) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12The results of analyzing the binding state of the RpBphP1 mutant (with Q502A and E513A introduced) and wild-type RpPpsR2 in the dark and under near-infrared light irradiation are shown in (b). The binding pattern of RpBphP1 and RpPpsR2 is Fig.12 The results of analyzing the binding state of wild-type RpBphP1 and RpPpsR2 mutant (with Q105T and L137E introduced) in the dark and under near-infrared light irradiation are shown. *1 indicates a point mutation that reduces the dark leakage signal. *2 indicates a point mutation that increases the NIR signal.

[0054] Fig.14 The functional analysis of the photoswitch protein composed of RpBphP1 and RpPpsR2 mutants with beneficial domain modification and point mutations. Fig.12 The results of analyzing the binding state of wild-type RpBphP1 and RpPpsR2 mutants with domain modification (D3-D2) and point mutation (introduction of L137E) under dark and near-infrared light irradiation are shown. Fig.13 The situation in b is the same, but the difference is that domain modification and point mutation were introduced into RpPpsR2. *2 indicates a point mutation that increases the NIR signal. *3 indicates a domain variant that reduces the dark leakage signal.

[0055] Fig.15 This is the functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 1). (a) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The results of analyzing the binding state of RpBphP1 mutants (introduced with Q367A or F518A) and RpPpsR2 domain variants (D3-D1-D2) in the dark and under near-infrared light irradiation are shown. (b) is the combination pattern of RpBphP1 and RpPpsR2. Fig.12 An example of combination 3. The results of analyzing the binding state of the RpBphP1 mutant (introduced D607E) and the RpPpsR2 mutant (introduced Q105S) in the dark and under near-infrared light irradiation are shown. *1 indicates a point mutation that reduces the dark leakage signal. *2 indicates a point mutation that increases the NIR signal. *3 indicates a domain variant that reduces the dark leakage signal.

[0056] Fig.16 This is the functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 2). (a) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The results of analyzing the binding state of RpBphP1 mutant (introduced with Q502A and E513A) and RpPpsR2 mutant (introduced with Q105S) in the dark and under near-infrared light irradiation are shown. (b) is the combination pattern of RpBphP1 and RpPpsR2. Fig.12 The results of analyzing the binding state of the RpBphP1 mutant (introduced with Q502A and E513A) and the RpPpsR2 mutant (Q105T) in the dark and under near-infrared light irradiation are shown. *1 indicates a point mutation that reduces the dark leakage signal. *2 indicates a point mutation that increases the NIR signal.

[0057] Fig.17 This is the functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 3). (a) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The example of combination 3 shows the results of analyzing the binding state of RpBphP1 mutant (introduced with D607E) and RpPpsR2 mutant (introduced with Q105S and L137E) in the dark and under near-infrared light irradiation. Fig.15 The situation in (b) is the same as that in (b), but two point mutations are introduced into RpPpsR2. (b) is the combination pattern of RpBphP1 and RpPpsR2. Fig.12 The results of analyzing the binding state of the RpBphP1 mutant (with D607E introduced) and the RpPpsR2 mutant (with Q105T and L137E introduced) in the dark and under near-infrared light irradiation are shown. *1 indicates a point mutation that reduces the dark leakage signal. *2 indicates a point mutation that increases the NIR signal.

[0058] Fig.18 This is the functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 4). (a) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The example of combination 4 shows the results of analyzing the binding state of RpBphP1 mutant (introduced with Q502A and E513A) and RpPpsR2 mutant (introduced with Q105S and L137E) in the dark and under near-infrared light irradiation. Fig.16 The situation in (a) is the same, but two point mutations have been introduced into RpPpsR2. (b) The combination pattern of RpBphP1 and RpPpsR2 is Fig.12The results of analyzing the binding state of the RpBphP1 mutant (introduced with Q502A and E513A) and the RpPpsR2 mutant (introduced with Q105T and L137E) in the dark and under near-infrared light irradiation are shown. *1 indicates a point mutation that reduces the dark leakage signal. *2 indicates a point mutation that increases the NIR signal.

[0059] Fig.19 This is a functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 5). The combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The results of analyzing the binding state of RpBphP1 mutants (with D609E, S525A or E327A introduced) and RpPpsR2 mutants with domain modification (D3-D2) and point mutation (with L137E introduced) in the dark and under near-infrared light irradiation are shown. Fig.17 The situation in b is the same, but the difference is that domain modification and point mutation were introduced into RpPpsR2. *1 indicates a point mutation that reduces dark leakage signal. *2 indicates a point mutation that increases NIR signal. *3 indicates a domain variant that reduces dark leakage signal.

[0060] Fig. 20 This is the functional analysis of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 (Part 6). This is the combination pattern of RpBphP1 and RpPpsR2 is Fig.12 The results of analyzing the binding state of the RpBphP1 mutant (introduced with Q502A and E513A) and the RpPpsR2 mutant with both domain modification (D3-D2) and point mutation (introduced with L137E) in the dark and under near-infrared light irradiation are shown. Fig.18 The situation in b is the same, but the difference is that domain modification and point mutation were introduced into RpPpsR2. *1 indicates a point mutation that reduces dark leakage signal. *2 indicates a point mutation that increases NIR signal. *3 indicates a domain variant that reduces dark leakage signal.

[0061] Fig.21 The confirmation of genomic gene activation in cells is achieved by the light switch protein of the present invention. ASCL1 , HBG1 , MYOD1 or IL1R2) as the target guide RNA and the essential component genes such as the light switch protein gene were introduced into HEK293T cells, and near-infrared light was irradiated to investigate the expression level of each genomic gene. a shows the expression level of each genomic gene in the dark and under near-infrared light irradiation. b shows the expression level of each genomic gene after near-infrared light irradiation. ASCL1 Time-dependent changes in mRNA expression. The enlarged image within 3 hours of near-infrared light irradiation is shown in the box. c shows the changes in mRNA expression due to the ON or OFF of near-infrared light irradiation. ASCL1 The results show how mRNA expression is affected.

[0062] Fig. 22 The light switch protein of the present invention is used to confirm gene activation in vivo in mice. ASCL1 The essential component genes such as the target gene) and the photoswitch protein gene were introduced into the mouse liver, and the mice were irradiated with light from outside the body using an LED panel (760nm, 300W / m 2 ). a shows the situation of near-infrared light irradiation experiment. b shows the analysis results using luminescent reporter gene. c (left) shows the results of the analysis of ASCL1 The liver removed from a mouse to determine the gene expression level. (Right) shows the expression of the gene in the liver of a mouse in the dark and under near-infrared light. ASCL1 The results of investigating the expression level of genes. DETAILED DESCRIPTION

[0063] Hereinafter, the mode for carrying out the present invention will be described.

[0064] The first embodiment is a proteome composed of two proteins that bind under near-infrared light irradiation (also described as "the proteome of the present embodiment" or "the photoprotein of the present embodiment").

[0065] The protein group is a group of RpBphP1 mutant and wild-type RpPpsR2, a group of wild-type RpBphP1 and RpPpsR2 mutant, or a group of RpBphP1 mutant and RpPpsR2 mutant.

[0066] The binding of this proteome was weak in the dark and / or strong under near-infrared light illumination compared to the binding of wild-type RpBphP1 to wild-type RpPpsR2.

[0067] As described above, since the wild-type RpBphP1 and RpPpsR2 protein pair binds to a certain extent even in the dark, that is, without light irradiation, it is difficult to reliably control the binding and dissociation of RpBphP1 and RpPpsR2 by turning on / off light irradiation.

[0068] Therefore, first, the present inventors attempted to produce a protein pair whose binding was weaker in the dark than the binding of wild-type RpBphP1 to wild-type RpPpsR2 and / or a protein pair whose binding was stronger under near-infrared light irradiation. Specifically, the inventors attempted to produce mutants whose binding of RpBphP1 mutant to wild-type RpPpsR2, binding of wild-type RpBphP1 to RpPpsR2 mutant, and binding of RpBphP1 mutant to RpPpsR2 mutant satisfied the following conditions 1 and / or 2.

[0069] 1. Weak in a state where near-infrared light is not irradiated (hereinafter also referred to as "in a dark place").

[0070] 2. Strong under near-infrared light.

[0071] Here, "near infrared light" refers to light having a wavelength of approximately 650 nm to 900 nm.

[0072] In addition, the binding of the protein group of the present embodiment is "weak" compared to the binding of the wild-type RpBphP1 and the wild-type RpPpsR2, which means that the binding strength evaluated in the reporter gene analysis described later is less than 0.6 times, preferably less than 0.4 times, and more preferably less than 0.2 times the binding strength of the wild-type RpBphP1 and the wild-type RpPpsR2. In addition, the binding of the protein group of the present embodiment is "strong" compared to the binding of the wild-type RpBphP1 and the wild-type RpPpsR2, which means that the binding strength of the wild-type RpBphP1 and the wild-type RpPpsR2 is more than 1.1 times, preferably more than 1.5 times.

[0073] As described above, in the case of the pair of wild-type RpBphP1 and wild-type RpPpsR2 proteins, the binding strength under near-infrared light irradiation is at most about 2 times relative to the binding strength in the dark. With this degree of change in binding strength, it is difficult to accurately control binding and dissociation by turning on / off near-infrared light irradiation. The difference in binding strength between the protein group of the present embodiment and the wild-type when paired with each other is greater, and its binding and dissociation can be more accurately controlled. Specifically, with respect to the protein group of the present embodiment, the binding strength under near-infrared light irradiation is preferably about 3 times or more, more preferably about 5 times or more, and further preferably about 7 times or more relative to the binding strength in the dark.

[0074] It should be noted that, in this specification, “about” refers to a numerical range of ±10%.

[0075] In the present embodiment, the wild-type RpBphP1 is a protein consisting of the amino acid sequence represented by the following sequence number 1 or sequence number 2, or a protein substantially identical to the protein consisting of the amino acid sequence represented by the sequence number 1 or sequence number 2. Here, "the protein substantially identical to the protein consisting of the amino acid sequence represented by the sequence number 1 or sequence number 2" refers to a protein consisting of an amino acid sequence having a sequence homology of 80% or more, 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 to the amino acid sequence represented by the sequence number 1 or sequence number 2. However, the RpBphP1 mutant of the present embodiment is not included.

[0076] Sequence No. 1:

[0077] MVAGHASGSPAFGTADLSNCEREEIHLAGSIQPHGALLVVSEPDHRIIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEYDGLMHRPPEGGLIIELERAGPPIDLSGTLAPALERIRTAGSLRALCDDTALLFQQCTGYDRVMVYRFDEQGHGEVFS ERHVPGLESYFGNRYPSSDIPQMARRLYERQRVRVLVDVSYQPVPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQYLKNMGVRATLVVSLVVGGKLWGLVACHHYLPRFIHFELRAICELLAEAIATRITALESFAQSQSELFVQRLEQRMIEAITREGDWRAAIFDTSQSILQPLHADGCALV YEDQIRTIGDVPSTQDVREIAGWLDRQPRAAVTSTASLGLDVPELAHLTRMASGVVAAPISDHRGEFLMWFRPERVHTTVWGDPKKPFTMGDTPADLSPRRSFAKWHQVVEGTSDPWTAADLAAARTIGQTVADIVLQFRAVRTLIAREQYEQFSSQVHASMQPVLITDAEGRILLMNDSF RDMLPGSPSAVHLDDLAGFFVESNDFLRNVAELIDHGRGWRGEVLLRGAGNRPLPLAVRADPVTRTEDQSLGFVLIFSDATDRRTADAARTRFLEGILASARPGVRLDSKSDLLHEKLLSALVENAQLAALEITYGVETGRIAELLEGVRQSMLRTAEVLGHLVQHAARTAGSDSSSNGSQN

[0078] serial number 2:

[0079] MAGHASGSPAFGTADLSNCEREEIHLAGSIQPHGALLVVSEPDHRIIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEYDGLMHRPPEGGLIIELERAGPPIDLSGTLAPALERIRTAGSLRALCDDTALLFQQCTGYDRVMVYRFDEQGHGEVFSERHVPGLESYFGNRYPSSDIPQMARRLYERQRVRVLVDVSYQVPPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQYLKNMGVRATLVVSLVVGGKLWGLVACHHYLPRFIHFELRAICELLAEIATRITALESFAQSQSELFVQRLEQRMIEAITREGDWRAAIFDTSQSILQPLHADGCALVY EDQIRTIDGDVPSTQDVREIAGWLDRQPRAAVTSTASLGLDVPELAHLTRMASGVVAAPISDHRGEFLMWFRPERVHTTVWGDPKKPFTMGDTPADLSPRRSFAKWHQVVEGTSDPWTAADLAAARTIGQTVADIVLQFRAVRTLIAREQYEQFSSQVHASMQPVLITDAEGRILLMNDSFRD MLPAGSPSAVHLDDLAGFFVESNDFLRNVAELIDHGRGWRGEVLLRGAGNRPLPLAVRADPVTRTEDQSLGFVLIFSDATDRRTADAARTRFQEGILASARPGVRLDSKSDLLHEKLLSALVENAQLAALEITYGVETGRIAELLEGVRQSMLRTAEVLGHLVQHAARTAGSDSSSNGSQNKK

[0080] It should be noted that the amino acid sequence of sequence number 2 is a sequence registered in the NCBI database with accession number WP_119846077. The amino acid sequence represented by sequence number 1 is the sequence used as the amino acid sequence of wild-type RpBphP1 in this example, and V is inserted between the first M and the next A of the amino acid sequence represented by sequence number 2, and the Q at position 641 of the amino acid sequence represented by sequence number 2 is changed to L. These mutations are performed in order to increase the expression rate of wild-type RpBphP1, etc., and the amino acid sequence of sequence number 1 and the amino acid sequence of sequence number 2 have a sequence homology of more than 99%, and it has been confirmed that the activity as RpBphP1 is also the same.

[0081] In addition, in this specification, the mutant of RpBphP1 is described by using the 3rd position A in SEQ ID NO: 1 (the 2nd position A in SEQ ID NO: 2) as amino acid number 1.

[0082] In the present embodiment, the wild-type RpPpsR2 is a protein consisting of the amino acid sequence represented by the sequence number 3 shown below, or a protein substantially identical to the protein consisting of the amino acid sequence represented by the sequence number 3. Here, "a protein substantially identical to the protein consisting of the amino acid sequence represented by the sequence number 3" refers to a protein consisting of an amino acid sequence having a sequence homology of more than 80% with the amino acid sequence represented by the sequence number 3, and more preferably a protein consisting of an amino acid sequence having a sequence homology of more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%. Among them, the RpPpsR2 mutant of the present embodiment is not included.

[0083] Sequence number 3:

[0084] MASKSVHADITLLLDMEGVIREATLSPTMAAAESVDGWLGRRWSDIAGAEGGDKVRRMVEDARRSGISAFRQINQPFPSGVEIPIEFTTMLLGDRTGMIAVGKNMQAVTELHSRLIA AQQAMERDYWRLRELETRYRLVFDAAADAVMIVSAGDMRIVEANRAAVNAISRVERGNDDLAGRDFLAEVAAADRDAVRDMLAQVRQRGTALSVLVHLGRYDRAWMLRGSLMSSER RQVFLLHFTPVTTTPAIDDVDDDAVLRGLIDRIPDGFVALDSEGVVRHANQAFLDLVQIGSKPAAVGRSLGVWMGRPGADLSSLLTLLRRYKTVRLFQTTIRGELGTETEVEVSAV DGEDDQYIGVLMRNVARRLDAADDHDALRQALGPISKQLGRSSLRKLVKNAVSIVEQHYVKEALLRSKGNRTATAELLGLSRQSLYAKLNSYGFDDKGVVASAADGAEGASDDAED

[0085] In the present embodiment, when a specific amino acid in an amino acid sequence is represented, the "specific amino acid (single-letter expression)" and "amino acid position number" in the amino acid sequence are recorded in sequence. For example, the glutamine at position 514 in the amino acid sequence is marked as Q514. In addition, the description of the point mutation in the present embodiment records the "amino acid", "amino acid position number" and "substituted amino acid" in the wild-type sequence in sequence. For example, the mutation in which the amino acid at position 514 of the wild-type RpBphP1 is substituted from Q to A is marked as Q514A, and a mutant having the mutation is recorded as an RpBphP1 mutant having a point mutation of Q514A, etc. Similarly, the mutation in which the amino acid at position 105 of the wild-type RpPpsR2 is substituted from Q to T is marked as Q105T, and a mutant having the mutation is recorded as an RpPpsR2 mutant having a point mutation of Q105T, etc. In the case of a mutant having multiple point mutations, for example, it is described as an RpBphP1 mutant having point mutations Q514A and D607E (a mutant in which Q at position 514 is substituted with A and D at position 607 is substituted with E).

[0086] For RpPpsR2 mutants, in addition to point mutants, mutants that replace the positions of the five domains constituting the wild-type RpPpsR2, mutants that partially or completely delete one or more of the five domains, and mutants that replace the positions of the five domains and partially or completely delete one or more of the five domains are also included in the present embodiment. Mutants involving these domains are also recorded as "domain variants". In this specification, the positions of the five domains of RpPpsR2 are defined as follows:

[0087] In the amino acid sequence represented by SEQ ID NO: 3,

[0088] Domain 1 (also referred to as D1): A2 to N103

[0089] Domain 2 (also referred to as D2): Q105 to A141

[0090] Domain 3 (also referred to as D3): V146 to T241

[0091] Domain 4 (also referred to as D4): D253 to N362

[0092] Domain 5 (also referred to as D5): A369 to D464

[0093] Here, the amino acid sequence between the domains, such as amino acid residues or amino acid sequences such as M104, A142-A145, P242-V252, V363-D368 (also referred to as linkers, etc.) are positioned to connect the domains to each other. The amino acid or amino acid linker. However, in addition to the amino acid sequences of each domain determined above, the domain in the present embodiment may also have a number of amino acid residues (e.g., about 1 to 10 residues) added or deleted at its N-terminus and / or C-terminus that do not impair the function of each domain. Specifically, for example, in the case of a domain variant described as D2-D1-D4, the polypeptide (D2) of Q105-A141, the polypeptide (D1) of A2-N103, and the polypeptide (D4) of D253-N362 of the amino acid sequence represented by sequence number 3 may be sequentially configured from the N-terminal side, and D2, D1 and D4 may be connected by a polypeptide consisting of 1 to several amino acids. Furthermore, a polypeptide consisting of one to several amino acids may be added to the C-terminal side of D4 or the N-terminal side of D2.

[0094] When one or both of the domains constituting the domain variant are missing, for example, they are described as D1-D2-D3 (del C 25), D1-D2 (del N 93), D1-D2 (del N 93) (del C 5), etc. D1-D2-D3 (del C 25) indicates a domain variant in which each domain of D1, D2, and D3 is sequentially connected from the N-terminal side, but 25 amino acids are deleted from the C-terminal side of D1-D2-D3. In addition, D1-D2 (del N 93) indicates a domain variant in which each domain of D1 and D2 is sequentially connected from the N-terminal side, but 93 amino acids are deleted from the N-terminal side of D1-D2. In addition, D1-D2 (del N93) (del C 5) indicates a domain variant in which the domains D1 and D2 are linked in sequence from the N-terminal side, but 93 amino acids are deleted from the N-terminal side of D1-D2 and 5 amino acids are deleted from the C-terminal side. It should be noted that the above-mentioned domain variant labels indicate either the domain variant itself or the amino acid sequence of the domain variant.

[0095] In this specification, "amino acid" is used in the broadest sense, and includes, in addition to natural amino acids, derivatives thereof and synthetic amino acids. As amino acids in this specification, for example, in addition to natural amino acids, non-natural amino acids are also included. In addition, amino acids whose main chain structures are different from the natural type, amino acids whose side chain structures are different from the natural type, amino acids whose side chains have additional methylene groups, and amino acids whose carboxylic acid functional groups in the side chains are substituted by sulfonic acid groups are also included in "amino acids".

[0096] The protein group of the present embodiment includes a group of RpBphP1 mutant and wild-type RpPpsR2, a group of wild-type RpBphP1 and RpPpsR2 mutant, or a group of RpBphP1 mutant and RpPpsR2 mutant.

[0097] The RpBphP1 mutants of the present embodiment include mutants having one or more of the following point mutations in which amino acids are substituted with other amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, namely:

[0098] E327, E332, E337, D339, E365, Q367, E429, R490, D498, Q502, F503, R504, R507, R512, E513, Q514, E516, F518, Q521, S525, D607, E613, R636, R638, F639, D654, H661, E678, Y681, E693, R696, H712,

[0099] More specifically, the RpBphP1 mutant of the present embodiment includes a mutant having an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2:

[0100] Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502 I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E51 3P, E516A, F518A, S525A, D607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D 607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A.

[0101] The above point mutations can be classified into the following 3 groups:

[0102] (1) A mutation in which the binding of a RpBphP1 mutant having at least one of the above mutations to wild-type RpPpsR2 is weaker in the dark than that of wild-type RpBphP1 to wild-type RpPpsR2, but the binding is almost the same under near-infrared light irradiation, that is:

[0103] Q514A, D607E, R638A, H661A, R696A

[0104] (2) A mutation in which the binding of a RpBphP1 mutant having at least one of the above mutations to wild-type RpPpsR2 is weaker in the dark and also weaker under near-infrared light irradiation than the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0105] E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502 T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E5 13D, E513P, E516A, F518A, S525A, D607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P

[0106] (3) A mutation in which the binding of a RpBphP1 mutant having at least one of the above mutations to wild-type RpPpsR2 is stronger under near-infrared light irradiation and also stronger in the dark than the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0107] R512A, E513V, E513A, E513L, E513T, E513M, Q521A

[0108] The RpPpsR2 mutant of the present embodiment includes a mutant having an amino acid sequence having one or more of the following point mutations in which amino acids are substituted with other amino acids in the amino acid sequence represented by SEQ ID NO: 3, namely:

[0109] K102, N103, Q105, V107, T108, L110, R113, L114, I115, Q119, M121, R123, D1 24. Y125, W126, R127, R129, E132, T133, R134, R136, L137, V138, F139, V146.

[0110] More specifically, the RpPpsR2 mutant of the present embodiment includes a mutant having an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 3, namely:

[0111] Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, V146A.

[0112] The above point mutations can be classified into the following 4 groups:

[0113] (1) A mutation in which the binding of a RpPpsR2 mutant having at least one of the above mutations to wild-type RpBphP1 is weaker in the dark than that of wild-type RpBphP1 to wild-type RpPpsR2, but the binding is almost the same under near-infrared light irradiation, that is:

[0114] Q105T, T133A, L137A.

[0115] (2) A mutation in which the binding of a RpPpsR2 mutant having at least one of the above mutations to wild-type RpBphP1 is weaker in the dark and also weaker under near-infrared light irradiation than the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0116] N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M12 1A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A.

[0117] (3) A mutation in which the binding of a RpPpsR2 mutant having at least one of the above mutations to wild-type RpBphP1 is stronger under near-infrared light irradiation than that of wild-type RpBphP1 to wild-type RpPpsR2, and the binding in the dark is almost the same, that is:

[0118] Q105V, Q105S.

[0119] (4) A mutation in which the binding of a RpPpsR2 mutant having at least one of the above mutations to wild-type RpBphP1 is stronger under near-infrared light irradiation and also stronger in the dark than the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0120] K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, V146A.

[0121] In addition, the RpPpsR2 mutant of the present embodiment includes a mutant having an amino acid sequence in which the amino acid sequence represented by SEQ ID NO: 3 is modified into any one of the following domain modification sequences, namely:

[0122] <h2 style=";text-align:left;direction:ltr">D1-D1-D2, D3-D1-D2, D1-D2(from N 77), D1-D2, D2-D1, D2-D2, D3-D2, D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2(from N 88), D1-D2(from N 89), D1-D2(from N 90)、D1-D2(del N91)、D1-D2(del N 92)、D1-D2(del N 93)、D1-D2(del N 94)、D1-D2(del N 95)、D1-D2(del N 96)、D1-D2(del N 97)、D1-D2(del C 5)、D1-D2(del C 15)、D1-D2(del N 93)(del C 5)、D1-D2(del N 93)(del C6)、D1-D2(del N 93)(del C 11)、D1-D2(del N 93)(del C 12)、D1-D2-D3(del C 25) D1-D2-D3 (C 41) D1-D2-D3 (C 49) D1-D2-D3 (C 70) D1-D2-D3 (C 87) D1-D2-D3 (C 96) D1-D2-D3 (C 104) D1-D2 (N 64)、D2-D3、D1-D2-D3、D2-D2-D3、D2-D3-D1、D2-D3-D3、D3 -D2-D3、D4-D2-D3、D1-D1-D2-D3、D1-D2-D3-D1、D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15)、D1-D2(del N 33) D1-D2 (from N 47) D1-D2-D3 (from C 9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0123] <h2 style=";text-align:left;direction:ltr"> Description:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0124] (1) A mutation in which the binding of the domain variant of RpPpsR2 to wild-type RpBphP1 is weaker in the dark than that of wild-type RpBphP1 to wild-type RpPpsR2, but the binding is almost the same under near-infrared light irradiation, that is:

[0125] D1-D1-D2, D3-D1-D2, D1-D2(del N 77)

[0126] (2) A mutation in which the binding of the domain variant of RpPpsR2 to wild-type RpBphP1 is weaker in the dark and also weaker under near-infrared light irradiation than the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0127] D1-D2, D2-D1, D2-D2, D3-D2, D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D 3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2(del N88), D1-D2(del N 89), D1-D2(del N 90), D1-D2(del N 91), D1-D2(del N 92), D1-D2(del N 93), D1-D2(del N 94), D1-D2(del N 95), D1-D2(del N96), D1-D2(del N 97), D1-D2(del C 5), D1-D2(del C 15), D1-D2(del N 93)(del C 5), D1-D2(del N 93)(del C 6), D1-D2(del N 93)(del C11), D1-D2(del N 93)(del C 12), D1-D2-D3(del C 25), D1-D2-D3(del C 41), D1-D2-D3(del C 49), D1-D2-D3(del C 70), D1-D2-D3(del C 87), D1-D2-D3(del C 96), D1-D2-D3(del C 104)

[0128] (3) A mutation in which the binding of the domain variant of RpPpsR2 to wild-type RpBphP1 is stronger under near-infrared light irradiation and the binding in the dark is almost the same as that of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0129] D1-D2 (del N 64)

[0130] (4) A mutation in which the binding of the domain variant of RpPpsR2 to wild-type RpBphP1 is enhanced under near-infrared light irradiation and in the dark, compared with the binding of wild-type RpBphP1 to wild-type RpPpsR2, that is:

[0131] D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3-D 2. D1-D2-D3-D3, D1-D2-D3-D4, D2-D1-D2-D3, D2-D2-D3-D4, D2-D3-D4-D4, D3-D1-D2-D3, D3-D2-D3-D4, D1-D2(del N 15), D1-D2(del N 33), D1-D2(delN 47), D1-D2-D3(del C 9)

[0132] Furthermore, the RpPpsR2 mutant of the present embodiment also includes mutants having both the above-mentioned point mutation and mutation based on domain modification.

[0133] That is, the RpPpsR2 mutant of the present embodiment comprises the amino acid sequence represented by SEQ ID NO: 3 modified to D1-D1-D2, D3-D1-D2, D1-D2 (del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2 (del N 88), D1-D2 (del N 89), D1-D2 (del N 90), D1-D2 (del N 91), D1-D2 (del N 92), 92), D1-D2(del N 93), D1-D2(del N94), D1-D2(del N 95), D1-D2(del N96), D1-D2(del N 97), D1-D2(del C 5), D1-D2(del C 15), D1-D2(del N 93)(del C 5), D1-D2(del N 93)(del C 6), D1-D2(del N 93)(del C 11), D1-D2(del N 93)(del C12), D1-D2-D3(del C 25), D1-D2-D3(del C 41), D1-D2-D3(del C 49), D1-D2-D3(del C 70), D1-D2-D3(del C 87), D1-D2-D3(del C 96), D1-D2-D3(del C 104), D1-D2(del N 64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15), D1-D2(del N 33), D1-D2(del N 47) A mutant having an amino acid sequence obtained by modification of any one of the structural domains D1-D2-D3 (del C 9), and having a point mutation in the amino acid sequence corresponding to any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 3, namely:

[0134] Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, V146A.

[0135] More specifically, as an RpPpsR2 mutant having both a domain modification and a point mutation, a mutant having both a D3-D2 modification mutation and an L137E point mutation is described as an example. The D3-D2 variant having the L137E point mutation refers to a D3-D2 variant in which the 137th position of the amino acid sequence represented by sequence number 3 is substituted by lysine with glutamic acid, and is labeled as D3-D2 (L137E) in this specification.

[0136] As the above-mentioned RpPpsR2 mutant having both domain modification and point mutation, the above-mentioned D3-D2 (L137E) can be cited. Compared with the binding of wild-type RpBphP1 to wild-type RpPpsR2, the binding of D3-D2 (L137E) to wild-type RpBphP1 is weaker in the dark and the binding is also weaker under near-infrared light irradiation.

[0137] The proteome of the present embodiment includes the following proteome, which is a group of wild-type RpPpsR2 and RpBphP1 mutants, wherein the RpBphP1 mutant has a residue selected from Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T , Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503 A. R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D607A, D607G, D607V, D60 7L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D6 A mutant consisting of an amino acid sequence of one or more point mutations in the point mutation group consisting of 07M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, and Q521A.

[0138] The proteome of the present embodiment includes the following proteome, which is a group of wild-type RpBphP1 and RpPpsR2 mutants, wherein the RpPpsR2 mutant has an amino acid sequence represented by SEQ ID NO: 3 and has a residue selected from Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119 A mutant consisting of an amino acid sequence of one or more point mutations in the point mutation group consisting of A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, and V146A.

[0139] The proteome of the present embodiment includes the following proteome, which is a group of wild-type RpBphP1 and RpPpsR2 mutants, wherein the RpPpsR2 mutant is modified with the amino acid sequence represented by SEQ ID NO: 3 to D1-D1-D2, D3-D1-D2, D1-D2(del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2(L137E), D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2(del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2(L137E), 88), D1-D2(del N 89), D1-D2(del N 90), D1-D2(del N91), D1-D2(del N 92), D1-D2(del N 93), D1-D2(del N 94), D1-D2(del N 95), D1-D2(del N96), D1-D2(del N 97), D1-D2(del C 5), D1-D2(del C 15), D1-D2(del N 93)(del C 5), D1-D2(del N 93)(del C 6), D1-D2(del N 93)(del C11), D1-D2(del N 93)(del C 12), D1-D2-D3(del C 25), D1-D2-D3(del C 41), D1-D2-D3(del C 49), D1-D2-D3(del C 70), D1-D2-D3(del C 87), D1-D2-D3(del C 96), D1-D2-D3(del C 104), D1-D2(del N 64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15), D1-D2(del N 33), D1-D2(del N 47), D1-D2-D3(del C 9).

[0140] The proteome of the present embodiment includes the following proteome, which is a group of RpBphP1 mutants and RpPpsR2 mutants, wherein the RpBphP1 mutant is composed of an amino acid sequence represented by sequence number 1 or sequence number 2 having a member selected from the group consisting of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, and Q502T. , Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E51 6A, F518A, S525A, D607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D6 07M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M and Q521A; and the RpPpsR2 mutant is a mutant having an amino acid sequence selected from the group consisting of Q105T, T133A, L137A, N103A, Q105A, Q105C, Q Any mutant consisting of an amino acid sequence of one or more point mutations in the point mutation group consisting of 105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E and V146A.

[0141] The proteome of the present embodiment includes the following proteome, which is a group of RpBphP1 mutants and RpPpsR2 mutants, wherein the RpBphP1 mutant has a residue selected from Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E339B, and E340A in the amino acid sequence represented by sequence number 1 or sequence number 2. 365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D607A, D607G, D607V, D607L , D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D607M , D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M and Q521A;Similarly, RpPpsR2 could be assigned to the D1-D1-D2 region of D3-D1-D2(N-D1-D2). 77)、D1-D2、D2-D1、D2-D2、D3-D2、D3-D2(L137E)、D4-D2、D1-D2-D1、D1-D2-D2、D1-D2-D4-D1、D2、D2 -D2、D2-D3-D4、D4-D1-D2、D2-D3-D4-D1、D2-D3-D4-D2、D2-D3-D4-D3、D2-D3-D4-D5、D1-D2(del N 88)、D1-D2(of N 89)、D1-D2(of N 90)、D1-D2(of N91)、D1-D2(of N 92)、D1-D2(of N 93)、D1-D2(of N(D 91-D2)、 95)、D1-D2(of N96)、D1-D2(of N 97)、D1-D2(of C 5)、D1-D2(of C 15)、D1-D2(of N 93)(of C 5)、D1-D2)(of N 93)、of N 93)(from C11)、D1-D2(from N 93)(from C 12)、D1-D2-D3(from C 25)、D1-D2-D3(from C 41)、D1-D2-D3(from C 49)、D10(D7)、D2-D3 87)、D1-D2-D3(of C 96)、D1-D2-D3(of C 104)、D1-D2(of N 64)、D2-D3、D1-D2-D3、D2-D2-D3、D2-D3-D1、D2-D3-D3、D3 -D2-D3、D4-D2-D3、D1-D1-D2-D3、D1-D2-D3-D1、D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D3-4D2、D3-D2-D2 N 15)、D1-D2 (of N 33)、D1-D2 (of N 47)or D1-D2-D3 (of C 9)

[0142] The binding strength between the proteins constituting the photoswitch protein of the present embodiment (i.e., RpBphP1 mutant and wild-type RpPpsR2, wild-type RpBphP1 and RpPpsR2 mutant, RpBphP1 mutant and RpPpsR2 mutant) can be easily measured by a person skilled in the art using a method known in the art or a method obtained by modifying the known method. Such a method includes, for example, the method using CPTS disclosed in the Examples.

[0143] Below, the working principle of CPTS (CRISPR-Cas9-based light-activated transcription system) is explained. CPTS is a light-manipulated tool that uses Cas9 that lacks DNA cleavage activity. It can be used for genomic gene activation and reporter gene analysis systems. Cas9 mutants that lack DNA cleavage activity (for example, Cas9D10A / H840A; dCas9) can form a complex with the guide RNA and bind to the DNA base sequence targeted by the guide RNA. Among the several loop structures of the guide RNA, if an aptamer sequence that binds to a specific protein (for example, denoted as "protein 1") is inserted into the loop exposed from dCas9, protein 1 can bind to dCas9 via the aptamer sequence.

[0144] A protein on one side of the photoswitch protein of the present embodiment (for example, "wild-type RpBphP1") is connected to protein 1, and a transcription-activating factor (for example, p65, HSF1, etc.) is connected to the protein on the other side (for example, "RpPpsR2 mutant"). That is, a protein 1-wild-type RpBphP1 linker and a RpPpsR2 mutant-transcription activator linker are made. In a state where a reporter gene having a DNA base sequence targeted by a guide RNA near the transcription start site, a complex of a protein 1-wild-type RpBphP1 linker and dCas9, and a RpPpsR2 mutant-transcription activator linker coexist, dCas9 binds to the vicinity of the targeted DNA base sequence of the guide RNA. Here, if light (near-infrared light in the present embodiment) is irradiated, the wild-type RpBphP1 binds to the RpPpsR2 mutant, and the transcription activator linked to the RpPpsR2 mutant is anchored near the transcription start site. As a result, transcription of the reporter gene is activated, mRNA transcription occurs, and protein translation occurs. On the other hand, when light irradiation is stopped, wild-type RpBphP1 and RpPpsR2 mutant dissociate, so the transcription activator moves away from the transcription start site, and expression of the gene stops.

[0145] When the above CPTS is used, the binding strength between RpBphP1 mutant and wild-type RpPpsR2, wild-type RpBphP1 and RpPpsR2 mutant, or RpBphP1 mutant and RpPpsR2 mutant can be quantitatively evaluated using the expression level of the reporter gene as an indicator.

[0146] In addition to the method using CPTS, the binding strength between proteins constituting the photoswitch protein of this embodiment can also be measured by methods such as reporter gene analysis using TetO-tetR, analysis using a fluorescence microscope using translocation to the cell membrane as an indicator, and the like.

[0147] Each protein constituting the photoswitch protein of the present embodiment (for example, wild-type RpBphP1 and RpPpsR2 mutant) can be bound or fused to "other substances (i.e., substances different from the proteins constituting the photoswitch protein)". The "other substances" are not particularly limited and can be any substances, and examples thereof include various biological substances such as proteins, lipids, carbohydrates, and nucleic acids. The photoswitch protein of the present embodiment and the "other substances" can be bound via a linker, etc., and when the "other substances" are proteins, they can also be fused.

[0148] In the above description of CPTS, "protein 1" and "transcription activator" correspond to "other substances".

[0149] Alternatively, as "other substances", the interacting proteins A and B can be bound or fused to each protein of the photoswitch protein of the present embodiment. In this case, if the photoswitch protein of the present embodiment is irradiated with near-infrared light, the interaction between protein A and protein B can be induced, and if the near-infrared light irradiation is stopped, protein A and protein B can be dissociated. Therefore, by using the photoswitch protein of the present embodiment, the interaction between proteins can be freely controlled by turning on / off the light irradiation. In addition to the examples shown here, the photoswitch protein of the present embodiment also has other useful uses (see non-patent document 1 and non-patent document 2, etc.).

[0150] The second embodiment is a nucleic acid (DNA or RNA) encoding the RpBphP1 mutant and the RpPpsR2 mutant among the proteins constituting the photoswitch protein of the present embodiment.

[0151] The nucleic acid of the present embodiment can be prepared by a method known in the art or by a method after appropriately modifying the method. For example, the desired mutation can be introduced into the DNA encoding the wild type to obtain the desired nucleic acid. Alternatively, it can be synthesized using an automatic synthesis device. When each protein constituting the optical switch of the present embodiment is fused with other proteins, the nucleic acid encoding such a fusion protein is also included in the nucleic acid of the present embodiment.

[0152] The wild-type RpBphP1, wild-type RpPpsR2, RpBphP1 mutant and RpPpsR2 mutant in this embodiment can be prepared by integrating the nucleic acids encoding them into appropriate expression vectors, transforming or transfecting appropriate host cells with the expression vectors, culturing them in appropriate culture media, and purifying the expressed proteins.

[0153] As host cells for protein expression, for example, bacterial cells (e.g., Escherichia coli B strain (EscherichiacoliB strain) , Escherichia coli K12 strain, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) , Corynebacterium glutamicum , Serratia liquefaciens (Serratia liquefaciens) , Streptomyces lividans , Pseudomonas putida (Pseudomonas putida) etc.), mold (e.g. Penicillium camembertii , Cephalosporium acremonium (Acremonium chrysogenum etc.), animal cells, plant cells, baculovirus / insect cells or yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris etc.

[0154] The expression vector for expressing protein can use a vector suitable for various host cells. As an expression vector, for example, pBR322, pBR325, pUC118, pET, etc. (Escherichia coli host), pEGFP-C, pEGFP-N, etc. (animal cell host), pVL1392, pVL1393, etc. (insect cell host, baculovirus vector), pG-1, Yep13 or pPICZ, etc. (yeast cell host) can be used. These expression vectors have replication initiation sites, selection markers and promoters suitable for each vector, and can have enhancers, transcription termination sequences (terminators), ribosome binding sites and polyadenylation signals, etc. as required. In addition, the base sequences for merging and expressing FLAG tags, His tags, HA tags and GST tags, etc. can be inserted in the expression vector to make the expressed polypeptide easy to purify.

[0155] The expression vector can be prepared by a method known to those skilled in the art, or by using a commercially available kit or the like as appropriate.

[0156] When the expressed protein is extracted from the cultured bacteria or cultured cells, after the culture, the bacteria or cultured cells are collected by a known method, suspended in an appropriate buffer, and the bacteria or cells are broken by ultrasound, lysozyme and / or freeze-thaw, and then centrifuged and filtered to obtain a soluble extract. In particular, when cultured cells are used as a host, it is preferred to obtain the protein expressed in the culture supernatant by recovering the supernatant. The target protein can be obtained from the obtained extract or culture supernatant by appropriately combining known separation and purification methods. As known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation, methods mainly utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration, SDS-PAGE, methods utilizing charge differences such as ion exchange chromatography, methods utilizing specific affinity such as affinity chromatography (for example, when expressing a polypeptide with a GST tag, a resin formed by binding glutathione to a carrier is used, when expressing a polypeptide with a His tag, a Ni-NTA resin or a Co-based resin is used, when expressing a polypeptide with an HA tag, an anti-HA antibody resin is used, when expressing a polypeptide with a FLAG tag, an anti-FLAG antibody-bound resin is used, etc.), methods utilizing hydrophobicity differences such as reversed-phase high performance liquid chromatography, and methods utilizing isoelectric point differences such as isoelectric focusing electrophoresis, etc. can be used.

[0157] When the present specification is translated into English and contains words in the singular form of "a", "an" and "the", it includes both the singular and the plural unless the context clearly indicates otherwise.

[0158] The present invention will be further described below with reference to examples. However, these examples are merely illustrative of the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0159] Example

[0160] 1. Experimental Methods

[0161] 1-1. Wild-type RpBphP1 gene and wild-type RpPpsR2 gene

[0162] The wild-type RpBphP1 gene (SEQ ID NO: 4) and the wild-type RpPpsR2 gene (SEQ ID NO: 5) were purchased from the artificial gene synthesis service of GenScript Inc. At this time, the codon optimization algorithm of the company was used to optimize the human codons.

[0163] 1-2. Introduction of point mutations

[0164] The introduction of point mutations into RpBphP1 and RpPpsR2 was performed using the overlap extension PCR method (Overlap Extension PCR, Ho, SN, et al., Gene 77: 51-59 1989) or the transfer PCR method (Transfer-PCR, Erijman, et al., J. Struct. Biol. 175: 171-177 2011). In both PCR methods, oligonucleotide DNA used as primers was purchased from Eurofins genomics. As a DNA polymerase, PrimeSTAR HSDNA polymerase from Takara Bio was used, and the PCR reaction conditions were in accordance with the operating instructions of the corresponding product. In the case of using the overlap extension PCR method to introduce point mutations, the obtained PCR product fragments were treated with restriction enzymes and ligated with a vector treated with the same restriction enzymes. Restriction enzymes used products from Takara Bio or New England Bio, and the ligation reaction used Ligation high Ver. 2 from TOYOBO. The respective reaction conditions were in accordance with the operating instructions of each product. As a vector, the mammalian cell expression vector pcDNA3.1-V5HisA of Invitrogen was used. The ligation product was mixed with the Mach1 chemically competent cells of Invitrogen and transformed by heat shock at 42°C. According to the conventional method of genetic engineering experiments, the plasmid was cloned from the transformed Escherichia coli. The obtained plasmid was sequenced by the sequencing commission service of Eurofins genomics. In the case of using the transfer PCR method to introduce point mutations, first prepare a plasmid in which the wild-type sequence is cloned into pcDNA3.1-V5HisA, and use it as a template for transfer PCR reaction. After the PCR reaction, the wild-type template plasmid was digested by using Dpn I of Takara Bio. The product after the digestion reaction was transformed into Mach1 chemically competent cells and cloned according to the conventional method of genetic engineering experiments. The obtained plasmid was sequenced by the sequencing commission service of Eurofins genomics.

[0165] 1-3. Domain Reorganization

[0166] The domain reorganization of RpPpsR2 is carried out as follows. First, restriction enzyme sites are added to the domains that are desired to be reorganized. This is achieved by using primers designed in a manner that includes restriction enzyme sites to perform PCR amplification using a common PCR method. The resulting domain fragments are cloned into pcDNA3.1-V5HisA and sequenced using the sequencing entrustment service of Eurofins genomics. The domain fragments after sequence confirmation are treated with restriction enzymes for cutting, connected with the desired combination, and cloned into pcDNA3.1-V5HisA.

[0167] 1-4. Preparation of domain deletion constructs

[0168] The domain deletion body of RpPpsR2 was prepared by conventional PCR as follows. Primers were designed to delete the N-terminal and C-terminal sides of the desired domain in a manner that deletion occurred at the 5' and 3' sides of the domain DNA. The deletion domain was amplified by conventional PCR. The resulting deletion domain fragment was cloned into pcDNA3.1-V5HisA and sequenced by Eurofins genomics' sequencing service.

[0169] 1-5. Introduction of mutants into the CPTS system

[0170] The functional evaluation of the mutants was carried out using a light-dependent gene activation tool based on the CRISPR-Cas9 system (CRISPR-Cas9-based light-activated transcription system; CPTS). CPTS is a tool developed by the inventors (Nihongaki et al., Nature Methods 14:963-9662017). The CPTS system consists of the following four components. (1) A mutant in which mutations are introduced into the 10th amino acid and the 840th amino acid of Cas9 to render the DNA cleavage activity deficient (Cas9D10A / H840A; dCas9), (2) a guide RNA into which an aptamer sequence that binds to the MS2 coat protein is inserted, (3) one of the pairs of photoswitch proteins connected to the MS2 coat protein, and (4) the other of the pairs of photoswitch proteins connected to the transcription activator (p65-HSF1). Since the light switch protein pair this time is the RpBphP1 mutant and the RpPpsR2 mutant, the RpBphP1 mutant was connected to p65-HSF1, and the RpPpsR2 mutant was connected to the MS2 coat protein. The DNA of p65-HSF1 was amplified by PCR using Addgene plasmid #61423 purchased from Addgene, and then subcloned, and the sequence was confirmed by the sequencing service of Eurofins genomics. The DNA of MS2 coat protein (MS2 delta FG) was amplified by PCR using Addgene plasmid #27122 purchased from Addgene, and then subcloned, and the sequence was confirmed by the sequencing service of Eurofins genomics. When connecting them to the light switch proteins, restriction enzyme sites were used for all of them. In addition, the N-terminus of each construct was linked to a sequence consisting of three consecutive nuclear localization signals (NLS) from SV40 (NLSx3; ASPKKKRKVEASASPKKKRKVEASASPKKKSKVEAS; sequence number 4). The nuclear localization signal was also linked using restriction enzyme sites. The resulting NLSx3-RpBphP1 mutant-p65-HSF1 and NLSx3-MS2-RpPpsR2 mutant were cloned into pcDNA3.1-V5HisA.

[0171] 1-6. Fabrication of other components of the CPTS system

[0172] (1) Preparation of dCas9

[0173] The DNA of Streptococcus pyogenes-derived Cas9 (SpCas9) optimized for human codons uses hSpCas9 purchased from Addgene plasmid #42230 from Addgene. According to the operating instructions, Agilent's Quick change Multi Site-Directed Mutagenesis Kit was used to introduce point mutations of D10A and H840A to eliminate the DNA cleavage activity of hSpCas9. In addition, the nuclear localization signal (NLS) from SV40 was added to the N-terminus and C-terminus respectively by PCR. After the NLS-dSpCas9-NLS obtained in this way was cloned into pcDNA3.1-V5HisA, the sequence was confirmed by the sequencing commission service of Eurofins genomics.

[0174] (2) Preparation of guide RNA with aptamer

[0175] The pSPgRNA vector (Addgene plasmid #47108) purchased from Addgene was used to express the guide RNA in mammalian cells. The aptamer sequence that binds to the MS2 coat protein was purchased from the artificial gene synthesis service of Eurofins genomics. The synthesized aptamer sequence was cloned into the Kpn I / Sac I site of the pSPgRNA vector. A sequence targeting the upstream activation sequence of the GAL4 / UAS system was inserted into the BbsI site of the prepared vector. The target sequence was prepared by annealing the oligonucleotide DNA purchased from Eurofins genomics. The completed guide RNA with aptamer was sequenced and confirmed by the sequencing service of Eurofins genomics.

[0176] 1-7. Preparation of luminescent reporter gene plasmid

[0177] In order to simply evaluate the function of the mutant, the increase or decrease of the luminescent signal is used as an indicator. Therefore, a reporter gene plasmid utilizing CPTS light irradiation dependency to activate the luminescent reporter gene is used. Promega's pGL4.31 equipped with the upstream activation sequence of the GAL4 / UAS system is used, and the luminescent reporter gene downstream thereof is replaced by a short-lived firefly luciferase gene with a normal lifespan firefly luciferase gene and used. The above-mentioned guide RNA with an aptamer is designed to bind to the upstream activation sequence.

[0178] 1-8. Cell Culture

[0179] The functional evaluation of the mutants was performed using HEK293T cells. HEK293T cells were obtained from the American Type Culture Collection (ATCC). The culture medium used Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS (Gibco), 100 units / mL penicillin and 100 μg / mL streptomycin (Gibco), and 0.584 g / L L-glutamine (Gibco), and cultured in an incubator at 37°C and 5% CO2.

[0180] 1-9. Irradiation with near-infrared light

[0181] As the near-infrared light source, a LED light source (760±20 nm) from CCS was used. The irradiation intensity was 10 W / m 2 In order to prevent the temperature in the incubator and the cell culture plate from rising, the near-infrared light irradiation was performed using an ON / OFF timer (WT-03N) from CUSTOM with a cycle of ON / OFF = 1 minute / 4 minutes. In addition, in order to irradiate the cell culture plate with near-infrared light at a uniform intensity, a light diffusion film (Scotchcal, transmittance 30%) from 3M was used.

[0182] 1-10. CPTS luminescent reporter gene analysis

[0183] (1) Cell inoculation

[0184] First, HEK293T cells were seeded into 96-well black-walled culture plates (Greiner Bio). The cell concentration was 2.3×10 4 100 μL of cell suspension was added to each well in the manner of cells / well. When biliverdin (BV; Frontier scientific company) was added, it was added to a final concentration of 25 μM. It should be noted that since BV is easily decomposed under light, the work was carried out in the dark. The culture plate inoculated with cells was cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0185] (2) Plasmid transfection

[0186] Plasmid transfection was performed by either the following method (A) or (B).

[0187] (A) Lipofectamine-based transfection

[0188] The gene introduction reagent used was Lipofectamine 3000 (Invitrogen), and the plasmid used in the experiment was introduced by liposome transfection. According to the operating instructions, Lipofectamine was added at 0.2 μL / well and P3000 was added at 0.2 μL / well. The total amount of DNA introduced was 0.1 μg per well. The plasmids encoding dSpCas9, guide RNA, MS2-RpPpsR2, RpBphP1-p65HSF1 and GAL4UAS-Firefly luciferase reporter genes were introduced at a ratio of 1:1:1:1:1. In addition, as a negative control, the plasmid encoding the pcDNA3.1V5 / His-A vector and the GAL4UAS-Firefly luciferase reporter gene were introduced at a ratio of 4:1. After the gene introduction, the culture plate was quickly wrapped with aluminum foil to avoid light and returned to the incubator.

[0189] (B) Transfection based on cationic polymer (PEI MAX)

[0190] The gene introduction reagent used was PEI MAX (Transfection Grade Linear Polyethylenimine Hydrochloride; Polysciences). PEI MAX was 0.6 μL / well, and the plasmids used in the experiment were introduced. The total amount of DNA introduced was 0.1 μg per well. The plasmids encoding SpCas9, guide RNA, MS2-RpPpsR2, RpBphP1-p65HSF1, and GAL4UAS-Firefly luciferase reporter genes were introduced at a ratio of 1:1:1:1:1. In addition, as a negative control, the plasmids encoding the pcDNA3.1 V5 / His-A vector and the GAL4UAS-Firefly luciferase reporter gene were introduced at a ratio of 4:1. After gene introduction, the culture plate was quickly wrapped with aluminum foil to avoid light and returned to the incubator.

[0191] (3) Near-infrared light irradiation

[0192] 24 hours after gene introduction, in order to prevent drying caused by evaporation of the culture medium, each well was supplemented with 100 μL of culture medium. The wells with BV (+) were supplemented with culture medium with 25 μM BV. At this time, the operation of the culture plate for light irradiation and the culture plate for dark conditions were performed in the dark. After the culture medium was supplemented, the culture plate for dark conditions was quickly wrapped with aluminum foil again to avoid light and placed in an incubator. The culture plate for light irradiation was irradiated with near-infrared light in the incubator.

[0193] (4) Determination of the luminescence intensity of the luminescent reporter gene

[0194] 24 hours after the start of light irradiation, the two culture plates were recovered. The culture medium was removed from each well of the recovered culture plate, and 100 μL of HBSS (Gibco) with 200 μM D-luciferin (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well. After incubation at room temperature for 30 minutes, a microplate luminescence detector (Centro XS3 LB 960 Plate Reader; Berthold Technologies) was used for bioluminescence measurement. The exposure time was set to 1 second / well.

[0195] 1-11. Confirmation of genomic gene activation by the photoswitchable protein of the present invention in cells and in vivo

[0196] 1-11-1. Preparation of guide RNA with aptamers targeting genomic genes

[0197] In order to target genomic genes, the following sequences were inserted into the Bbs I site of the guide RNA with aptamer, respectively.

[0198] People ASCL1 In the case of a target gene: GCAGCCGCTCGCTGCAGCAG (SEQ ID NO: 7)

[0199] People HBG1 When the gene is the target, GGCTAGGGATGAAGAATAAA (SEQ ID NO: 8)

[0200] People MYOD1 When the gene is the target, it is GCCTGGGCTCCGGGGCGTTT (SEQ ID NO: 9)

[0201] People IL1R2 When the gene is the target, it is GTCCTTGGCCACTTCCCCATC (SEQ ID NO: 10)

[0202] Mouse ASCL1 When the gene is the target, it is AGCTGAGGAGGTGGGGGA AG (SEQ ID NO: 11)

[0203] These target sequences were prepared by annealing oligonucleotide DNA purchased from Eurofins genomics, Inc. The completed guide RNA with aptamers was sequenced by Eurofins genomics' outsourced sequencing service.

[0204] 1-11-2. Activation of genomic genes in cells based on CPTS

[0205] (1) Cell inoculation

[0206] First, HEK293T cells were seeded in 96-well culture plates (Thermo Fisher Scientific). The cell concentration was 2.3×10 4 100 μL of cell suspension was added to each well in the manner of cells / well. When BV was added, it was added to a final concentration of 25 μM. It should be noted that since BV is easily decomposed under light, the operation was performed in the dark. The culture plate inoculated with cells was cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0207] (2) Plasmid transfection

[0208] A combination of RpBphP1 mutants (introduced with Q502A and E513A) and wild-type RpPpsR2 was used as a photoswitch protein. Lipofectamine 3000 (Invitrogen) was used as the gene introduction reagent, and the plasmids used in the experiment were introduced by lipofectamine transfection. According to the operating instructions, add Lipofectamine at 0.2 μL / well and P3000 at 0.2 μL / well. The total amount of introduced DNA is 0.1 μg per well. Plasmids encoding dSpCas9, guide RNA, MS2-RpPpsR2, and RpBphP1-p65HSF1 were introduced at a ratio of 1:1:1:1. In addition, as a negative control, a guide RNA plasmid without a target sequence was used. After gene introduction, the culture plate was quickly wrapped with aluminum foil to avoid light and returned to the incubator.

[0209] (3) Near-infrared light irradiation

[0210] 24 hours after gene introduction, in order to prevent drying caused by evaporation of the culture medium, each well was supplemented with 100 μL of culture medium. The wells of BV(+) were supplemented with culture medium with 25 μM BV. At this time, both the light irradiation culture plate and the dark condition culture plate were operated in the dark. After the culture medium was supplemented, the dark condition culture plate was quickly wrapped with aluminum foil again to avoid light and placed in an incubator. The light irradiation culture plate was irradiated with near-infrared light in the incubator.

[0211] (4) Evaluation of genomic gene expression based on real-time PCR

[0212] After 24 hours from the start of light irradiation, two culture plates were recovered. CellAmpDirect RNAPrep Kit from Takara Bio was used to extract mRNA from the cells of the recovered culture plates. The extraction steps were in accordance with the operating instructions. Next, the extracted mRNA was reverse transcribed into cDNA using Superscript IV VILO Master Mix (Thermo Fisher Scientific). The steps were in accordance with the operating instructions. After reverse transcription, the amount of cDNA was evaluated by real-time PCR. TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assay (Thermo Fisher Scientific) were used as reagents for real-time PCR. The ID of the TaqMan probe used is as follows:

[0213] people ASCL1 Gene: Hs04187546_g1

[0214] people HBG1 Gene: Hs00361131_g1

[0215] people MYOD1 Gene: Hs02330075_g1

[0216] people IL1R2 Gene: Hs01030384_m1

[0217] people GAPDH Gene: Hs99999905_m1

[0218] The thermal cycler and software for real-time PCR used the StepOnePlus system (Thermo Fisher Scientific). The standard ΔΔCt method was used in the assay to determine the relative mRNA expression level relative to the mRNA of untransfected HEK293T cells.

[0219] 1-11-3. CPTS-based gene activation in mice

[0220] (1) Preparation of mice for experiment

[0221] Animal experiments using mice were performed in accordance with the Animal Experiment Implementation Manual and Experimental Guidelines established by the University of Tokyo. Six-week-old female BALB / c mice (Sankyo Labo Service Co., Ltd.) were used.

[0222] (2) Gene transfer into mouse liver

[0223] A combination of RpBphP1 mutant (introduced with Q502A and E513A) and wild-type RpPpsR2 was used as a photoswitch protein. Genes were introduced into mouse liver cells using the hydrodynamic tail vein injection method. TransIT-EE hydrodynamic injection solution from Mirus Bio was used, and the steps for preparing the solution followed the operating instructions. The total amount of plasmid DNA was 150 μg per mouse. The ratio of plasmid DNA for gene introduction was 1:1:1:1:1 for dSpCas9, guide RNA, MS2-RpPpsR2, RpBphP1-p65HSF1, and GAL4UAS-Akaluc reporter gene. The prepared solution was injected into the tail vein of the mouse using a 27G injection needle from Terumo. At this time, pay attention to complete the injection in about 5 to 6 seconds. After the injection, the mouse was returned to the cage and raised for 8 hours.

[0224] (3) Near-infrared light irradiation of mice

[0225] Eight hours after injection, mice were irradiated with near-infrared light ( Fig. 22 a). Near infrared light at 760±20nm, 300W / m 2 After 12 hours of light irradiation, the irradiation was temporarily stopped and the cages were cleaned.

[0226] (4) Bioluminescence imaging of mouse liver cells

[0227] Bioluminescence imaging of mouse liver cells was performed 25 hours after injection. 200 μL of 10 mM Akalumine-HCl (Fujifilm Wako Pure Chemical Industries, Ltd.) was injected into the peritoneal cavity of the mouse. After injection, the mouse was anesthetized with isoflurane (Fujifilm Wako Pure Chemical Industries, Ltd.). After anesthesia, the bioluminescence images of the mouse were taken using the Lumazone bioluminescence imaging system (Shoshin) equipped with an Evolve 512EMCCD camera (Photometrics). The data were analyzed using SlideBook 4.2 software (Intelligent Innovations).

[0228] (5) Genomic gene activation in mouse liver cells

[0229] Using mice ASCL1The guide RNA used as the target gene was used to introduce dSpCas9, guide RNA, MS2-RpPpsR2, and RpBphP1-p65HSF1 at a ratio of 1:1:1:1 using hydrodynamic tail vein injection. The total amount of plasmid DNA was 120μg per mouse. Light irradiation was the same as that of the above-mentioned luminescent reporter gene. After light irradiation, mRNA was extracted from the mouse liver. The mouse liver was crushed using a Precellys Evolution tissue homogenizer (Bertin Instruments). The extracted mRNA was reverse transcribed into cDNA using Superscript IV VILO Master Mix (Thermo Fisher Scientific). The steps were in accordance with the operating instructions. After reverse transcription, the amount of cDNA was evaluated by real-time PCR. As reagents for real-time PCR, Luna Universal Probe qPCR Master Mix (New England Biolabs) and TaqMan Gene Expression Assay (Thermo Fisher Scientific) were used. The ID of the TaqMan probe used is as follows:

[0230] Mouse ASCL1 Gene: Mm03058063_m1

[0231] Mouse GAPDH Gene: Mm99999915_g1

[0232] The thermal cycler and software for real-time PCR used the StepOnePlus system (Thermo Fisher Scientific). The relative mRNA expression level was determined relative to the mRNA of untransfected mouse liver cells using the standard ΔΔCt method.

[0233] 2. Results

[0234] 2-1. Confirmation of the function of the photoswitch protein composed of wild-type RpBphP1 and wild-type RpPpsR2

[0235] Wild-type RpBphP1 and wild-type RpPpsR2 were loaded into the CRISPR-Cas9 system-based light-dependent gene activation tool (CPTS). The working principle of CPTS is explained below (refer to Figure 1 a).

[0236] The mutant (Cas9D10A / H840A; dCas9) in which the DNA cleavage activity is lost by introducing mutations to the 10th amino acid and the 840th amino acid of Cas9 can form a complex with the guide RNA and bind to the base sequence of the genomic DNA targeted by the guide RNA. For example, it is about 100 to 500bp upstream of the transcription start site of the gene whose expression is desired to be light-controlled. The guide RNA is cleverly designed. In several loop structures of the guide RNA, other RNA sequences can be inserted into the loop exposed from dCas9. Therefore, an aptamer sequence that binds to a specific protein is inserted. Here, an aptamer sequence that binds to the coat protein possessed by the bacteriophage MS2 is inserted. Moreover, one of the optical switch proteins is connected to the MS2 coat protein. The optical switch protein on the other side is connected to the factor that activates transcription (p65 and HSF1). Thus, if light is irradiated, the optical switch protein binds, and as a result, the transcription activator is anchored near the transcription start site. Thus, the transcription of the gene is activated, mRNA transcription occurs, and translation to protein occurs. On the other hand, if the light irradiation stops, the photoswitch protein dissociates, so the transcription activator moves away from the transcription start site and the expression of the gene stops. In this way, the gene encoded on the genome and the luminescent reporter gene on the luminescent reporter gene plasmid can be switched on / off by light.

[0237] Wild-type RpBphP1 and wild-type RpPpsR2 bound to each other even in the dark, indicating that gene activation occurred ( Figure 1 b, WT). Due to the “dark leakage signal ( Figure 1 The "Dark" of WT in b) is very high, so the gene activation based on light irradiation increased by at most 1.8 times. In this way, the generation of "dark leakage signal" is a fatal flaw as a photoswitch protein, and it cannot be put into practical use in this way.

[0238] 2-2. Functional analysis of photoswitch proteins composed of RpBphP1 mutant and wild-type RpPpsR2

[0239] By introducing point mutations into RpBphP1 to create RpBphP1 mutants, we investigated how the RpBphP1 mutants bind to wild-type RpPpsR2 in the dark and under near-infrared light irradiation. As a result, we found that the mutants ( Figure 2 a, b and c). In mutants with significantly reduced dark leakage signals, gene activation based on light irradiation can be greatly increased. For example, a 15.7-fold increase was obtained in the D607A mutant ( Figure 2 a), a 12.4-fold increase was obtained in the Q502T mutant ( Figure 2b), a 9.7-fold increase was obtained in the D607S mutant ( Figure 2 c). However, there is also a trend that the signals under near-infrared light irradiation (NIR signals) also decrease accordingly. On the other hand, there are also amino acid mutants such as D607E that have the effect of reducing the dark leakage signal without reducing the NIR signal ( Figure 2 c) The RpBphP1 mutation D607E is a promising candidate amino acid mutation for improving the function of RpBphP1 by combining with other mutations.

[0240] In addition, a point mutation was introduced into RpBphP1, and a mutant of the photoswitch protein composed of this mutant and wild-type RpPpsR2 was found to have a higher NIR signal ( Figure 3 a and b). These mutations are also promising candidates for amino acid mutations in improving the function of RpBphP1 by combining with other mutations.

[0241] 2-3. Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 mutants

[0242] Next, we introduced point mutations into RpPpsR2 to create RpPpsR2 mutants, and investigated the binding state of wild-type RpBphP1 and the RpPpsR2 mutants in the dark and under near-infrared light irradiation. As a result, we found a mutant that reduced the dark leakage signal compared to wild-type RpPpsR2 (WT). Figure 4 a and b). In mutants with significantly reduced dark leakage signals, gene activation based on light irradiation can be greatly increased. For example, a 13.4-fold increase was obtained in the L110A mutant ( Figure 4 a). In addition, there are also amino acid mutants such as Q105T that have the effect of reducing dark leakage signals without reducing NIR signals ( Figure 4 b) The mutation Q105T is a promising candidate amino acid mutation for improving the function of RpPpsR2 by combining with other mutations.

[0243] In addition, a point mutation was introduced into RpPpsR2, and a mutant of the photoswitch protein composed of this mutant and wild-type RpBphP1 was found to have an enhanced NIR signal ( Figure 5 a~c). In particular, the photoswitch protein composed of the RpPpsR2 mutant with a mutation at Q105S and the wild-type RpBphP1 had no effect on the dark leakage signal, and the NIR signal became higher ( Figure 5 b) The mutation Q105S is a promising candidate amino acid mutation for improving the function of RpPpsR2 by combining with other mutations.

[0244] 2-4. Functional analysis of photoswitch proteins composed of wild-type RpBphP1 and RpPpsR2 domain variants

[0245] A method was tried to reduce the dark leakage signal without relying on amino acid point mutation. The three-dimensional structure of wild-type RpPpsR2 is composed of 5 domains. The inventors found that the binding property with RpBphP1 can be changed by removing some of the 5 domains, changing the connection order, and changing the number (reorganizing the domains). Figure 6 For example, in the photoswitch protein composed of the domain-shuffled RpPpsR2 domain variant D4-D1-D2 and the wild-type RpBphP1, the dark leakage signal was greatly reduced, and as a result, the light-dependent activation increased by 7.2 times ( Figure 6 b). In addition, in the D3-D1-D2 domain variant, the NIR signal was not reduced, but the dark leakage signal was reduced ( Figure 6 b) Domain shuffling such as D3-D1-D2 has the potential to improve the function of RpPpsR2 by combining with other mutations.

[0246] Mutants that increased NIR signal were also found in domain shuffling ( Figure 7 These domain shufflings may also be promising mutational candidates for improving the function of RpPpsR2 by combining with other mutations.

[0247] The present inventors have found that the binding property to RpBphP1 can be altered by deleting the N-terminal and C-terminal sides of the RpPpsR2 domain (domain deletion). For example, the N-terminal and C-terminal sides of one of the domain shuffles (D1-D2) of RpPpsR2 were deleted (see Figure 8 a) Domain variants have altered binding to RpBphP1. For example, domain variant D1-D2 (del C 15) significantly reduced dark leakage signal and increased light-dependent activation by 9.6-fold ( Figure 8 b). In addition, the domain variant D1-D2 (del N 77) did not reduce the NIR signal but reduced the dark leakage signal ( Figure 8 b) Such domain modifications involving amino acid deletions are expected to improve the function of RpPpsR2 by combining with other mutations.

[0248] In addition, mutants with increased NIR signal were also found in domain variants containing deletions ( Fig. 9 a and b). In particular, D1-D2 (del N 64) did not change the dark leakage signal, while the NIR signal increased ( Fig. 9b) Such domain variants have the potential to improve the function of RpPpsR2 by combination with other mutations.

[0249] Next, for D1-D2 (del N 93), one of the domain variants containing a deletion ( Figure 8 b) From (del N88) to (del N97), the surrounding amino acid deletions were investigated one by one (refer to Fig.10 a). As a result, it can be seen that the surrounding missing bodies reduce the dark leakage signal ( Fig.10 b) Domain variants containing such deletions are expected to improve the function of RpPpsR2 by combination with other mutations.

[0250] In addition, for one of the domain variants containing a deletion, D1-D2 (del N 93), a deletion variant in which the C-terminal side was deleted was investigated (refer to Fig.11 As a result, it was found that the dark leakage signal was further reduced by deleting the C-terminal side ( Fig.11 b) Domain variants containing such deletions are expected to improve the function of RpPpsR2 by combination with other mutations.

[0251] 2-5. Functional analysis of photoswitch proteins composed of RpBphP1 mutants and / or RpPpsR2 mutants with multiple beneficial mutations (point mutations or domain modifications, etc.)

[0252] Functional analysis of photoswitch proteins consisting of RpBphP1 mutants and / or RpPpsR2 mutants having multiple beneficial mutations of amino acid point mutants or domain variants (domain shuffles and domain deletions) that have been functionally analyzed so far was performed. Fig.12 The figure shows what combinations are possible when RpBphP1 and RpPpsR2 have multiple mutations and modifications that reduce dark leakage signals and mutations and modifications that increase NIR signals. There are a total of 12 combinations of patterns. As a result of such combinations, it was found that photoswitch proteins with multiple beneficial mutations can accumulate the effects of mutations to further improve their functions. The following is a specific explanation.

[0253] We investigated how the properties of the photoswitch protein changed when RpBphP1 or RpPpsR2 had both a point mutation or domain modification that reduced dark leakage signals and a point mutation or domain modification that increased NIR signals. First, we tried to combine RpBphP1 and RpPpsR2 in a pattern of Fig.12An example of combination 2. If a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are simultaneously introduced into RpBphP1, the dark leakage signal can be reduced without almost reducing the NIR signal of the photoswitch protein ( Fig.13 a). Next, try the combination pattern of RpBphP1 and RpPpsR2 as Fig.12 An example of combination 7. If a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are simultaneously introduced into RpPpsR2, the dark leakage signal can be reduced without almost reducing the NIR signal of the photoswitch protein ( Fig.13 b) In addition, the combination pattern of RpBphP1 and RpPpsR2 was tried as follows Fig.12 Combination 7 but with both domain modification and point mutation in RpPpsR2. That is, a RpPpsR2 mutant having both domain modification (D3-D2) that reduces dark leakage signal and point mutation (L137E) that increases NIR signal was prepared and combined with wild-type RpBphP1. As a result, the ratio of NIR signal intensity of photoswitch protein to dark leakage signal intensity can be improved ( Fig.14 ).

[0254] In summary, it is shown that the function of the photoswitch protein can be improved by introducing multiple mutations (point mutations or domain modifications, etc.) that have beneficial effects into RpBphP1 or RpPpsR2.

[0255] Next, we investigated the properties of photoswitch proteins when beneficial mutations were introduced into both RpBphP1 and RpPpsR2 ( Fig.15 , Fig.16 , Fig.17 , Fig.18 ). First, try the combination pattern of RpBphP1 and RpPpsR2 as Fig.12 An example of combination 9. The RpBphP1 mutant into which a point mutation that reduces dark leakage signals was introduced was combined with the RpPpsR2 mutant that underwent domain shuffling to reduce dark leakage signals. As a result, the dark leakage signals were reduced without substantially reducing the NIR signals of the photoswitch protein. Fig.15 a). Next, try the combination pattern of RpBphP1 and RpPpsR2 as follows Fig.12 An example of combination 3. The RpBphP1 mutant with a point mutation that reduces the dark leakage signal was combined with the RpPpsR2 mutant with a point mutation that increases the NIR signal. As a result, the dark leakage signal can be reduced without almost reducing the NIR signal of the photoswitch protein ( Fig.15 b).

[0256] Next, we tried the combination pattern of RpBphP1 and RpPpsR2. Fig.12 An example of combination 4 is shown. That is, a RpBphP1 mutant into which a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are introduced is combined with an RpPpsR2 mutant into which a point mutation that increases the NIR signal is introduced. As a result, the dark leakage signal can be reduced without almost reducing the NIR signal of the switch protein ( Fig.16 a). Next, we tried the combination pattern of RpBphP1 and RpPpsR2. Fig.12 An example of combination 10 is shown. That is, a RpBphP1 mutant into which a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are introduced is combined with an RpPpsR2 mutant into which a point mutation that reduces the dark leakage signal is introduced. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig.16 b).

[0257] In addition, the combination pattern of RpBphP1 and RpPpsR2 was tried as follows Fig.12 Combination 3 but with multiple point mutations that increase the NIR signal in RpPpsR2. That is, the RpPpsR2 mutant with two point mutations that increase the NIR signal is combined with the RpBphP1 mutant with one point mutation that reduces the dark leakage signal. As a result, the dark leakage signal can be reduced without almost reducing the NIR signal of the photoswitch protein ( Fig.17 a). Next, we tried to make the combination pattern of RpBphP1 and RpPpsR2 Fig.12 In the case of combination 11, one point mutation that reduces dark leakage signal and one point mutation that increases NIR signal are introduced into RpPpsR2, and one point mutation that reduces dark leakage signal is introduced into RpBphP1. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig.17 b).

[0258] In addition, the combination pattern of RpBphP1 and RpPpsR2 was tried as Fig.12 Combination 4 but with multiple point mutations that increase the NIR signal in RpPpsR2. That is, the RpPpsR2 mutant into which two point mutations that increase the NIR signal are introduced is combined with the RpBphP1 mutant into which one point mutation that reduces the dark leakage signal and one point mutation that increases the NIR signal are introduced. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig.18a). In addition, the combination pattern of RpBphP1 and RpPpsR2 was tried as follows Fig.12 An example of combination 12 is shown. That is, a RpBphP1 mutant into which a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are introduced is combined with an RpPpsR2 mutant into which a point mutation that reduces the dark leakage signal and a point mutation that increases the NIR signal are introduced. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig.18 b). Fig.18 A combination of Fig.18 The combination of b can induce more than 10-fold light-dependent activation (ie, the ratio of NIR signal intensity to dark signal intensity is greater than 10). The above results indicate that the function of photoswitch proteins can be improved by introducing multiple beneficial mutations into RpBphP1 and RpPpsR2.

[0259] Next, the combination pattern of RpBphP1 and RpPpsR2 is Fig.12 Combination 11, but the RpPpsR2 mutant with both domain modification and point mutation was combined with the RpBphP1 mutant that reduced the dark leakage signal. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig.19 ).

[0260] In addition, the combination pattern of RpBphP1 and RpPpsR2 is Fig.12 Combination 12, but the RpPpsR2 mutant having both domain modification and point mutation is combined with the RpBphP1 mutant having multiple point mutations. That is, it is combined with the RpBphP1 mutant into which one point mutation for reducing the dark leakage signal and one point mutation for increasing the NIR signal are introduced. As a result, in this case, the ratio of the NIR signal intensity of the photoswitch protein to the dark leakage signal intensity can be improved ( Fig. 20 ).

[0261] The above results indicate that the function of photoswitch proteins can be improved by combining all the RpBphP1 point mutations with beneficial effects with domain modification and point mutations of RpPpsR2.

[0262] 2-6. Confirmation of gene activation by the photoswitchable protein of the present invention in cells and in vivo

[0263] 2-6-1. Confirmation of genomic gene activation in cells

[0264] The function of the photoswitch protein of the present invention in cultured cells is analyzed using a near-infrared light manipulation tool (the aforementioned CRISPR-Cas9 system-based gene expression light manipulation tool; CPTS).

[0265] The light-controlled tools were redesigned with guide RNA to target human genes ( ASCL1 , HBG1 , MYOD1 or IL1R2 ). The four genes were introduced into human cell lines and irradiated with near-infrared light. As a result, all four human genes were activated simultaneously ( Fig.21 a). To investigate how long it takes for the near-infrared light manipulation tool to activate genes after the start of near-infrared light irradiation. Human cell lines into which the light manipulation tool has been introduced were irradiated with light, and the degree of activation of the light manipulation tool was quantitatively evaluated at a specified time. As a result, it was found that gene activation could be induced 18-fold in 1.5 hours after light irradiation ( Fig.21 b). In addition, it was investigated whether gene activation based on the light control tool stops when light irradiation stops. As a result, it was found that gene activation can be stopped when light irradiation stops, and gene activation can be restarted when light irradiation starts again ( Fig.21 c).

[0266] 2-6-2. Confirmation of gene activation in vivo

[0267] CPTS was used to analyze the function of the photoswitch protein of the present invention in vivo in mice.

[0268] The genes of the components of CPTS were introduced into the liver of mice, and light was irradiated from outside the mouse body using an LED panel (760nm, 300W / m 2 ).

[0269] When CPTS for luminescent reporter gene was used, no luminescent signal was observed from mice kept in the dark (Dark), but a strong luminescent signal was observed from the liver of mice irradiated with near-infrared light (NIR). Fig. 22 b) Next, using mice ASCL1 In the case of CPTS gene, the liver was removed from the mouse after near-infrared light irradiation ( Fig. 22 c left figure), mRNA was extracted and quantitatively analyzed. As a result, it was confirmed that the liver of mice irradiated with near-infrared light (NIR) was significantly higher than that of mice kept in the dark (Dark). ASCL1 The gene was activated 47.8 times ( Fig. 22 c right figure).

[0270] Industrial Applicability

[0271] The present invention provides a photoswitch protein that can precisely control binding and dissociation by turning on / off near-infrared light irradiation. The photoswitch protein of the present invention introduced into a living body can be used for the treatment of diseases and the like because its function can be controlled by near-infrared light from outside the body, and is expected to be used in the medical field and the like.

Claims

1. A protein group consisting of two proteins that bind under near-infrared light irradiation, the protein group is a group of RpBphP1 mutant and wild-type RpPpsR2, a group of wild-type RpBphP1 and RpPpsR2 mutant, or a group of RpBphP1 mutant and RpPpsR2 mutant, and the binding of the protein group is weaker in the dark and / or stronger under near-infrared light irradiation than the binding of wild-type RpBphP1 and wild-type RpPpsR2.

2. The protein group according to claim 1, wherein The wild-type RpBphP1 is a protein composed of the amino acid sequence represented by sequence number 1 or sequence number 2, or a protein having a sequence homology of more than 90% with the amino acid sequence represented by sequence number 1 or sequence number 2, and the wild-type RpPpsR2 is a protein composed of the amino acid sequence represented by sequence number 3, or a protein having a sequence homology of more than 90% with the amino acid sequence represented by sequence number 3.

3. The protein group according to claim 2, wherein: The protein group is a group of wild-type RpPpsR2 and RpBphP1 mutant, The RpBphP1 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, The point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, It is composed of E513P, E516A, F518A, S525A, D607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, and Q521A.

4. The protein group according to claim 2, wherein The protein group is a group of wild-type RpBphP1 and RpPpsR2 mutant, The RpPpsR2 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 3, The point mutation group consists of Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, and V146A.

5. The protein group according to claim 2, wherein: The protein group is a group of wild-type RpBphP1 and RpPpsR2 mutant, The RpPpsR2 mutant is modified with the amino acid sequence represented by SEQ ID NO: 3 to be D1-D1-D2, D3-D1-D2, D1-D2 (del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2 (L137E), D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2 (del N 88), D1-D2 (del N 89), D1-D2 (del N 90), D1-D2 (del N 91), 91), D1-D2(del N 92), D1-D2(del N 93), D1-D2(del N94), D1-D2(delN 95), D1-D2(delN 96), D1-D2(delN 97), D1-D2(delC 5), D1-D2(delC 15), D1-D2(del N 93)(delC 5), D1-D2(del N93)(delC 6), D1-D2(delN 93)(delC 11), D1-D2(del N 93)(delC12), D1-D2-D3(delC25), D1-D2-D3(delC 41), D1-D2-D3(delC 49), D1-D2-D3(del C 70), D1-D2-D3(del C87), D1-D2-D3(del C 96), D1-D2-D3(delC 104), D1-D2(del N 64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15), D1-D2(del N 33), D1-D2 (del N 47) or D1-D2-D3 (delC 9).

6. The protein group according to claim 2, wherein: The protein group is a group of RpBphP1 mutants and RpPpsR2 mutants, The RpBphP1 mutant is any one of mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C , Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D 607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D60 7Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A; The RpPpsR2 mutant is any one of the mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 3, wherein the point mutation group consists of Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A , L114A, I115A, Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R134A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, and V146A.

7. The protein group according to claim 2, wherein: The protein group is a group of RpBphP1 mutants and RpPpsR2 mutants, The RpBphP1 mutant is any one of mutants consisting of the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence having one or more point mutations selected from the following point mutation group in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutation group consists of Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C , Q502M, Q502P, F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D 607A, D607G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D60 7Q, D607F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A; For RpPpsR2, the same sequence as D1-D1-D2 and D3-D1-D2(from D1-D2) 77)、D1-D2、D2-D1、D2-D2、D3-D2、D3-D2(L137E)、D4-D2、D1-D2-D1、D1-D2-D2、D1-D2-D4-D1、D2、D2 -D2、D2-D3-D4、D4-D1-D2、D2-D3-D4-D1、D2-D3-D4-D2、D2-D3-D4-D3、D2-D3-D4-D5、D1-D2(del N 88)、D1-D2(of N 89)、D1-D2(of N 90)、D1-D2(of N 91)、D1-D2(of N 92)、D1-D2(of N 93)、D1-D2(of N91-D20、 95)、D1-D2(delN 96)、D1-D2(delN 97)、D1-D2(delC 5)、D1-D2(delC 15)、D1-D2(del N 93)(delC 5)、del D1-D)2(del N16)、del C N93 93)(delC 11)、D1-D2(del N 93)(delC12)、D1-D2-D3(delC25)、D1-D2-D3(delC 41)、D1-D2-D3(delC 49)、D1-del(D1)D70(delC2-D3 C87)、D1-D2-D3(of C 96)、D1-D2-D3(of C 104)、D1-D2(of N 64)、D2-D3、D1-D2-D3、D2-D2-D3、D2-D3-D1、D2-D3-D3、D3 -D2-D3、D4-D2-D3、D1-D1-D2-D3、D1-D2-D3-D1、D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D3-4D2、D3-D2-D2 N 15)、D1-D2 (of N 33)、D1-D2 (of N 47) or D1-D2-D3 (of C 8. An RpBphP1 mutant, comprising an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, wherein the point mutations are Q514A, D607E, R638A, H661A, R696A, E327A, E332A, E337A, D339A, E365A, Q367A, E429A, R490A, D498A, Q502A, Q502G, Q502V, Q502L, Q502I, Q502S, Q502T, Q502E, Q502K, Q502R, Q502H, Q502N, Q502F, Q502Y, Q502W, Q502C, Q502M, Q502P , F503A, R504A, R507A, E513G, E513D, E513P, E516A, F518A, S525A, D607A, D60 7G, D607V, D607L, D607I, D607S, D607T, D607K, D607R, D607H, D607N, D607Q, D6 07F, D607W, D607C, D607M, D607P, E613A, R636A, F639A, D654A, E678A, Y681A, E 693A, H712D, H712E, H712P, R512A, E513V, E513A, E513L, E513T, E513M, Q521A.

9. An RpPpsR2 mutant, comprising an amino acid sequence having any one or more of the following point mutations in the amino acid sequence represented by SEQ ID NO: 3, wherein the point mutations are Q105T, T133A, L137A, N103A, Q105A, Q105C, Q105H, V107A, T108A, L110A, R113A, L114A, I115A , Q119A, M121A, M121E, R123A, D124A, Y125A, Y125E, W126A, R127A, R129A, E132A, R1 34A, R136A, Q105V, Q105S, K102A, Q105P, Q105R, L137E, V138A, V138E, F139E, V146A.

10. An RpPpsR2 mutant, wherein the amino acid sequence represented by SEQ ID NO: 3 is modified to D1-D1-D2, D3-D1-D2, D1-D2 (del N 77), D1-D2, D2-D1, D2-D2, D3-D2, D3-D2 (L137E), D4-D2, D1-D2-D1, D1-D2-D2, D1-D2-D4, D2-D1-D2, D2-D3-D4, D4-D1-D2, D2-D3-D4-D1, D2-D3-D4-D2, D2-D3-D4-D3, D2-D3-D4-D5, D1-D2 (del N 88), D1-D2 (del N 89), D1-D2 (del N 90), D1-D2 (del N 91), 91), D1-D2(del N 92), D1-D2(del N93), D1-D2(delN 94), D1-D2(delN 95), D1-D2(delN 96), D1-D2(delN 97), D1-D2(delC 5), D1-D2(delC 15), D1-D2(del N 93)(del C 5), D1-D2(delN 93)(delC 6), D1-D2(delN 93)(delC 11), D1-D2(delN 93)(delC 12), D1-D2-D3(delC 25), D1-D2-D3(delC 41), D1-D2-D3(del C 49), D1-D2-D3(del C 70), D1-D2-D3(del C 87), D1-D2-D3(delC 96), D1-D2-D3(delC 104), D1-D2(del N 64), D2-D3, D1-D2-D3, D2-D2-D3, D2-D3-D1, D2-D3-D3, D3-D2-D3, D4-D2-D3, D1-D1-D2-D3, D1-D2-D3-D1, D1-D2-D3 -D2、D1-D2-D3-D3、D1-D2-D3-D4、D2-D1-D2-D3、D2-D2-D3-D4、D2-D3-D4-D4、D3-D1-D2-D3、D3-D2-D3-D4、D1-D2(del N 15), D1-D2(del A mutant having an amino acid sequence obtained from any one of delN 33), D1-D2 (delN 47), and D1-D2-D3 (delC 9).

11. A nucleic acid encoding any one of the RpBphP1 mutants of claim 8.

12. A nucleic acid encoding any one of the RpPpsR2 mutants of claim 9.

13. A nucleic acid encoding any one of the RpPpsR2 mutants of claim 10.

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