Novel polypeptides exhibiting fluorescent properties and uses thereof

Abstract

Provided are a novel fluorescent protein and a use for the same. A polypeptide according to the present invention has fluorescent properties and is any one of (1) to (3):　(1) a polypeptide having the amino acid sequence of SEQ ID NO: 1; (2) a polypeptide having an amino acid sequence obtained by substituting, deleting, inserting, and / or adding 1-32 amino acids in the amino acid sequence of SEQ ID NO: 1; or (3) a polypeptide having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1.

Description

[Technical Field]

[0001] The present invention relates to novel polypeptides that exhibit fluorescent properties and uses thereof. [Background technology]

[0002] Fluorescent proteins have become indispensable tools for visualizing cells, tissues, and individual organisms.

[0003] In recent years, various bioimaging techniques using fluorescent proteins have been developed, and various modified versions of known fluorescent proteins have been reported (Non-Patent Documents 1 to 5). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Shaner, NC et al., Nature Methods 5, 545-551, 2008 [Non-patent document 2] Shaner, NC, Methods in Cell Biology 123, 95-111,2014 [Non-patent document 3] Bindels, DS et al. Nature Methods 14, 53-56, 2017 [Non-patent document 4] Zhong, S. et al. Journal of Neuroscience Methods 313, 68-76, 2019 [Non-patent document 5] Shaner, NC et al. Nature Methods 10, 407-409, 2013 Summary of the Invention [Problem to be solved by the invention]

[0005] The photostability of fluorescent dyes is one of the most pressing issues in bioimaging technology. Photobleaching makes it difficult to observe fluorescent signals from molecules of interest with low copy numbers for a sufficiently long period of time. Photobleaching is particularly critical in single-molecule imaging or imaging where the amount of fluorescent dye introduced is limited. Photobleaching also makes it difficult to quantitatively evaluate the target phenomenon. In this situation, there is a need to develop fluorescent proteins with superior photostability compared to previously reported fluorescent proteins.

[0006] The present inventors have succeeded in isolating a novel green fluorescent protein from the pearl jellyfish (Cytaeis uchidae).

[0007] That is, an object of the present invention is to provide novel fluorescent proteins that exhibit useful properties and uses thereof. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention includes any one of the following aspects.

[0009] <1> A polypeptide having fluorescent properties, as defined in any one of (1) to (3) below: (1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) A polypeptide having an amino acid sequence in which 1 to 32 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1; (3) A polypeptide having a sequence identity of 85% or more to the amino acid sequence set forth in SEQ ID NO:1. <2> exhibiting higher photostability than EGFP or brighter fluorescence than EGFP, <1> The polypeptide described in <3> The 168th amino acid of the amino acid sequence set forth in SEQ ID NO: 1 is alanine, and the amino acid sequence shows 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. <1> or <2> The polypeptide described in <4> A polynucleotide according to any one of (1) to (3) below: (1) a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) a polynucleotide encoding a polypeptide having fluorescent properties, which has an amino acid sequence in which 1 to 32 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1; (3) A polynucleotide that has 85% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and encodes a polypeptide that has fluorescent properties. <5> (a) an expression control region that is functional in the expression host; and (b) <4> An expression cassette comprising the polynucleotide described in <6> <4> or a polynucleotide according to <5> A vector comprising the expression cassette described in . <7> <4> a polynucleotide according to <5> or an expression cassette according to <6> A transformant having the vector described in . <8> a non-human transgenic organism, <7> The transformant described in <9> <1> ~ <3> A fusion polypeptide comprising any one of the polypeptides described above and another polypeptide. <10> Two or more connected <1> ~ <3> The polypeptide according to any one of <9> The fusion polypeptide according to claim 1. <11> <1> ~ <3> The polypeptide according to any one of <4> a polynucleotide according to item <5; an expression cassette according to item <5; <6> The vector according to <7> or <8> or <9> or <10> A kit comprising the fusion polypeptide described in . <12> <1> ~ <3> or a polypeptide according to any one of <9> or <10> A method for fluorescence observation comprising a production step of producing the fusion polypeptide described in claim 1 in a cell, an excitation light irradiation step of irradiating the cell with excitation light, and an observation step of observing fluorescence derived from the polypeptide or fusion polypeptide. [Effects of the Invention]

[0010] The present invention provides a fluorescent polypeptide having extremely high photostability, which has the advantage that it can be used in many fields such as molecular biology. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows the fluorescence excitation and emission spectra observed in C. uchidae in an example of the present invention. [Figure 2] 1 shows the absorption spectrum of StayGold (CU17S / V168A) in an example of the present invention. [Figure 3] 1 shows the fluorescence excitation and emission spectra of StayGold (CU17S / V168A) in an example of the present invention. [Figure 4] 1 shows an alignment of the amino acid sequences of the StayGold, CU17S and EGFP proteins of the present invention. [Figure 5] 1 shows a simple comparison of changes in fluorescence intensity over time for five types of green fluorescent proteins when purified proteins are used in an example of the present invention. [Figure 6] 1 shows normalized bleaching curves of five types of fluorescent proteins when purified proteins are used in an example of the present invention. [Figure 7] 1 shows normalized bleaching curves of 16 types of fluorescent proteins in an example of the present invention. [Figure 8] 1 shows a simple comparison of changes in fluorescence intensity over time of five types of green fluorescent proteins in living cells in an example of the present invention. [Figure 9] 1 shows normalized bleaching curves of five types of green fluorescent proteins in living cells in an example of the present invention. [Figure 10] FIG. 1 shows a comparison of photobleaching between StayGold-expressing cells and EGFP-expressing cells, and a comparison of photobleaching between StayGold-expressing cells and mNeonGreen-expressing cells, in an example of the present invention. [Figure 11]1 shows the results of labeling microtubules with tdStayGold in an example of the present invention. [Figure 12] 1 shows the results of labeling Golgi membranes with tdStayGold(long) in an example of the present invention. [Figure 13] 1 shows the results of labeling with a fusion protein of tdoxStayGold and the postsynaptic protein PSD-95 in an example of the present invention. [Figure 14] 1 shows the results of labeling the endoplasmic reticulum membrane with tdStayGold alpha in an example of the present invention. [Figure 15] 1 shows the results of labeling the endoplasmic reticulum lumen with er-(n2)oxStayGold(c4) in an example of the present invention. [Figure 16] 1 shows the results of labeling mitochondria with mt(n1)-StayGold in an example of the present invention. [Figure 17] 1 shows the results of pseudo-native PAGE for StayGold variants into which the L155T or Y187A mutation has been introduced in an example of the present invention. [Figure 18] 1 shows the results of pseudo-native PAGE for StayGold variants in which a mutation was further introduced into a StayGold variant having the L155T mutation in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Definitions of terms, etc.] As used herein, "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule." Unless otherwise specified, "polynucleotide" encompasses polynucleotides containing known analogs of naturally occurring nucleotides that can function in a similar manner to naturally occurring nucleotides. Furthermore, "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence." Unless otherwise specified, "base sequence" refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides. Furthermore, polynucleotides may be single-stranded or double-stranded, and if single-stranded, they may be the sense strand or the antisense strand.

[0013] As used herein, a "gene" refers to a "polynucleotide" that encodes a protein.

[0014] As used herein, the "expression control region" of a gene refers to a "polynucleotide" that controls the expression of the gene. Examples of the "expression control region" include a promoter region and an enhancer region.

[0015] As used herein, "expression cassette" refers to an expression unit comprising an expression control region functional in an expression host and a polynucleotide operably linked to the expression control region. In an expression cassette, the polynucleotide is preferably a gene or a gene fragment. An example of an expression cassette is one in which the expression control region and the polynucleotide are genetically linked. "Operably linked" refers to a state in which expression of the polynucleotide is controlled by an expression control sequence. The expression cassette may be in the form of an expression vector.

[0016] As used herein, "polypeptide" can be alternatively referred to as "protein." A "polypeptide" includes a structure formed by peptide bonds between amino acids, and may further include structures such as sugar chains or isoprenoid groups. Unless otherwise specified, a "polypeptide" also includes polypeptides containing known analogs of naturally occurring amino acids that can function in a similar manner to naturally occurring amino acids.

[0017] As used herein, the term "fluorescent polypeptide" refers to a polypeptide that has fluorescent properties. A polypeptide that has the property of emitting fluorescence when irradiated with excitation light of a specific wavelength.

[0018] In this specification, "A and / or B" is a concept that includes both A and B and A or B, and can also be expressed as "at least one of A and B."

[0019] 1. Polypeptides with fluorescent properties The polypeptide according to the present invention is a polypeptide having fluorescent properties (fluorescent polypeptide) shown in any one of the following (1) to (3).

[0020] (1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) A polypeptide having an amino acid sequence in which 1 to 32 amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1. The number of substituted, deleted, inserted, and / or added amino acids is preferably 1 to 26, more preferably 1 to 21, more preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, and particularly preferably 1 to 4. Hereinafter, amino acid substitutions, deletions, insertions, and / or additions may be collectively referred to as amino acid mutations.

[0021] (3) A polypeptide having 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The sequence identity is preferably 88% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, and particularly preferably 98% or more or 99% or more.

[0022] The origin of the fluorescent polypeptide is not limited, and it may be, for example, chemically synthesized or produced using recombinant DNA technology. More specifically, the fluorescent polypeptide includes isolated and purified polypeptides, chemically synthesized polypeptides, and polypeptides produced from host cells based on recombinant DNA technology. Host cells are described in detail in the section explaining "transformants."

[0023] An example of a fluorescent polypeptide according to the present invention is derived from the pearl jellyfish (Cytaeis uchidae). The present inventors have succeeded in isolating a novel green fluorescent protein from the pearl jellyfish (Cytaeis uchidae). Furthermore, by modifying this novel fluorescent protein, they have succeeded in obtaining a variant that is bright and does not fade for a long time. An example of a fluorescent polypeptide is the fluorescent polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 and is called "StayGold." StayGold has bright fluorescence and is significantly more photostable than any of the conventionally known fluorescent proteins currently available.

[0024] The main fluorescent properties of StayGold are as follows: Maximum excitation wavelength (nm): 496 Maximum fluorescence wavelength (nm): 505 (green) Molar extinction coefficient (M -1 cm -1 ): 159000 (at 496 nm) Quantum yield (%): 93 Fluorescence lifetime (ns): 2.81 The fluorescent polypeptides shown in (2) or (3) above can be considered to be variants of the fluorescent polypeptide shown in (1) above. The fluorescent polypeptides shown in (2) or (3) can be obtained, for example, by expressing a polynucleotide encoding the fluorescent polypeptide shown in (1) above, in which mutations have been artificially introduced using site-directed mutagenesis. Examples of site-directed mutagenesis methods include the Kunkel method (Kunkel et al. (1985): Proc. Natl. Acad. Sci. USA, vol. 82, p. 488-).

[0025] One example of the fluorescent polypeptide described in (2) or (3) above is a fluorescent polypeptide having the amino acid sequence set forth in SEQ ID NO: 4. The fluorescent polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 is one of the fluorescent protein clones isolated from Acanthus pulcherrimus and is referred to as "CU17S." StayGold is a variant of CU17S obtained by modifying CU17S, in which the 168th amino acid, valine (V), in the amino acid sequence set forth in SEQ ID NO: 4 (the amino acid sequence of CU17S) is replaced with alanine (A).

[0026] That is, in another embodiment, the polypeptide according to the present invention also includes a fluorescent polypeptide shown in any one of (4) to (6) below.

[0027] (4) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 4. (5) A polypeptide having an amino acid sequence in which 1 to 32 amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 4. The number of substituted, deleted, inserted, and / or added amino acids is preferably 1 to 26, more preferably 1 to 21, more preferably 1 to 10, still more preferably 1 to 8, even more preferably 1 to 6, and particularly preferably 1 to 4.

[0028] (6) A polypeptide having 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. The sequence identity is preferably 88% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, and particularly preferably 98% or more or 99% or more.

[0029] An example of the fluorescent polypeptide shown in (2) or (3) above is an amino acid sequence in which the 168th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is alanine and which has an amino acid sequence that shows 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0030] An example of the fluorescent polypeptide shown in (2) or (3) above is a fluorescent polypeptide having the amino acid sequence set forth in SEQ ID NO:6.

[0031] The fluorescent polypeptide described in (2) or (3) above may exhibit fluorescent properties equivalent to those of the fluorescent polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 1. Here, "equivalent fluorescent properties" refers to having at least one or more of the following: an excitation wavelength, a fluorescence wavelength, a pH sensitivity, photostability, a molar extinction coefficient, a fluorescence quantum efficiency, an excitation spectrum or an emission spectrum shape, an excitation wavelength maximum and an emission wavelength maximum, an excited state lifetime, and a chromophore maturation rate equivalent to those of the fluorescent polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 1. "Equivalent fluorescent properties" preferably refer to photostability and fluorescence brightness when introduced into cells, as well as an equivalent quantum yield.

[0032] Examples of fluorescent polypeptides having an excitation wavelength equivalent to that of StayGold include, but are not limited to, those with a maximum excitation wavelength in the range of 486 nm to 506 nm, and those with a maximum fluorescence wavelength equivalent to that of StayGold include, but are not limited to, those with a maximum fluorescence wavelength in the range of 495 nm to 515 nm.

[0033] The fluorescence of fluorescent polypeptides is observed to fade over time, and as this fade progresses, it becomes difficult to observe the fluorescence. The fluorescent polypeptides of the present invention have high photostability. The photostability of fluorescence can be evaluated using resistance to fading as an indicator. Higher photostability can have the effect of extending the observable time.

[0034] The fluorescent polypeptides of the present invention are resistant to fading. One example of the fluorescent proteins of the present invention is less susceptible to fading than known fluorescent proteins, and maintains high fluorescence intensity for a long period of time. For example, the fluorescent polypeptides of the present invention preferably take 1,000 seconds or more, more preferably 5,000 seconds or more, for the number of photons emitted per molecule per second to halve from 1,000 to 500, based on a standard method for evaluating photostability.

[0035] The fluorescent polypeptides of the present invention are resistant to bleaching and can therefore be suitably used, for example, for single-molecule imaging or for observing fluorescence in cells expressing a small number of copies of the fluorescent polypeptide.

[0036] Furthermore, the fluorescent polypeptides of the present invention exhibit bright fluorescence. Absolute fluorescence brightness can be evaluated using the product of the absolute molar extinction coefficient and the fluorescence quantum efficiency as an index. Meanwhile, actual fluorescence brightness can be evaluated using the product of the effective molar extinction coefficient and the fluorescence quantum efficiency, taking into account the maturation speed of the chromophore.

[0037] The absolute molar extinction coefficient (M -1 cm -1 ) is 100,000 or more, preferably 120,000 or more, more preferably 130,000 or more, even more preferably 140,000 or more, and particularly preferably 150,000 or more. In addition, the fluorescence quantum efficiency Φ is 0.75 (75%) or more, preferably 0.80 (80%) or more, and more preferably 0.90 (90%) or more.

[0038] As will be shown in the Examples, this quantum yield value is significantly higher than that of conventional green fluorescent proteins. The higher the fluorescence quantum yield, the greater the fluorescence intensity and generally the brighter the fluorescence, making it more suitable for use in fluorescence observation, etc.

[0039] An example of a fluorescent polypeptide of the present invention exhibits greater photostability than EGFP or is brighter in fluorescence than EGFP.

[0040] In fluorescent polypeptides, the amino acid sequence XYG (X represents any amino acid) is known to form a chromophore. In the fluorescent polypeptide shown in (1) above, the amino acid sequence at positions 57 to 59 in SEQ ID NO: 1 is GYG. Therefore, in the fluorescent polypeptides shown in (2) or (3) above, the amino acid at position 57 in SEQ ID NO: 1 may be substituted, but it is preferable that the amino acids at positions 58 to 59 remain unchanged, and it is even more preferable that all of the amino acids at positions 57 to 59 remain unchanged.

[0041] Furthermore, the fluorescent polypeptide may be either a monomer or a polymer. For example, when labeling a molecule or when using it as a probe for FRET (fluorescence resonance energy transfer), it is preferable that the fluorescent polypeptide is a monomer. Both the leucine (L) at position 155 and the tyrosine (Y) at position 187 in the amino acid sequence set forth in SEQ ID NO: 1 are presumed to be particularly involved in the formation of polymers of the fluorescent polypeptide.

[0042] Furthermore, in the amino acid sequence set forth in SEQ ID NO: 1, leucine (L) at position 135, proline (P) at position 136, glutamic acid (E) at position 138, isoleucine (I) at position 142, arginine (R) at position 144, leucine (L) at position 155, cysteine ​​(C) at position 165, glutamic acid (E) at position 167, tyrosine (Y) at position 187, and tryptophan (W) at position 189 are all involved in the dimerization of the fluorescent polypeptide, i.e., form an interface. Therefore, monomerization can be achieved by replacing at least one of these amino acids with any other amino acid.

[0043] To make the fluorescent polypeptide monomeric, it is preferable to have an amino acid substitution of at least one of the leucine (L) at amino acid position 155 and the tyrosine (Y) at amino acid position 187 in the amino acid sequence set forth in SEQ ID NO: 1. In particular, to make the fluorescent polypeptide monomeric, it is preferable to have a substitution of leucine (L) at amino acid position 155 with threonine (T) and a substitution of tyrosine (Y) at amino acid position 187 with alanine (A) in SEQ ID NO: 1. Examples of such fluorescent polypeptides include the amino acid sequence set forth in SEQ ID NO: 29, which has a substitution of leucine (L) at amino acid position 155 with threonine (T), and the amino acid sequence set forth in SEQ ID NO: 27, which has a substitution of tyrosine (Y) at amino acid position 187 with alanine (A). The nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 29 is set forth in SEQ ID NO: 30, and the nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 27 is set forth in SEQ ID NO: 28.

[0044] In addition to the substitution of leucine (L) at amino acid position 155 and tyrosine (Y) at amino acid position 187 in the amino acid sequence set forth in SEQ ID NO: 1, it is further preferred that there be an amino acid substitution at one or more amino acids selected from the group consisting of leucine (L) at amino acid position 135, proline (P) at amino acid position 136, glutamic acid (E) at amino acid position 138, isoleucine (I) at amino acid position 142, arginine (R) at amino acid position 144, cysteine ​​(C) at amino acid position 165, glutamic acid (E) at amino acid position 167, and tryptophan (W) at amino acid position 189 in the amino acid sequence set forth in SEQ ID NO: 1. These amino acids are present at the protein interface of StayGold, with their side chains facing outward from the protein, and are presumed to be sites that particularly affect multimerization. Therefore, substitution of these amino acids enables StayGold to be more stably monomerized. Furthermore, the amino acids asparagine (N) at position 132, proline (P) at position 151, and lysine (K) at position 162 are also present at the protein interface and may influence multimerization.

[0045] The amino acid substitutions predicted to affect the multimerization of the above-mentioned proteins are not particularly limited to the amino acids after substitution, as long as they can produce the desired effect. For example, the amino acid substitutions may be one or more selected from the group consisting of a substitution of leucine (L) at amino acid position 135 with threonine (T), a substitution of proline (P) at amino acid position 136 with tyrosine (Y), a substitution of glutamic acid (E) at amino acid position 138 with glutamine (Q), a substitution of isoleucine (I) at amino acid position 142 with threonine (T), a substitution of arginine (R) at amino acid position 144 with threonine (T), a substitution of cysteine ​​(C) at amino acid position 165 with glutamine (Q), a substitution of glutamic acid (E) at amino acid position 167 with alanine (A), and a substitution of tryptophan (W) at amino acid position 189 with tyrosine (Y) in the amino acid sequence set forth in SEQ ID NO: 1.

[0046] The number of mutations for monomerizing the fluorescent polypeptide is not particularly limited. For example, the fluorescent polypeptide may contain one or more of the above-mentioned mutations, two or more, or three or more. Furthermore, the fluorescent polypeptide may contain six or less of the above-mentioned mutations, five or less, or four or less.

[0047] Furthermore, the fluorescent polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 is a variant of the fluorescent polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 (the H169Y / C174I / C208I variant of StayGold), which has a substitution of histidine (H) at amino acid position 169 with tyrosine (Y) (H169Y), a substitution of cysteine ​​(C) at amino acid position 174 with isoleucine (I) (C174I), and a substitution of cysteine ​​(C) at amino acid position 208 with isoleucine (I) (C208I). Because this variant contains fewer cysteines, it exhibits more stable folding in the oxidized state and is therefore useful, for example, for labeling the lumen of the endoplasmic reticulum. Furthermore, variants with the H169Y, C174I, or C208I substitutions are also useful in that they enhance the fluorescence of fluorescent proteins. A mutant having an H169Y, C174I or C208I substitution may also have an L155T or Y187A mutation.

[0048] 2. Polynucleotides Encoding Fluorescent Polypeptides The polynucleotide of the present invention encodes any of the above fluorescent polypeptides.

[0049] Specifically, the polynucleotide encoding the fluorescent polypeptide is any one of the polynucleotides (1) to (3) below.

[0050] (1) a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) a polynucleotide encoding a polypeptide having fluorescent properties, which has an amino acid sequence in which 1 to 32 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1; (3) A polynucleotide encoding a polypeptide having fluorescent properties and having 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. This polynucleotide preferably has 88% or more sequence identity to the base sequence of the polynucleotide set forth in (1) above, more preferably 90% or more sequence identity, preferably 95% or more sequence identity, more preferably 96% or more sequence identity, even more preferably 97% or more sequence identity, and particularly preferably 98% or more or 99% or more sequence identity.

[0051] The polynucleotide of the present invention may exist in the form of RNA or DNA. An example of the RNA form is mRNA. An example of the DNA form is cDNA or genomic DNA. The DNA may be double-stranded or single-stranded.

[0052] The base sequences shown in SEQ ID NOs: 2 and 3, which are examples of polynucleotides according to the present invention, are cDNAs encoding the fluorescent polypeptides shown in SEQ ID NO: 1, and the base sequences shown in SEQ ID NOs: 5 and 7 are cDNAs encoding the fluorescent polypeptides shown in SEQ ID NOs: 4 and 6, respectively. Furthermore, polynucleotides according to the present invention may contain additional sequences such as sequences of untranslated regions (UTRs).

[0053] The method for obtaining (isolating) the polynucleotide of the present invention is not particularly limited, but for example, a probe that specifically hybridizes with a portion of the base sequence of the polynucleotide may be prepared and used to screen a genomic DNA library or a cDNA library. Alternatively, the polynucleotide of the present invention may be synthesized according to a nucleic acid synthesis method such as the phosphoramidite method.

[0054] Another method for obtaining the polynucleotide of the present invention is to use a nucleic acid amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and these primers are used to perform PCR or the like using genomic DNA or cDNA as a template to amplify the DNA region between the two primers. This allows for the large-scale production of DNA fragments containing the polynucleotide of the present invention.

[0055] 3. Vectors and Expression Cassettes The polynucleotide (e.g., DNA) of the present invention may be inserted into an appropriate vector and used as a vector. The type of vector may be an autonomously replicating vector such as a plasmid, or may be a vector that, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosomes of the host cell.

[0056] The vector is preferably an expression vector. In the expression vector, the polynucleotide of the present invention is functionally linked to elements necessary for transcription, such as a promoter sequence. The promoter sequence is a DNA sequence that exhibits transcriptional activity in host cells. The type of promoter sequence to be used may be appropriately selected depending on the type of host cell and the purpose for which the fluorescent polypeptide of the present invention is used. Examples of types of host cells include those described in [4. Transformants and methods for producing transformants].

[0057] Examples of promoter sequences operable in the host cell include those derived from the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene. Examples of promoters include promoters of the phage lambda (protease inhibitor) gene; PR or PL promoters; Escherichia coli lac promoter, trp promoter, and tac promoter; polyhedrin promoter, P10 promoter, Autographa californica polyhedrosis basic protein promoter, baculovirus immediate early gene 1 promoter, baculovirus 39K delayed early gene promoter, promoters derived from yeast glycolysis genes, alcohol dehydrogenase gene promoter, TPI1 promoter, ADH2-4c promoter, ADH3 promoter, tpiA promoter, cauliflower mosaic virus 35S promoter, SV40 promoter, MT-1 (metallothionein gene) promoter, cytomegalovirus promoter, and adenovirus 2 major late promoter.

[0058] In the expression vector, the polynucleotide of the present invention may be functionally linked to a suitable terminator (e.g., a polyadenylation signal, a mammalian growth hormone terminator, a TPI1 terminator, or an ADH3 terminator) as needed. The type of suitable terminator may be selected appropriately depending on the type of host cell.

[0059] The vector of the present invention may further comprise elements such as a transcription enhancer sequence.

[0060] The vector of the present invention may further comprise a DNA sequence that enables replication of the vector in a host cell. When the host cell is a mammalian cell that expresses a large T antigen, such a DNA sequence may include the SV40 replication origin.

[0061] The vectors of the present invention may further have a selection marker. Examples of the selection marker include drug resistance genes for drugs such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, or hygromycin. These selection markers can be used for any type of vector.

[0062] The expression cassette of the present invention refers to an expression cassette comprising (a) an expression control region functional in an expression host; and (b) a polynucleotide of the present invention. The expression cassette of the present invention may be in the form of the above-mentioned expression vector.

[0063] 4. Transformants and methods for producing transformants (Transformants and methods for producing transformants) A transformant having the polynucleotide of the present invention, the expression cassette of the present invention, or the vector of the present invention can be produced by introducing the polynucleotide of the present invention, the expression cassette of the present invention, or the vector of the present invention into an appropriate host cell. The produced transformant contains the full-length polynucleotide of the present invention or at least a portion of the polynucleotide and is capable of expressing any of the fluorescent polypeptides of the present invention. Similarly, progeny of the transformant obtained using the transformant of the present invention also contain the full-length polynucleotide of the present invention or at least a portion of the polynucleotide and are capable of expressing any of the fluorescent polypeptides of the present invention. In the produced transformant or its progeny, it is preferred that the full-length or a portion of the polynucleotide of the present invention is integrated into the genome.

[0064] In the following description, the polynucleotide of the present invention, the expression cassette of the present invention, and the vector of the present invention are collectively referred to as the "foreign nucleic acid molecule" of the present invention. The method for introducing the foreign nucleic acid molecule of the present invention into a host cell may be selected depending on the type of host cell, as exemplified below. Furthermore, the method for obtaining offspring of the transformant of the present invention may also be selected depending on the type of transformant.

[0065] Host cells include, for example, bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Higher eukaryotic cells include, for example, plant cells and animal cells. Animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells. Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces; and Gram-negative bacteria such as Escherichia coli. Examples of yeast cells include cells belonging to the genus Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae or Saccharomyces kluyveri. Examples of fungal cells other than yeast cells include cells of filamentous fungi. Examples of filamentous fungal cells include cells of filamentous fungi belonging to the genus Aspergillus, Neurospora, Fusarium, or Trichoderma. Examples of insect cells include silkworm cells, etc. Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, and CHO cells.

[0066] Transformation of host cells can be performed by any method appropriately selected depending on the type of host cell, etc., and can be performed by, for example, the protoplast method, a method using competent cells, electroporation, spheroblast method, lithium acetate method, calcium phosphate method, lipofection method, Agrobacterium method, and particle gun method. Other methods for transforming host cells include methods that involve obtaining host cells in which the foreign nucleic acid molecule of the present invention has been integrated into the host chromosome. Integration of the foreign nucleic acid molecule into the host chromosome can be achieved, for example, by homologous recombination or heterologous recombination. Still other methods for transforming host cells include co-transfecting the foreign nucleic acid molecule of the present invention and a baculovirus into host cells to obtain a recombinant baculovirus in the culture supernatant of the host cells, and then infecting the host cells with the recombinant baculovirus to cause the host cells to produce the fluorescent polypeptide of the present invention. Examples of co-transfection methods include the calcium phosphate method and lipofection method.

[0067] The transformants are cultured or grown under conditions that allow expression of the introduced foreign nucleic acid molecule.

[0068] The form of the transformant is not limited to cells. That is, the transformant may be, for example, a tissue, organ, or individual transformed with the foreign nucleic acid molecule of the present invention. However, transformants other than cells may preferably be of non-human origin, and in particular, individuals of non-human origin are preferred. Transformed individuals of non-human origin are referred to as non-human transgenic organisms.

[0069] (Non-human transgenic organisms and methods for producing same) The non-human transgenic organisms of the present invention are, for example, higher organisms. Examples of transgenic plants include dicotyledonous plants such as Arabidopsis thaliana; and monocotyledonous plants such as Brachypodium distachyon, rice, wheat, and barley. Examples of transgenic animals include transgenic zebrafish, mice, rats, and pigs.

[0070] The method for producing a non-human transgenic organism according to the present invention may be selected depending on the type of transgenic organism. Examples of methods for producing transgenic animals include introducing the foreign nucleic acid molecule according to the present invention into a fertilized egg collected from a donor organism ex vivo using microinjection or the like; or infecting early-developing embryonic cells derived from a donor organism ex vivo with a viral vector such as a retrovirus. To produce a transgenic plant, the foreign nucleic acid molecule according to the present invention may be introduced into a plant cell using, for example, the Agrobacterium method, particle gun method, or electroporation method, followed by a callus formation process, if necessary, to obtain a transformed plant.

[0071] Furthermore, the method for obtaining offspring of the non-human transgenic organism of the present invention may also be selected depending on the type of the non-human transgenic organism. In the case of higher organisms, for example, methods for obtaining offspring by mating may be used. In the case of plants among higher organisms, offspring may be obtained using asexual reproduction techniques appropriate for the type of plant.

[0072] (Cloning of non-human transgenic organisms and methods for producing clones) The present invention also encompasses, for example, the creation of clones using the non-human transgenic organisms of the present invention. The clones created, like the original non-human transgenic organisms, contain the full length of the polynucleotides of the present invention or at least a portion of the polynucleotides in their genomes and are capable of expressing any of the fluorescent polypeptides of the present invention. The term "clone" encompasses both germ cell clones and somatic cell clones.

[0073] An example of a method for producing a clone is nuclear transplantation, in which a donor cell nucleus is transplanted into a recipient unfertilized egg from which the nucleus has been removed. Here, the donor cell nucleus can be 1) the somatic cell nucleus of the original non-human transgenic organism, or 2) an embryonic cell nucleus derived from the original non-human transgenic organism. The donor cell nucleus contains the full length of the polynucleotide of the present invention in its genome, or at least a portion of the polynucleotide.

[0074] The method for nuclear transplantation of a donor cell nucleus is not particularly limited, and examples include: 1) a method of cell fusion between an enucleated unfertilized egg and a donor cell; and 2) a method of introducing a donor cell into an enucleated unfertilized egg without cell fusion.

[0075] 5. Recombinant antibodies in which any antibody protein is genetically linked to the fusion polypeptide or fluorescent polypeptide of the present invention (fusion polypeptide) Fusion polypeptides comprising the fluorescent polypeptide of the present invention and another polypeptide (hereinafter referred to as the fusion polypeptide of the present invention) are also within the scope of the present invention. Examples of fusion polypeptides include fusion proteins produced by expressing the expression cassette and / or vector of the present invention; fusion proteins in which any protein is labeled with the fluorescent polypeptide of the present invention; fusion proteins in which the fluorescent polypeptide of the present invention is fused with a specific peptide sequence for stabilizing fluorescence; and FRET probes comprising the fluorescent polypeptide of the present invention and another fluorescent polypeptide. In other words, the type of other polypeptide to be fused with the fluorescent polypeptide of the present invention is not particularly limited.

[0076] The present invention also encompasses fusion polypeptides in which any other polypeptide is linked to both the N-terminus and C-terminus, or to either the N-terminus or C-terminus, of the fluorescent polypeptide of the present invention. The length and sequence of the linked polypeptide are not limited.

[0077] In one example of the fusion protein of the present invention, both the N-terminus and C-terminus, or either the N-terminus or C-terminus, of the fluorescent polypeptide of the present invention are linked to another polypeptide via an insertion sequence consisting of any amino acid sequence. Another example of the fusion protein of the present invention has an insertion sequence consisting of any amino acid sequence at the insertion position anywhere between the first and fifth amino acids at the N-terminus of the fluorescent polypeptide of the present invention.

[0078] Any of these insertion sequences can be amino acid sequences of 3 or more, 5 or more, 7 or more, or 10 or more amino acids, or the insertion sequence can be an amino acid sequence of 30 or less, 20 or less, or 15 or less amino acids.

[0079] For example, in a fusion protein formed by linking the C-terminus of a fluorescent polypeptide of the present invention to any other polypeptide, the C-terminus of the fluorescent polypeptide of the present invention and the other polypeptide are linked via an amino acid sequence consisting of 10 amino acids set forth in SEQ ID NO: 8 (referred to as insertion sequence c4).

[0080] Furthermore, fusion proteins formed by linking either the N-terminus or C-terminus of a fluorescent polypeptide of the present invention to any other polypeptide include those in which an amino acid sequence consisting of nine amino acids set forth in SEQ ID NO: 9 or SEQ ID NO: 10 (referred to as insertion sequence n1 or n2, respectively) is inserted between the first and fifth amino acids of the N-terminus of the fluorescent polypeptide of the present invention. Examples include those in which insertion sequence n1 (SEQ ID NO: 9) is inserted between the fourth and fifth amino acids of the N-terminus of a fluorescent polypeptide of the present invention, or those in which insertion sequence n2 (SEQ ID NO: 10) is inserted between the third and fourth amino acids of the N-terminus of a fluorescent polypeptide of the present invention. Fusion proteins also include those containing both insertion sequence c4 and insertion sequence n1 or n2. The presence of such insertion sequences can improve the photostability of the fusion protein.

[0081] Furthermore, when both the N-terminus and C-terminus, or either the N-terminus or C-terminus, of the fluorescent polypeptide of the present invention are linked to any other polypeptide via an insertion sequence consisting of any amino acid sequence, more stable fluorescence can be obtained than when any other polypeptide is directly linked to the fluorescent polypeptide of the present invention.

[0082] Further examples of fusion proteins include two or more linked fluorescent polypeptides of the present invention, either of the same or different types. Specifically, these include tandem dimers of two linked fluorescent polypeptides of the present invention, either of the same or different types, and fusion proteins of three or more linked fluorescent polypeptides. In such tandem dimers of fluorescent polypeptides of the present invention and fusion proteins of three or more linked fluorescent polypeptides, the fluorescent polypeptides may be linked via an insertion sequence having any sequence. An insertion sequence consisting of any amino acid sequence may be inserted anywhere between the first and fifth amino acids at the N-terminus of the fluorescent polypeptide of the present invention. The amino acid sequence and length of the insertion sequence are not limited, but may be, for example, 10 to 150 amino acids. The insertion sequence may be as described above. Furthermore, any other polypeptide may be added to both the N-terminus and C-terminus of the linked fluorescent polypeptides, or to either the N-terminus or C-terminus. The amino acid sequence and length of the added polypeptide are not limited, but may be, for example, 5 to 20 amino acids.

[0083] In tandem dimers and fusion proteins in which three or more proteins are linked, the amino acid sequences of each protein may be linked via a linker sequence known as an insertion sequence. For example, depending on the purpose, each amino acid sequence may be linked using an EV linker, an alpha linker, or the like.

[0084] Examples of tandem dimers in which two fluorescent polypeptides of the present invention are linked include those having the amino acid sequence of SEQ ID NO: 13 (referred to as tdStayGold), those having the amino acid sequence of SEQ ID NO: 15 (referred to as tdoxStayGold), those having the amino acid sequence of SEQ ID NO: 17 (referred to as tdoxStayGold), and those having the amino acid sequence of SEQ ID NO: 19 (referred to as tdStayGold alpha). The nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 13 is shown in SEQ ID NO: 14, the nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 15 is shown in SEQ ID NO: 16, the nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 17 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 19 is shown in SEQ ID NO: 20. Other examples of tandem dimers in which two fluorescent polypeptides of the present invention are linked include those having amino acid sequences that show 85% or more, 88% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequences of the tandem dimers listed above.

[0085] The fusion polypeptide of the present invention may be chemically synthesized or produced using recombinant gene technology in the same manner as the fluorescent polypeptide of the present invention.

[0086] A fusion protein in which a tandem dimer formed by linking two fluorescent polypeptides of the present invention as described above is fused to a target protein for visualization can be suitably used for dynamic analysis of the target protein by observing its intracellular fluorescence.

[0087] (antibody) The present invention also encompasses recombinant antibodies in which any antibody protein is genetically linked to the fluorescent polypeptide of the present invention. The antibody may be an antibody fragment that retains its specific binding ability to an antigen.

[0088] 6. Pluripotent Stem Cells and Methods for Producing Pluripotent Stem Cells The present invention also encompasses a method for producing pluripotent stem cells from cells of the non-human transgenic organism of the present invention. The method for producing pluripotent stem cells comprises a step (reprogramming step) of producing pluripotent stem cells by introducing reprogramming factors into cells (sometimes referred to as starting cells) collected from the non-human transgenic organism of the present invention or by treating the cells with reprogramming factors. The non-human transgenic organism is, for example, a non-human transgenic higher animal, particularly a non-human transgenic mammal.

[0089] (Resulting pluripotent stem cells) The obtained pluripotent stem cells exhibit at least multipotency, and more preferably exhibit a state exhibiting pluripotency or a state prior to that. In the present invention, multipotency refers to the ability to differentiate into certain cell types, such as those of the nervous system or hematopoietic system. In the present invention, pluripotency refers to the ability to differentiate into all of the cells and tissues that constitute an individual, but not to constitute the individual itself. One example of the pluripotent stem cells obtained is the so-called "induced pluripotent stem cell." "Induced pluripotent stem cells" are cells with properties similar to those of embryonic stem cells (ES cells), and more specifically, include undifferentiated cells that have pluripotency and undifferentiated proliferation ability depending on the culture conditions.

[0090] 7. Fluorescence Observation Methods The uses of the fluorescent polypeptide or fusion polypeptide of the present invention are not particularly limited, and can be widely used for fluorescence observation. A fluorescence observation method includes a production step of producing the polypeptide or fusion polypeptide of the present invention in cells, an excitation light irradiation step of irradiating the cells with excitation light, and an observation step of observing the fluorescence derived from the polypeptide or fusion polypeptide.

[0091] The production step can be carried out, for example, by the method described in the section "4. Transformants and Methods for Producing Transformants" above. The fluorescence observation step is a step of observing the fluorescence emitted from the polypeptide or fusion polypeptide of the present invention. The observation step is carried out by detecting the fluorescence emitted from the polypeptide or fusion polypeptide of the present invention. The fluorescence detection method is not particularly limited, but examples include fluorescence detection means such as a UV transilluminator or LED transilluminator, a fluorescence microscope, a fluorescence detector, or flow cytometry. Most preferred methods include a fluorescence microscope, single-molecule imaging (total internal reflection TIRFM), and a light-sheet microscope. Less preferred methods include a structured illumination microscope (SIM) and a Nipkow disk confocal (multifocal) microscope. Using the above-mentioned fluorescence detection means, the presence or absence of fluorescence, the distribution of fluorescence, or the fluorescence intensity can be measured temporarily or over time.

[0092] As a method of irradiating the excitation light in the excitation light irradiation step, for example, Kohler illumination is preferably used to illuminate the object using a microscope objective lens, as this method provides a higher effect of improving the photostability of the fluorescent polypeptide of the present invention than does critical illumination.

[0093] In the observation step, the fluorescence derived from the polypeptide or fusion polypeptide may be observed by taking two-dimensional or three-dimensional images or videos.

[0094] Furthermore, the captured fluorescence images or videos may be analyzed or processed using appropriate information processing technology. For example, it is possible to accumulate a large amount of image or video information and use artificial intelligence (AI) to perform machine learning.

[0095] Another example of fluorescence observation is a method comprising a step of introducing the fluorescent polypeptide or fusion polypeptide of the present invention into cells (introduction step) and the above-mentioned "fluorescence observation step." Examples of methods for introducing a fluorescent polypeptide or the like into cells include the microinjection method, in which a purified fluorescent polypeptide or the like is injected into cells.

[0096] One purpose of fluorescence observation is to analyze the localization or dynamics of a polypeptide. By using a fusion polypeptide obtained by genetically fusing the fluorescent polypeptide of the present invention with another polypeptide (referred to as polypeptide X), it becomes possible to visualize and analyze the localization or dynamics of polypeptide X within a cell. The type of polypeptide X is not particularly limited, and examples thereof include proteins localized within cells, proteins specific to intracellular organelles, and targeting signals. Examples of targeting signals include nuclear localization signals, mitochondrial localization signals, plasma membrane localization signals, and endoplasmic reticulum localization signals.

[0097] Another purpose of fluorescence observation is to analyze the expression of a target gene. By performing the above production process under the control of the expression control sequence of the target gene, the activity of the expression control sequence can be measured. The activity of the expression control sequence of the target gene reflects the expression level of the target gene.

[0098] 8. Method for producing fluorescent polypeptides The present invention also provides methods for producing mutant fluorescent polypeptides based on a fluorescent polypeptide having any of the amino acid sequences set forth in SEQ ID NOs: 1, 4 or 6.

[0099] That is, one embodiment of the method for producing a fluorescent polypeptide according to the present invention comprises: i) preparing a mutant polypeptide in which at least one amino acid mutation has been introduced into an amino acid other than positions 58 and 59 in the amino acid sequence set forth in SEQ ID NO: 1, 4, or 6; ii) comparing the fluorescent properties of the mutant polypeptide with the fluorescent properties of the polypeptide before mutation; iii) a selection step of selecting mutant polypeptides whose fluorescence properties have changed in the comparison step compared to before the mutation was introduced.

[0100] In the process of preparing the mutant polypeptide, -YG of the amino acid sequence XYG (X represents any amino acid) that forms the chromophore is fixed, and the other amino acids are mutated. This makes it possible to efficiently produce mutant polypeptides that retain fluorescent properties. Note that for SEQ ID NOS: 1, 4, and 6, the number of amino acids to be mutated and the method for mutating the amino acids can be found, for example, in the above section [1. Polypeptides with fluorescent properties].

[0101] Furthermore, in the above selection step, mutant polypeptides that have fluorescent properties but whose fluorescent properties have been altered are selected, and mutant polypeptides that have completely lost their fluorescent properties are excluded.

[0102] The above-described method for producing mutant fluorescent polypeptides can also be considered as a method for screening mutant fluorescent polypeptides.

[0103] 9. Kit of the Present Invention (kit) The kit of the present invention comprises at least one selected from the group consisting of 1) the fluorescent polypeptide of the present invention, 2) a polynucleotide encoding the fluorescent polypeptide of the present invention, 3) the expression cassette of the present invention, 4) the vector of the present invention, 5) the transformant of the present invention, and 6) the fusion polypeptide of the present invention. When the polynucleotide of 2) above is RNA, the kit can be applied to individual organisms such as humans as a transient expression kit that does not involve recombination into a genomic gene.

[0104] The kit of the present invention can be prepared using materials and techniques known in the art. Reagents such as fluorescent polypeptides or polynucleotides can be prepared in a form suitable for storage by dissolving them in a suitable solvent. Examples of solvents that can be used include water, ethanol, and various known buffer solutions.

[0105] The kit of the present invention may further include at least one of various reagents and tools (buffer solutions, test tubes, pipettes, etc.) and instructions for use of the kit, as needed. The instructions for use of the kit may include, for example, details of the detection method of the present invention, as explained above in the section "7. Observation using fluorescent polypeptides, etc." The kit may be used, for example, as a reagent or for diagnostic purposes.

[0106] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0107] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples. [Example]

[0108] [1. Cloning of genes from the pearl jellyfish] (material and method) RNA was extracted from the giant jellyfish Cytaeis uchidae (Rees, 1962) (Phylum Cnidaria, Hydrozoa, Anthomedusae, Cytaeidae, Cytaeis (Cnidaria, Hydrozoa, Anthomedusae, Cytaeidae, Cytaeis)). Total RNA from C. uchidae was used for RNA sequencing and subsequent anchored PCR analysis. Details are as follows.

[0109] We discovered that Unigene (#1784) encodes a polypeptide containing a GFP-like domain. This polypeptide contained two GYG sequences thought to be involved in chromophore synthesis. 5'-RACE-PCR using this polypeptide as a template with the CU17 5' RACE primer revealed an N-terminal extension containing an additional GYG sequence. Next, RT-PCR using the CU17 1st_Fwd and CU17 Rev primers yielded two RNA transcripts encoding distinct polypeptides (designated CU17L and CU17S).

[0110] CU17L corresponds to the protein product of #1784 and appears to be a three-domain fluorescent protein. However, CU17L itself did not fluoresce in the expression system used by the inventors. Furthermore, when expressed separately, no fluorescent domains were observed. On the other hand, CU17S appears to be a single-domain fluorescent protein. Interestingly, the N-terminal region of CU17S (approximately three-quarters of the total protein length) shared 84.8% sequence identity with the corresponding region of the first repeat of CU17L. Furthermore, the C-terminal region of CU17S (approximately one-quarter of the total protein length) shared 93.3% sequence identity with the corresponding region of the third repeat of CU17L. To examine the presence of the CU17S transcript, the RT-PCR product was extended at the 3' end using the CU17 3' RACE primer and at the 5' end using the CU17 5' RACE-5 primer. These anchored PCR and RT-PCR analyses were performed using total RNA prepared from C. uchidae strain #17.

[0111] (result) As a result of the above, a transcript encoding a fluorescent protein having the peptide GYG that forms a chromophore at an appropriate position was identified. This clone was named CU17S. The amino acid sequence of CU17S is shown in SEQ ID NO: 4, and the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 5. CU17S had an entirely novel primary structure. The sequence identity with ObeCFP, the homolog with the highest sequence identity to CU17S, was only 15.2%. According to the amino acid sequence alignment (see also FIG. 4 described later), CU17S is considered to have a β-can structure similar to other fluorescent proteins.

[0112] [2. Expression of CU17S Protein in Escherichia coli] (Materials and Methods) ><Expression, Culture, and Protein Purification of Escherichia coli Expression Vector in Escherichia coli> After inserting the cDNA into an expression vector for Escherichia coli (pRSETB), it was introduced into Escherichia coli (JM109(DE3)) and cultured to obtain colonies. The obtained colonies were irradiated with light using a UV illuminator, a blue LED, and a green LED, respectively, to confirm whether fluorescence was emitted.

[0113] Also, a plasmid DNA for Escherichia coli expression with a histidine tag added to the N-terminus of the CU17S protein was constructed, transformed into Escherichia coli, and purified in one step using a Ni-NTA column. Next, buffer exchange was performed using a Sephadex G-25 column.

[0114] (Results) Colonies emitting green fluorescence were obtained, and a clone of the fluorescent protein was obtained from these colonies. As a result of expressing the CU17S protein in Escherichia coli, sufficient purified protein was obtained.

[0115] [3. Analysis of Fluorescent Properties of CU17S] (Materials and Methods) ><Measurement of Absorption Spectrum, Fluorescence Spectrum, and Quantum Yield of CU17S> To analyze the fluorescent properties of CU17S obtained in [1.] and [2.] above, the absorption spectrum, fluorescence spectrum, and quantum yield were measured.

[0116] The excitation and fluorescence spectra were measured using a spectrofluorometer F-4500 (Hitachi High-Technologies Corporation) at excitation wavelength 475 nm and emission wavelength 550 nm. The absorption spectrum was measured using a spectrophotometer U-2910 (Hitachi High-Technologies Corporation). The quantum yield was measured using an absolute PL quantum yield measurement system Quantaurus-QY (Hamamatsu Photonics K.K.) at excitation wavelengths of 470 nm and 480 nm.

[0117] (result) Figure 1 shows the fluorescence excitation and emission spectra observed in C. uchidae. The fluorescence excitation spectrum of CU17S showed a shoulder around 450 nm and a major peak at 496 nm, which was identical to the fluorescence characteristics observed in C. uchidae (Figure 1).

[0118] 4. Photostability Analysis of CU17S (material and method) HeLa cells were transfected with the CU17S gene using Lipofectamine 2000 reagent. One day after transfection, photostability was examined under the following conditions.

[0119] Buffer: HBSS containing 15 mM HEPES-NaOH (pH 7.4); cooled CCD camera (ORCA-AG, Hamamatsu Photonics); Fluorescence filter cube: Exciter: 488.0 IF 10 (488 ± 5 nm) (Cheshire Optical); Dichroic mirror: DM500 (Olympus); Emitter: BA520IF (520 nm <) (Olympus) combined with NDX001 (1% transmittance) (Asahi Spectra). Objective lens: 60x objective lens (UPlanSApo 60x oil, NA 1.35)

[0120] (result) When CU17S was expressed in HeLa cells, the green fluorescence of CU17S was localized in the cytoplasmic and nuclear compartments. Quantitative observations using a fluorescence microscope revealed that the green fluorescence of CU17S was not substantially bleached in both E. coli and mammalian cells, despite the incomplete maturation of the chromophore.

[0121] However, since it is generally believed that there is a negative correlation between the brightness (maturity) and photostability of fluorescent proteins, it was thought that it would be difficult to improve the maturity of the chromophore while maintaining the excellent photostability of CU17S.

[0122] 5. Preparation of variants based on CU17S (material and method) The CU17S variant was generated by random mutation. The primers used to generate the variant are shown in SEQ ID NOs: 11 and 12.

[0123] (result) A modified protein was obtained in which the 168th amino acid in the amino acid sequence of CU17S was substituted from valine to alanine.

[0124] The CU17S / V168A protein was expressed in E. coli and purified in the same manner as for CU17S described above. Large amounts of the CU17S / V168A protein were successfully produced and purified. The CU17S / V168A variant was designated StayGold. The amino acid sequence of StayGold is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene-coding region is shown in SEQ ID NO: 2. The nucleotide sequence of StayGold with humanized codon usage is shown in SEQ ID NO: 3.

[0125] 6. Analysis of Fluorescence Properties of StayGold (CU17S / V168A Modified Form) (material and method) The obtained fluorescent protein, StayGold (CU17S / V168A variant), was used to analyze its fluorescent properties in the same manner as described in [3.].

[0126] (Results) Figure 2 shows the absorption spectrum of StayGold (CU17S / V168A modified form). The absorption spectrum of CU17S / V168A has a high peak at 496 nm, and the absolute extinction coefficient at this wavelength is 159,000 M -1 ·cm -1 It was.

[0127] Figure 3 shows the fluorescence excitation and fluorescence emission spectra of StayGold (CU17S / V168A variant). The two graphs in Figure 3 show the excitation spectrum (shown by the dotted line) and fluorescence emission spectrum (shown by the solid line) of StayGold, respectively.

[0128] The fluorescence excitation and emission spectra of CU17S / V168A were similar to those of CU17S, and the quantum yield Φ was 0.93. The V168A mutation improved both protein expression and chromophore maturation.

[0129] FIG. 4 shows an alignment of the amino acid sequences of the StayGold, CU17S and EGFP proteins.

[0130] Table 1 shows the measurement results for various fluorescent proteins.

[0131] [Table 1] Subscripts a, λab: absorption peak wavelength (nm) Subscripts b and λem: maximum fluorescence wavelength (nm) Subscripts c and ε: molar extinction coefficient (M -1 cm -1 ) The molar absorption coefficient at the absorption peak wavelength is shown. The value in parentheses is the molar absorption coefficient at 488 nm (the center wavelength of the excitation bandpass filter). Subscript d, QY f : Fluorescence quantum yield (%) Absolute values ​​were measured using an absolute PL quantum yield measurement instrument C9920-02 (Hamamatsu Photonics). Subscript e: ε×QY f A numerical value that represents the absolute brightness of a fluorescent protein, calculated by Subscript f, fluorescent protein brightness in HeLa cells 30 hours after cDNA transfection. The values ​​for each green fluorescent protein were corrected by the mCherry value and normalized by the EGFP value. eCE: equimolar co-expression of green fluorescent protein and mCherry by using a bicistronic expression system. Subscript g, time required for the fluorescence emission rate to decrease from 1,000 photons / s per molecule to 500. Subscript h, total amount of purified protein recovered from 1 liter of E. coli culture. mNG: mNeonGreen These results demonstrate that StayGold exceeds known bright green fluorescent proteins such as EGFP or mNeonGreen in brightness, and StayGold has excellent photostability.

[0132] 7. Photostability Analysis of StayGold (Purified Protein) (7-1. Comparison of green fluorescent proteins) We compared the photostability of StayGold with that of four previously reported green fluorescent proteins: EGFP, SiriusGFP, mNeonGreen, and mClover3. SiriusGFP is a recently reported variant of EGFP with improved photostability. SiriusGFP exhibits a two-fold increase in photostability but a three-fold decrease in brightness compared to EGFP.

[0133] (material and method) Five fluorescent proteins were treated in parallel under identical conditions. When the fluorescent proteins were expressed and purified in E. coli, StayGold produced the highest yield. The soluble fraction was ~200 mg per liter of culture medium (Table 1).

[0134] <Measurement of protein concentration> Measurement was performed using a Bradford assay kit.

[0135] <Observation of photofading> A 1 μM solution of fluorescent protein dispersed in a polyacrylamide gel was prepared. The solution / gel mixture was sandwiched between two cover glasses and exposed to arc lamp irradiation (5.6 W / cm). 2 ) was used in a bleaching imaging experiment.

[0136] The evaluation method employed was the standard method for evaluating fluorescent proteins. First, the molar extinction coefficients of EGFP, SiriusGFP, mNeonGreen, and mClover3 were calculated at a central wavelength of 488 nm. The irradiance (5.6 W / cm) was 2 ) was used to calculate the density of photons reaching the sample. Furthermore, normalized bleaching curves were obtained for five types of fluorescent proteins, taking into account the quantum yield of fluorescence of the fluorescent proteins.

[0137] Changes in fluorescence intensity due to photobleaching were measured using a fluorescence microscope (IX81, Olympus) and an image analyzer (AQUACOSMOS, Hamamatsu Photonics). A xenon lamp (75W) was used as the excitation light source. The excitation wavelength was adjusted to a center wavelength of 488 nm and a half-width of 10 nm using a bandpass filter. This excitation light was irradiated onto a fluorescent protein solution sample placed on a stage through an objective lens (UPlanSApo 40x, NA 0.95). Fluorescence images of the sample were captured using a cooled CCD camera (ORCA-AG, Hamamatsu Photonics) through a 520 nm longpass filter. The excitation light was continuously irradiated for 90 minutes, and fluorescence images were acquired every 6 seconds. The average intensity of a circular area (115 pixels in diameter) in the center of each image was calculated, and the fluorescence intensity from a sample without fluorescent protein was subtracted from this as background light. The fluorescence photobleaching decay curve was obtained by plotting the intensity of the images at each time point.

[0138] (result) Figure 5 shows a simple comparison of the changes in fluorescence intensity over time for five types of green fluorescent proteins, showing that StayGold was brighter and significantly more photostable than the other fluorescent proteins.

[0139] Figure 6 shows the normalized bleaching curves of five types of fluorescent proteins when using purified proteins. For each fluorescent protein, the time it takes for the fluorescence emission rate to decrease from 1,000 photons / s per molecule at the start of measurement to 500 (t 1 / 2 ) was calculated (Table 1). Among other known fluorescent proteins, EGFP has the highest t of 700 s. 1 / 2 However, StayGold far exceeded this, showing a value of over 10,000 s. These results indicate that StayGold is more than 10 times more photostable than other fluorescent proteins, meaning that it can emit more than 10 times as many photons before bleaching.

[0140] (7-2. Comparison of various fluorescent proteins) We further compared the photostability of StayGold with that of 15 fluorescent proteins: EGFP, SiriusGFP, mNeonGreen, mClover3, TagRFP-T, mOrange2, mScarlet-H, mCardinal, mCherry, mScarlet-I, mTFP1, Venus, Achilles, mGold, and mVenus.

[0141] In the last 20 years, several powerful fluorescent proteins have been developed, such as TagRFP-T, mOrange2, and mScarlet-H. The photostability of red and orange fluorescent proteins has been improved. Although direct comparisons between fluorescent proteins of different colors are not possible, standard methods have been implemented to measure the photostability of fluorescent proteins. 1 / 2 Based on the calculation, it is possible to quantitatively compare and evaluate the fading.

[0142] (material and method) This was done in the same manner as in (7-1. Comparison of green fluorescent proteins).

[0143] (result) Figure 7 shows the normalized bleaching curves of StayGold and 15 fluorescent proteins, a total of 16 types of fluorescent proteins. As shown in Figure 7, StayGold had superior performance among the fluorescent proteins investigated.

[0144] 8. Photobleaching Analysis of StayGold (Living Cells) Using live cells, we compared the photobleaching of StayGold with four green fluorescent proteins: EGFP, SiriusGFP, mNeonGreen, and mClover3.

[0145] (material and method) HeLa cells were transfected with lentiviral vectors to stably express each fluorescent protein throughout the cell. The cells were cultured in 35-mm glass-bottom dishes. Changes in fluorescence intensity due to bleaching were measured using a fluorescence microscope (IX81, Olympus) and an image analyzer (AQUACOSMOS, Hamamatsu Photonics). A 75W xenon lamp was used as the excitation light source. The excitation wavelength was adjusted to a center wavelength of 488 nm and a half-width of 10 nm using a bandpass filter. This excitation light was irradiated onto a cell sample placed on a stage through an objective lens (UPlanSApo 40x, numerical aperture 0.95). Fluorescence images of the cells were captured using a cooled CCD camera (ORCA-AG, Hamamatsu Photonics) through a 520-nm longpass filter. The excitation light was continuously irradiated for 60 minutes, and fluorescence images were captured every 6 seconds. The average brightness of 20 × 20 pixels within the cells was calculated from the resulting image, and the brightness of areas without cells was subtracted as background light. The decay curve due to fluorescence bleaching was obtained by plotting the cell brightness at each time.

[0146] We investigated the photostability of five types of green fluorescent proteins, taking into account the brightness of the fluorescence, from cultured living cells expressing each protein.

[0147] Figure 8 shows a simple comparison of the changes in fluorescence intensity over time for five types of green fluorescent proteins in living cells.

[0148] Figure 9 shows the normalized bleaching curves of five types of green fluorescent proteins in living cells. Note that HBSS in Figures 8 and 9 means Hank's balanced salt solution.

[0149] This result also demonstrated that StayGold has obvious photostability and excellent brightness. 1 / 2 was 9,919 s, the highest t of any other fluorescent protein. 1 / 2The Sirius GPF was 522s (Table 1). Although not shown, the same tendency was observed when live cells were incubated with DMEM instead of HBSS. Specifically, the Sirius GPF was the highest among other fluorescent proteins. 1 / 2 The Sirius GPF was 558 s, whereas StayGold exceeded 10,000 s. Thus, StayGold was found to be one or even two orders of magnitude more photostable than any other currently available fluorescent proteins.

[0150] 9. Analysis of StayGold photobleaching (cell observation) To directly compare photobleaching in a single field, StayGold-expressing cells and EGFP-expressing cells were observed together, and a similar head-to-head comparison was also performed between StayGold-expressing cells and mNeonGreen-expressing cells.

[0151] (material and method) HeLa cells were transfected with lentiviral vectors to stably express each fluorescent protein throughout the cell. StayGold-expressing cells and EGFP-expressing cells, or StayGold-expressing cells and mNeonGreen-expressing cells, were mixed and cultured in 35-mm glass-bottom dishes. The fluorescence bleaching process was recorded using a fluorescence microscope (IX81, Olympus) and an image analyzer (AQUACOSMOS, Hamamatsu Photonics). A xenon lamp (75W) was used as the excitation light source. The excitation wavelength was adjusted to a center wavelength of 488 nm with a half-width of 10 nm using a bandpass filter. This excitation light was irradiated onto a cell sample placed on a stage through an objective lens (UPlanSApo 60x, numerical aperture 1.35). Fluorescence images of the cells were captured using a cooled CCD camera (ORCA-AG, Hamamatsu Photonics) through a 520-nm longpass filter. The excitation light was continuously irradiated for 30 minutes, and fluorescence images were acquired every 6 seconds.

[0152] (result) FIG. 10 shows a comparison of photobleaching between StayGold-expressing cells and EGFP-expressing cells, and a comparison of photobleaching between StayGold-expressing cells and mNeonGreen-expressing cells.

[0153] The results in Figure 10 demonstrate that StayGold is brighter and more photostable than EGFP.

[0154] It should be noted that in comparative experiments between StayGold- and mNeonGreen-expressing cells, the two cell populations are indistinguishable at t = 0, since their initial fluorescence intensities are identical. In separate cell culture experiments using cDNA transfection, we also observed that StayGold matures at a rate virtually identical to that of mNeonGreen, the brightest fluorescent protein to date.

[0155] Molecular oxygen is often considered a double-edged sword for fluorescent proteins. On the one hand, chromophore maturation requires an oxidation reaction caused by molecular oxygen attack, and thus a high degree of oxygen binding contributes to a substantial increase in brightness. On the other hand, decomposition of the fluorescent chromophore is due to molecular oxygen attack that occurs while the fluorescent chromophore is in the excited state, so a high degree of oxygen binding acts to reduce photostability. In addition to normal oxygen conditions, we also performed fluorescence bleaching experiments using fluorescent protein-expressing cells under anoxic and hyperoxic conditions (not shown).The results showed that StayGold, like EGFP, SiriusGFP, mClover3, and mNeonGreen, is oxygen-sensitive.

[0156] 10. Construction of StayGold Modified Tandem Dimers (material and method) Based on the StayGold sequence, a StayGold variant tandem dimer was prepared.

[0157] (result) A tandem dimer protein was obtained in which two StayGold variant amino acid sequences were linked. Two types of tandem dimer proteins with different structures were obtained.

[0158] The two tandem dimer proteins were expressed in E. coli and purified in the same manner as the above-mentioned CU17S and StayGold. Large amounts of each tandem dimer protein were successfully produced and purified. These two proteins were designated tdStayGold and tdStayGold(long), respectively. The amino acid sequence of tdStayGold is shown in SEQ ID NO: 13, and the nucleotide sequence of the coding region of tdStayGold is shown in SEQ ID NO: 14. The amino acid sequence of tdStayGold(long) is shown in SEQ ID NO: 15, and the nucleotide sequence of the coding region of tdStayGold(long) is shown in SEQ ID NO: 16. Both tandem dimer proteins are linked via a 132 amino acid insertion sequence between the two StayGold variant sequences, and tdStayGold(long) has an additional c4 (10 amino acids, SEQ ID NO: 8) at the C-terminus of tdStayGold.

[0159] It has been found that tdStayGold is preferably used for fusing to the C-terminus of a certain protein X. In this case, the fusion protein has a structure of protein X-tdStayGold. On the other hand, it has been found that tdStayGold(long) is preferably used for fusing to the N-terminus of a certain protein X. In this case, the fusion protein has a structure of tdStayGold(long)-protein X.

[0160] 12. Microtubule labeling with StayGold variant tandem dimers

[0161] An expression construct was prepared encoding a fusion protein in which the N-terminus of tdStayGold, which has the amino acid sequence of SEQ ID NO: 13, was linked to the C-terminus of human wild-type tau four-repeat (hereinafter simply referred to as "Tau"), a microtubule-binding protein, using a Gly-Gly linker.

[0162] HeLa cells were transfected with 1 μg of plasmid DNA (Tau-GG-tdStayGold / pcDNA3) using Lipofectamine 2000. One day after transfection, live cells were observed with fluorescence. Observations were performed using a confocal laser scanning microscope (FV3000). The objective lens was a UPLSAPO 60XS / 1.3 NA, the sampling speed was 2.0 μs / pixel, the integration was 3 lines, the zoom was 3x, the CA was 222 nm, the laser was 488 nm, 1.5%, the PMT voltage was 520 V, and the detection was 500-600 nm.

[0163] (result) Figure 11 shows the results of labeling microtubules with tdStayGold (scale bar: 10 μm). As shown in Figure 11, tdStayGold brightly labeled intracellular microtubules. Furthermore, the fluorescence of tdStayGold did not fade, demonstrating that tdStayGold enabled clear observation of microtubule dynamics in live cells.

[0164] 13. Labeling of Golgi membrane proteins with modified StayGold tandem dimers An insertion sequence c4 (10 amino acids, SEQ ID NO: 8) was added to the C-terminus of the amino acid sequence of the StayGold tandem dimer used in Example 12 to prepare tdStayGold(long) having the amino acid sequence of SEQ ID NO: 15. The nucleotide sequence of the coding region of tdStayGold(long) having the amino acid sequence of SEQ ID NO: 15 is shown in SEQ ID NO: 16.

[0165] An expression construct was created that encodes a fusion protein that targets the Golgi membrane. The C-terminus of tdStayGold(long), which has the amino acid sequence of SEQ ID NO: 15, is linked to the N-terminus of the Golgi membrane protein Giantin (amino acid sequence 3131-3259 of the human Giantin protein). The construct was introduced into HeLa cells, and the fluorescent protein was transiently expressed, allowing for fluorescence observation of live cells.

[0166] HeLa cells were transfected with 1 μg of plasmid DNA (tdStayGold(long)-Coupler-Giantin / pcDNA3) using Lipofectamine 2000. One day after transfection, the cells were observed using a SpinSR10 super-resolution microscope. The camera was an ORCA-Flash 4.0, the objective was an UPLAPO OHR 100x lens with a 1.5 numerical aperture, the CSU disk was a SoRa lens, the magnification was 3.2x, the laser was 488 nm, 1%, the exposure time was 100 msec, the Z steps were 0.25 μm / slice, 46 slices, the DM was D405 / 488 / 561 / 640, the CH1 filter wheel was B525 / 50, and the buffer was HBSS, 10 mM HEPES-NaOH (pH 7.4).

[0167] (result) Figure 12 shows the results of labeling the Golgi membrane with tdStayGold (long) (scale bar: 5 μm). As shown in Figure 12, tdStayGold (long) brightly labeled the Golgi membrane within the cell, enabling the dynamics of the Golgi membrane in live cells to be observed at high resolution, high speed, and continuously without bleaching.

[0168] 14. Labeling of postsynaptic protein PSD-95 with StayGold modified tandem dimers A StayGold variant (oxStayGold) was prepared by further adding the mutations H169Y, C174I, and C208I to StayGold contained in the StayGold tandem dimer having the amino acid sequence of SEQ ID NO: 13. Based on the oxStayGold sequence, an oxStayGold tandem dimer (tdoxStayGold) having the following structure was prepared. tdoxStayGold: (n1)oxStayGold(c4)-EV linker-(n1)oxStayGold The amino acid sequence of tdoxStayGold is shown in SEQ ID NO: 17, and the nucleotide sequence of the coding region of tdoxStayGold is shown in SEQ ID NO: 18.

[0169] We created an expression construct encoding a fusion protein (PSD-95-Coupler-tdoxStayGold) in which the N-terminus of tdoxStayGold was linked to the C-terminus of the postsynaptic protein PSD-95. We then expressed the fusion protein in primary cultured neurons from fetal rat hippocampus and performed fluorescent imaging of live cells. The Coupler linker is a linker consisting of three repeats of the sequence Gly-Gly-Gly-Gly-Ser.

[0170] Primary cultured neurons from fetal rat hippocampus (DIV4: Day 4 in vitro culture) were transfected with 4 μg of plasmid DNA (PSD95-Coupler-tdoxStayGold / pcDNA3) using the calcium phosphate method. Primary cultured neurons were observed under a SpinSR10 microscope at DIV25 (Day 25 in vitro culture). The camera was ORCA-Flash 4.0, the objective lens was UPLAPO OHR 100x, NA=1.5, CSU disk: 50um, Zoom: x1, Laser: 488nm, 10%, Excitation time: 500msec, Z steps: 0.25um / slice, 30 slices, DM: D405 / 488 / 561 / 640, CH1 Filter Wheel: B525 / 50, B447 / 60, Culture medium used during imaging: DMEM / F12 (1:1), 2% FBS, N2-supplement (x1), B-27 (x1.5).

[0171] (result) Figure 13 shows the results of labeling the protein PSD-95 with tdoxStayGold (scale bar 20 μm in Figure 13). As shown in Figure 13, labeling with tdoxStayGold enabled the clear observation of PSD-95 localized to glutamate receptors and signaling molecules that regulate signal plasticity as a scaffolding structure.

[0172] 15. Endoplasmic reticulum membrane labeling with StayGold modified tandem dimers Based on the StayGold sequence, a modified tandem dimer (tdStayGold alpha) having the following structure was prepared. tdStayGold alpha: (n1)StayGold(c4)-alpha linker-(n1)StayGold The amino acid sequence of tdStayGold alpha is shown in SEQ ID NO: 19, and the nucleotide sequence of the coding region of tdStayGold alpha is shown in SEQ ID NO: 20.

[0173] We constructed an expression construct encoding a fusion protein targeted to the endoplasmic reticulum (ER) membrane by linking tdStayGold alpha to a protein called cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM). The construct was transfected into HeLa cells, where it transiently expressed a fluorescent protein, allowing for fluorescence imaging of live cells.

[0174] HeLa cells were transfected with 1 μg of plasmid DNA (CytERM-tdStayGold alpha / pcDNA3) using Lipofectamine 2000. Images were observed using a widefield microscope (IX83 P2ZF) equipped with an ORCA-Fusion camera, a UPLXAPO 40x objective with a 0.95 numerical aperture, a U-FBNA cube (excitation: 470-495, emission: 510-550, DM: 505), 5% excitation light, a 10% neutral density filter, a 500-msec excitation time, and HBSS, 10 mM HEPES-NaOH (pH 7.4) buffer.

[0175] (result) FIG. 14 shows the results of labeling the membrane of the endoplasmic reticulum with tdStayGold alpha (in FIG. 14, the scale bar is 20 μm). When a fluorescent protein is attached to the C-terminus of CytERM, it marks the ER by penetrating the membrane from the cytoplasmic side. If the fluorescent protein forms a multimer, the membranes overlap, forming a vortex-like shape (bright spot). When the C-terminus of CytERM was connected to the N-terminus of (n1) StayGold, which was obtained by inserting the insertion sequence n1 into the amino acid sequence of StayGold of SEQ ID NO: 1, several such swirls (bright spots) appeared (not shown). On the other hand, as shown in Figure 14, when tdStayGold alpha was connected, almost no swirls appeared.

[0176] 16. Endoplasmic reticulum lumen labeling with modified StayGold A StayGold variant ((n2)oxStayGold(c4)) was prepared by inserting the insertion sequences n2 and c4 into the amino acid sequence of oxStayGold. Based on (n2)oxStayGold(c4), a StayGold variant (er-(n2)oxStayGold(c4)) having the following structure was prepared. er-(n2)oxStayGold(c4): SP(CRT)-(n2)StayGold(c4)-KDEL The amino acid sequence of er-(n2)oxStayGold(c4) is shown in SEQ ID NO: 21, and the nucleotide sequence of the coding region of er-(n2)oxStayGold(c4) is shown in SEQ ID NO: 22. Here, SP(CRT) is the sequence of the calreticulin signal peptide, and KDEL is the sequence of the ER retention signal. A construct encoding er-(n2)oxStayGold(c4) was prepared, and the fusion protein was transiently expressed in HeLa cells, followed by fluorescence observation of live cells.

[0177] Plasmid DNA was transfected into HeLa cells using PEI. The cells were observed using a super-resolution microscope, N-SIM S. The camera was ORCA-Fusion, the objective lens was SR HP Plan Apo 100X / 1.35 Sil λS, the image acquisition mode was 3D-SIM, the laser was 488 nm, 50%, the excitation time was 15 msec, and the buffer was HBSS, 10 mM HEPES-NaOH (pH 7.4).

[0178] (result) Figure 15 shows the results of labeling the endoplasmic reticulum lumen with er-(n2)oxStayGold(c4) (scale bar: 10 μm). As shown in Figure 15, er-(n2)oxStayGold(c4) brightly labeled the endoplasmic reticulum lumen within cells, enabling high-resolution, high-speed, continuous observation of the dynamics of the endoplasmic reticulum lumen in live cells without bleaching.

[0179] 17. Mitochondrial membrane labeling with StayGold modifications mt-StayGold was prepared using StayGold having the amino acid sequence of SEQ ID NO: 1. mt-StayGold has the following structure. mt-StayGold:CoxVIII×2-StayGold

[0180] The amino acid sequence of mt-StayGold is shown in SEQ ID NO: 23, and the nucleotide sequence of the coding region of mt-StayGold is shown in SEQ ID NO: 24. Here, CoxVIII (cytochrome c oxidase subunit VIII) was a sequence transported to mitochondria. mt-StayGold was produced using an expression construct encoding a fusion protein in which an amino acid sequence consisting of two repeats of the CoxVIII sequence (72 amino acids, amino acids 1 to 72 of SEQ ID NO: 23) was linked to the N-terminus of the StayGold amino acid sequence (amino acids 75 to 291 of SEQ ID NO: 23). The amino acids 73 and 74 of SEQ ID NO: 23 are derived from the BamHI sequence used in the production of mt-StayGold.

[0181] This gene was inserted into a cell expression plasmid (CSII-EF-MCS) and transfected into HeLa cells to express mt-StayGold. As a result, it was found that mt-StayGold emitted fluorescence in mitochondria (not shown). Furthermore, HeLa cells stably expressing mt-StayGold were generated using lentivirus. Fluorescent mt-StayGold was obtained even when lentivirus was used (not shown).

[0182] As a fluorescent protein with improved fluorescence intensity of mt-StayGold, an insertion sequence n1 (9 amino acids) was inserted between the fourth and fifth amino acids of the amino acid sequence of StayGold (sequence number 1) in the amino acid sequence of sequence number 23, to produce mt-(n1)StayGold with the following structure. mt-(n1)StayGold:CoxVIII×2-(n1)StayGold The amino acid sequence of mt-(n1)StayGold is shown in SEQ ID NO: 25, and the nucleotide sequence of the coding region of mt-(n1)StayGold is shown in SEQ ID NO: 26.

[0183] Plasmid DNA was transfected into HeLa cells using lentivirus. Images of mt-(n1)StayGold-transfected HeLa cells were captured using a fluorescence microscope (IX81, Olympus) and an image analyzer (AQUACOSMOS, Hamamatsu Photonics). Excitation wavelength: 460-495 nm, Emission wavelength: 510 nm long pass, Dichroic mirror: 505 nm, Objective lens: UPlanSApo 60x, NA 1.35, Cooled CCD camera: ORCA-AG, Binning: 2 x 2, Exposure time: 90 ms.

[0184] (result) Figure 16 shows the results of labeling mitochondria with mt-(n1)StayGold (scale bar in Figure 16: 20 μm). mt-(n1)StayGold enabled bright labeling of mitochondria, enabling faster and longer imaging.

[0185] 18. Monomerization of StayGold Modified Forms (1) Examination of Y187A and L155T mutations Mutations Y187A or L155T were added to StayGold of SEQ ID NO: 1 to generate StayGold variants (StayGold Y187A, StayGold L115T). The amino acid sequence of StayGold Y187A is shown in SEQ ID NO: 27, and the nucleotide sequence of the coding region of StayGold Y187A is shown in SEQ ID NO: 28. The amino acid sequence of StayGold L115T is shown in SEQ ID NO: 29, and the nucleotide sequence of the coding region of StayGold L115T is shown in SEQ ID NO: 30.

[0186] As shown in Table 2, recombinant proteins were produced in E. coli JM109 DE3 using expression constructs encoding StayGold variants, StayGold (dimer), and EGFP (monomer). Proteins were purified using Ni-NTA followed by pseudo-native PAGE. The separating gel was 10% acrylamide, 0.1% SDS. The sample buffer contained 2% SDS and 100 mM DTT. No heat treatment was performed. [Table 2]

[0187] (result) The results are shown in Figure 17. As shown in Figure 17, it was suggested that StayGold Y187A and StayGold L155T are likely to be monomers.

[0188] (2) Further investigation of mutations A variant of StayGold ((n1)oxStayGold) was prepared by further inserting the insertion sequence n1 into the oxStayGold prepared in Example 14. Mutations were introduced into (n1)oxStayGold / pRSET by site-directed mutagenesis to prepare a variant of (n1)oxStayGold ((n1)oxStayGold L155T) having the amino acid sequence of SEQ ID NO: 31. The nucleotide sequence of the region encoding (n1)oxStayGold L155T is shown in SEQ ID NO: 32. Further mutations were introduced into (n1)oxStayGold L155T based on the crystal structure of StayGold. Table 3 shows the mutations introduced into (n1)oxStayGold L155T and the fluorescent proteins used as comparative examples (AzamiGreen (tetramer), EGFP (monomer), and StayGold (dimer)). The positions of the amino acid substitutions shown in Table 3 correspond to the amino acid positions in the amino acid sequence of StayGold (SEQ ID NO: 1) without the n1 insertion sequence. Furthermore, the (n1)oxStayGold variants 1, 2, and 7 to 12 in the table are variants containing mutations in the amino acid sequence of StayGold (SEQ ID NO: 1) in which asparagine at position 132 is replaced with aspartic acid (N132D), proline at position 151 is replaced with threonine (P151T), and lysine at position 162 is replaced with glutamic acid (K162E). Table 3 also lists the reasons for selecting the mutation added to (n1)oxStayGold L155T. As shown in Table 3, recombinant proteins were produced in Escherichia coli JM109 DE3 using expression constructs encoding the (n1)oxStayGold variants, AzamiGreen (tetramer), EGFP (monomer), and StayGold (dimer). Proteins were purified using Ni-NTA and then subjected to pseudo-native PAGE. The separating gel was 10% acrylamide, 0.1% SDS. The sample buffer contained 2% SDS and 100 mM DTT. No heat treatment was performed. [Table 3]

[0189] (result) The results are shown in Figure 18. As shown in Figure 18, the (n1)oxStayGold L155T variant shown in Table 2 was likely to be a monomer, and in particular, mutations 1, 2, 7, 8, 9, 10, and 11 in Table 2 and Figure 18 were suggested to be useful mutations for monomerization.

[0190] [19. Summary] As shown above, the StayGold variants exhibited bright fluorescence and remarkably excellent photostability. Until now, improvements in the photostability of fluorescent proteins have always been accompanied by a decrease in brightness. The fluorescent properties of StayGold differ from those of conventional fluorescent proteins, which are incompatible with each other in terms of brightness and photostability. [Industrial Applicability]

[0191] An example of the fluorescent protein according to the present invention has bright fluorescence and excellent photostability, and is therefore useful in a wide range of fields, including molecular biology, biochemical analysis, and medicine.

Claims

1. A polypeptide having fluorescent properties, as defined in any one of (1) to (3) below: (1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) A polypeptide having an amino acid sequence in which the 168th amino acid of the amino acid sequence set forth in SEQ ID NO: 1 is alanine, and in which 1 to 21 amino acids in the amino acid sequence set forth in SEQ ID NO: 1 have been substituted, deleted, inserted, and / or added. (3) A polypeptide in which the 168th amino acid of the amino acid sequence set forth in SEQ ID NO: 1 is alanine and which has a sequence identity of 90% or more to the amino acid sequence set forth in SEQ ID NO:

1.

2. The polypeptide of claim 1, which exhibits higher photostability than EGFP or has brighter fluorescence than EGFP.

3. A polynucleotide according to any one of (1) to (3) below: (1) a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (2) A polynucleotide encoding a polypeptide having fluorescent properties, wherein the 168th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is alanine, and wherein 1 to 21 amino acids in the amino acid sequence set forth in SEQ ID NO: 1 have been substituted, deleted, inserted, and / or added. (3) A polynucleotide encoding a polypeptide having fluorescent properties, in which the 168th amino acid in the amino acid sequence set forth in SEQ ID NO: 1 is alanine and which has a sequence identity of 90% or more to the amino acid sequence set forth in SEQ ID NO:

1.

4. (a) an expression control region functional in the expression host; and (b) the polynucleotide of claim 3; An expression cassette comprising:

5. A vector comprising the polynucleotide of claim 3 or the expression cassette of claim 4.

6. A transformant (excluding individuals of human origin) having the polynucleotide according to claim 3, the expression cassette according to claim 4, or the vector according to claim 5.

7. The transformant according to claim 6, which is a non-human transgenic organism.

8. A fusion polypeptide comprising the polypeptide of claim 1 or 2 and another polypeptide.

9. The fusion polypeptide according to claim 8, comprising two or more linked polypeptides according to claim 1 or 2.

10. A kit comprising a polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, an expression cassette according to claim 4, a vector according to claim 5, a transformant according to claim 6 or 7, or a fusion polypeptide according to claim 8 or 9.

11. a producing step in which the polypeptide of claim 1 or 2 or the fusion polypeptide of claim 8 or 9 is produced in a cell; an excitation light irradiation step of irradiating the cells with excitation light; and an observation step of observing the fluorescence derived from the polypeptide or fusion polypeptide.

Images