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WO2018100803A1 - Complexe de protéine fluorescente et procédé électroluminescent de protéine fluorescente - Google Patents

Complexe de protéine fluorescente et procédé électroluminescent de protéine fluorescente Download PDF

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Publication number
WO2018100803A1
WO2018100803A1 PCT/JP2017/028174 JP2017028174W WO2018100803A1 WO 2018100803 A1 WO2018100803 A1 WO 2018100803A1 JP 2017028174 W JP2017028174 W JP 2017028174W WO 2018100803 A1 WO2018100803 A1 WO 2018100803A1
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Prior art keywords
fluorescent protein
wavelength
fluorescence
excitation
xaa
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Japanese (ja)
Inventor
晃尚 清水
行大 白鳥
克紀 堀井
巌 和賀
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NEC Solution Innovators Ltd
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NEC Solution Innovators Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a fluorescent protein complex and a fluorescent protein luminescence method.
  • Fluorescent proteins such as GFP (Green Fluorescent Protein) and YFP (Yellow Fluorescent Protein) are generally used (Non-patent Document 1).
  • the fluorescent protein is used for various purposes such as research.
  • each of the fluorescent proteins has a specific excitation wavelength and a maximum excitation wavelength, and a fluorescence wavelength and an emission maximum wavelength, each of the fluorescent proteins emits light by excitation and detects its emission. Use may be optically limited depending on excitation conditions and luminescence detection conditions.
  • an object of the present invention is to provide a new fluorescent protein that can expand optical conditions depending on the fluorescent protein, for example.
  • the fluorescent protein complex of the present invention comprises: Including a first fluorescent protein and a second fluorescent protein, The first fluorescent protein and the second fluorescent protein are linked;
  • the combination of the first fluorescent protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap. It is characterized by that.
  • the fluorescent protein luminescence method of the present invention comprises: Irradiating the fluorescent protein complex of the present invention with light having an excitation wavelength of the first fluorescent protein in the fluorescent protein complex, By irradiating the first fluorescent protein with light having an excitation wavelength of the first fluorescent protein, the first fluorescent protein is caused to emit light, The second fluorescent protein in the fluorescent protein complex is excited by the light emission of the first fluorescent protein to cause the second fluorescent protein to emit light.
  • the gene encoding the fluorescent protein complex of the present invention is: A first nucleic acid sequence encoding a first fluorescent protein and a second nucleic acid sequence encoding a second fluorescent protein; The first nucleic acid sequence and the second nucleic acid sequence are linked so as to be expressed as a complex in which the first fluorescent protein and the second fluorescent protein are linked; The combination of the first fluorescent protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap. It is characterized by.
  • the expression vector of the present invention is characterized by including a gene encoding the fluorescent protein complex of the present invention.
  • the transformant of the present invention is characterized by including the gene encoding the fluorescent protein complex of the present invention or the expression vector.
  • the present invention by combining the first fluorescent protein and the fluorescent protein under the above-described conditions, for example, when the excitation light from the outside is different from the excitation conditions of the second fluorescent protein or maximum excitation Even when the wavelength deviates from the wavelength, the second fluorescent protein can emit light by excitation due to light emission of the first fluorescent protein. For this reason, according to this invention, compared with the case where a fluorescent protein is used independently, the setting of a wider optical condition is attained, for example.
  • FIG. 1 is a graph showing fluorescence intensity at an excitation wavelength of 400 nm and a fluorescence detection wavelength of 516 nm in Example 2.
  • 2A is a graph showing the fluorescence intensity in the excitation wavelength of 400 nm and a predetermined fluorescence wavelength region in Example 3
  • FIG. 2B is a graph showing the maximum fluorescence intensity in the predetermined fluorescence wavelength region. is there.
  • FIG. 3 is a graph showing the fluorescence intensity at the excitation wavelength and the fluorescence wavelength in Example 3.
  • 4A is a graph showing the fluorescence intensity in the excitation wavelength of 400 nm and a predetermined fluorescence wavelength range in Example 4
  • FIG. 4B is a graph showing the maximum fluorescence intensity in the predetermined fluorescence wavelength range. is there.
  • FIG. 5 is a graph showing the fluorescence intensity at the excitation wavelength and the fluorescence wavelength in Example 4.
  • the fluorescent protein complex of the present invention includes, for example, the emission intensity (I2 E1 ) when the second fluorescent protein excites the second fluorescent protein with the excitation wavelength (E1) of the first fluorescent protein,
  • the fluorescence intensity (I2 L1 ) when the second fluorescent protein is excited at the fluorescence wavelength (L1) of one fluorescent protein is a protein that satisfies the relationship of I2 E1 ⁇ I2 L1 .
  • the first fluorescent protein and the second fluorescent protein are different proteins.
  • the maximum excitation wavelength of the first fluorescent protein is different from the maximum excitation wavelength of the second fluorescent protein.
  • the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein include wavelengths of 350 to 650 nm.
  • the fluorescence wavelength (L1) of the first fluorescent protein includes the maximum excitation wavelength (E2 max ) of the second fluorescent protein.
  • the fluorescence wavelength (L2) of the second fluorescent protein includes a range of 490 to 650 nm.
  • the first fluorescent protein and the second fluorescent protein are linked via a linker.
  • the linker is a peptide linker.
  • the peptide linker includes a glycine serine linker region.
  • the glycine serine linker region has a peptide unit consisting of the amino acid sequence of SEQ ID NO: 48 (GGGGS).
  • the fluorescent protein complex of the present invention is, for example, a linker in which 1 to 3 peptide units are linked to the glycine serine linker region.
  • the luminescent protein luminescence method of the present invention further includes, for example, a step of detecting luminescence of the second fluorescent protein.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked via a nucleic acid sequence encoding a linker.
  • the fluorescent protein complex of the present invention as described above, Including a first fluorescent protein and a second fluorescent protein, The first fluorescent protein and the second fluorescent protein are linked;
  • the combination of the first fluorescent protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap. It is characterized by that.
  • the fluorescent protein complex of the present invention excites the second fluorescent protein by the light emission of the first fluorescent protein. Therefore, it can also be referred to as, for example, a FRET type fluorescent protein complex.
  • the present invention is not limited by the function of so-called “FRET (fluorescence energy transfer)”.
  • the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap.
  • the other conditions are not particularly limited.
  • the “excitation wavelength of a fluorescent protein” refers to the wavelength of excitation light capable of emitting the fluorescent protein
  • the “fluorescence wavelength of the fluorescent protein” refers to the wavelength of the fluorescence emitted from the fluorescent protein by excitation. That's it.
  • the fluorescent protein of interest is the second fluorescent protein
  • the fluorescent protein that emits light having a fluorescent wavelength that overlaps the excitation wavelength of the second fluorescent protein is The first fluorescent protein.
  • the FRET type fluorescent protein complex of the present invention for example, it is possible to obtain luminescence with stronger fluorescence intensity than when the first fluorescent protein or the second fluorescent protein is used alone.
  • the second fluorescent protein is excited by light emission of the first fluorescent protein.
  • the second fluorescent protein is, for example, a protein that is excited at the fluorescence wavelength of the first fluorescent protein.
  • the second fluorescent protein may be a protein that can be excited by the light emission of the first fluorescent protein and can emit light.
  • the second fluorescent protein may emit light or emit light by excitation light irradiated on the first fluorescent protein. It does not have to be.
  • the FRET fluorescent protein complex of the present invention for example, light emission with a stronger light emission intensity can be obtained as compared with the case where the first fluorescent protein and the second fluorescent protein are excited individually to emit light. You can also.
  • the second fluorescent protein includes, for example, the emission intensity (I2 E1 ) when the second fluorescent protein is excited with the excitation wavelength (E1) of the first fluorescent protein, and the fluorescence of the first fluorescent protein.
  • Examples include proteins satisfying the relationship of I2 E1 ⁇ I2 L1 with the emission intensity (I2 L1 ) when the second fluorescent protein is excited at the wavelength (L1). If it is such a second fluorescent protein, the FRET fluorescent protein complex of the present invention has, for example, the first fluorescent protein more than the case where the second fluorescent protein is excited with the excitation wavelength (E1) of the first protein.
  • the second fluorescent protein is indirectly excited by the fluorescence wavelength (L1) emitted from the fluorescent protein, and can emit more intense light.
  • the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein only need to include overlapping wavelengths as described above.
  • the overlapping wavelengths are not particularly limited, and can be appropriately set according to the types of the first fluorescent protein and the second fluorescent protein.
  • the fluorescence wavelength (L1) of the first fluorescent protein may include, for example, the maximum excitation wavelength (E2 max ) of the second fluorescent protein.
  • E2 max the maximum excitation wavelength of the second fluorescent protein.
  • the second fluorescent protein is caused to emit light more efficiently and with high intensity by excitation of the second fluorescent protein by light emission of the first fluorescent protein. be able to.
  • the fluorescence maximum wavelength (L1 max ) of the first fluorescent protein is, for example, 511 to 519 nm.
  • the maximum excitation wavelength (E2 max ) of the second fluorescent protein is, for example, 501 to 509 nm and 506 to 514 nm.
  • the fluorescence maximum wavelength (L2 max ) of the second fluorescent protein is, for example, 511 to 519 nm or 516 to 524 nm.
  • the first fluorescent protein and the second fluorescent protein may be, for example, the same protein or different proteins.
  • the first fluorescent protein and the second fluorescent protein are in the same excitation wavelength range, for example, the first fluorescent protein is excited with a predetermined excitation wavelength (E1) included in the excitation wavelength range, and the first fluorescent protein The fluorescent protein is allowed to emit light, and the second fluorescent protein (same as the first fluorescent protein) is excited at the fluorescent wavelength (L1) included in the excitation wavelength range, and can be caused to emit light.
  • the difference between the maximum excitation wavelength (E1 max ) of the first fluorescent protein and the maximum excitation wavelength (E2 max ) of the second fluorescent protein is not particularly limited.
  • the maximum excitation wavelength (E1 max ) of the first fluorescent protein is not particularly limited and is, for example, in the range of 396 to 404 nm, 401 to 409 nm.
  • the excitation wavelength (E1) of the first fluorescent protein is not particularly limited, and includes, for example, a range of 350 to 650 nm.
  • the maximum excitation wavelength (E2 max ) of the second fluorescent protein is not particularly limited, and is as described above, for example.
  • the excitation wavelength (E2) of the second fluorescent protein is not particularly limited.
  • the first fluorescent protein and the second fluorescent protein may each have one fluorescent peak or two or more fluorescent peaks obtained by changing the excitation wavelength, for example.
  • the excitation wavelength can be set in a range in which emission of a fluorescence wavelength including a desired peak is generated.
  • the first fluorescent protein and the second fluorescent protein may be directly connected or indirectly connected.
  • the first fluorescent protein and the second fluorescent protein are linked via, for example, a linker.
  • the linker is not particularly limited as long as the first fluorescent protein and the second fluorescent protein can independently maintain their properties, for example.
  • the linker is, for example, a peptide linker.
  • the peptide linker is not particularly limited, and examples thereof include a peptide linker including a glycine serine linker region.
  • a glycine serine linker is a peptide comprising a glycine residue and a serine residue.
  • the peptide linker may include only the glycine serine linker region or may include the glycine serine linker region.
  • the glycine serine linker is hereinafter also referred to as “GS linker”, and the glycine serine linker region is hereinafter also referred to as “GS linker region”.
  • the glycine serine linker region has, for example, a peptide unit consisting of the amino acid sequence of SEQ ID NO: 48 (GGGGS).
  • the number (n) of the peptide units is not particularly limited, and n is a positive integer, for example, 1 to 3, and 1 to 2.
  • the glycine serine linker region is represented by (GGGGS) n , for example.
  • the peptide linker may include the glycine serine linker region, and in this case, the region other than the glycine serine linker region is not particularly limited.
  • the FRET fluorescent protein complex of the present invention is prepared, for example, by genetic engineering, it can be synthesized using the expression vector of the present invention described later.
  • the expression vector includes, for example, a nucleic acid sequence encoding the first fluorescent protein, a nucleic acid sequence encoding the second fluorescent protein, and optionally a nucleic acid sequence encoding the glycine serine linker region, and a restriction enzyme site. It can be constructed by linking to a vector serving as a backbone.
  • the other region in the peptide linker may be, for example, a peptide region derived from a restriction enzyme site for linking the nucleic acid sequences.
  • other regions other than the glycine serine linker region are not particularly limited.
  • the length (number of amino acid residues) is 1 to 3, 1 to 2.
  • the position of the other region is not particularly limited, and may be, for example, the C-terminal side, the N-terminal side, or both the C-terminal side and the N-terminal side of the glycine serine linker. It may be.
  • the length (number of amino acid residues) of the peptide linker is not particularly limited, and the lower limit thereof is, for example, 1, 5, 8, 10, 13, and the upper limit thereof, for example, 18, 15, 13, 10 , 8.
  • the FRET type fluorescent protein complex of the present invention may further have a peptide addition region, for example, upstream of the first fluorescent protein.
  • the FRET fluorescent protein complex of the present invention is prepared, for example, by genetic engineering, it can be synthesized using the expression vector of the present invention described later.
  • the expression vector includes, for example, a nucleic acid sequence encoding the first fluorescent protein, a nucleic acid sequence encoding the second fluorescent protein, and optionally a nucleic acid sequence encoding the glycine serine linker region, and a restriction enzyme site. It can be constructed by linking to a vector serving as a backbone.
  • a peptide region derived from a restriction enzyme site for linking to the expression vector may be added as the peptide addition region.
  • the length (number of amino acid residues) of the peptide addition region is not particularly limited, and is, for example, 1 to 4, 1 to 3, or 1 to 2.
  • the FRET type fluorescent protein complex of the present invention can excite the second fluorescent protein by the light emission of the first fluorescent protein and cause the second fluorescent protein to emit light.
  • a protein excited by black light is the second fluorescent protein, and is excited by UV light and emits light in the wavelength range of black light.
  • the combination which uses protein as said 1st fluorescent protein can be illustrated.
  • the first fluorescent protein is a fluorescent protein that is excited by UV light and emits light having a fluorescent wavelength included in the black light wavelength region when excited, the first fluorescent protein is UV-excited.
  • the first fluorescent protein can emit light.
  • the emitted light is a fluorescence wavelength included in the black light wavelength range, the emitted light can excite the second fluorescent protein without using a black light.
  • Examples of such a combination of the first fluorescent protein and the second fluorescent protein include the following combinations.
  • the first fluorescent protein and the second fluorescent protein are directly or indirectly linked and exist as a constituent element of the FRET fluorescent protein complex. For this reason, for example, when the FRET fluorescent protein complex is expressed by genetic engineering, the start codon of the complex encodes the first methionine residue of the first fluorescent protein, and thus the second fluorescent protein The first methionine residue of the protein may be deleted.
  • examples of the combination of the first fluorescent protein, the glycine serine linker region in the peptide linker, and the second fluorescent protein include the following combinations.
  • the number n of GS linker region (GGGGGS) n repeating units in the peptide linker is not particularly limited, and is, for example, 1 to 3, and amino acids in other regions N m in the peptide linker Residue N is not particularly limited and may be any amino acid (Xaa). For example, all may be the same or different, and the number m of repeating units is not particularly limited, and the peptide addition region and It is the same.
  • the FRET type fluorescent protein complex in the combination shown in Table 4 can be represented by the following sequence, for example.
  • the first fluorescent protein and the second fluorescent protein can be used in combination with, for example, the following fluorescent proteins in addition to those described above.
  • (F1) consists of the amino acid sequence of SEQ ID NO: 1, in the amino acid sequence of the SEQ ID NO: 1, is 52 amino acid Xaa 52 is any amino acid, 133 amino acid Xaa 133 is any amino acid, 154 amino acid Xaa 154 is, in 52 th amino acid Xaa 52, 133 amino acid Xaa 133, and 154 amino acid sequences other than the amino acid Xaa 154 protein (F2) wherein any amino acid (F1), 1 or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequences, and proteins (F3) said with fluorescent activity (F1) of 52 amino acid Xaa 52, 133 amino acid Xaa 133, Amino acids other than amino acid Xaa 154 at position 154 A protein having an amino acid sequence having 80% or more identity to the amino acid sequence and having fluorescence activity
  • the 52nd, 133rd, and 154th amino acids interact with, for example, the chromophore of the protein.
  • the protein can adjust the fluorescent activity of the protein by, for example, making the 52nd, 133rd, and 154th amino acids arbitrary amino acids.
  • relatively low wavelength excitation light for example, ultraviolet light ( The same applies to the following)
  • a protein that emits stronger fluorescence and a protein that emits stronger fluorescence when excited with excitation light of the same intensity can be obtained.
  • amino acids (X) surrounded by three squares correspond to the first amino acid corresponding to Xaa 52 , the second amino acid corresponding to Xaa 133 , and the third amino acid corresponding to Xaa 154 .
  • the first amino acid is the amino acid corresponding to Xaa 198
  • the second amino acid is the amino acid corresponding to Xaa 205 .
  • Xaa 52 is an arbitrary amino acid.
  • the arbitrary amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably Is C, F, H, K, M, or T.
  • it is more preferably C, M, or T because it emits stronger fluorescence even in excitation light having a relatively low wavelength.
  • Xaa 52 emits stronger fluorescence when excited with excitation light having the same intensity, and is more preferably H.
  • Xaa 133 is an arbitrary amino acid.
  • the arbitrary amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably Is L, Q, S, or T.
  • Xaa 133 emits stronger fluorescence when excited with excitation light having the same intensity, and is more preferably T.
  • Xaa 154 is an arbitrary amino acid.
  • the arbitrary amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably Is A, H, I, K, L, Q, V, or Y.
  • A, I, L, V, or Y since it emits stronger fluorescence even in excitation light having a relatively low wavelength, A, I, L, V, or Y.
  • Xaa 154 emits stronger fluorescence when excited with excitation light having the same intensity, and is more preferably K.
  • the combination of Xaa 52 , Xaa 133 , and Xaa 154 is not particularly limited, and for example, Xaa 52 is, for example, C, F, H, K, M, or T; Xaa 133 is, for example, L, Q, S, or T; Xaa 154 is, for example, A, H, I, K, L, Q, V, or Y.
  • Xaa 52 is, for example, C, M, or T
  • Xaa 133 is, for example, L, Q, S, or T
  • Xaa 154 is, for example, A, I, L, V, or Y.
  • (F1) is also referred to as (B1).
  • (F1) ) Is (B1), the (F2) and (F3) are also referred to as (B2) and (B3), respectively.
  • (B2) and (B3) are, for example, (F1) in (B1), (F2) in (B2) in (F2) and (F3), ( F3) can be read as (B3), and the description can be used.
  • the combination of Xaa 52 , Xaa 133 , and Xaa 154 is, for example, any one of the following (aa1) to (aa11) and (aa12). Further, in the protein, a combination of Xaa 52, Xaa 133, and Xaa 154 can, for example, even in the excitation light of the relatively low wavelength, since the emit stronger fluorescence, the following (aa1), (aa3), ( aa6), (aa9), (aa10), or (aa12) are preferred, and the following combinations (aa1) or (aa9) are more preferred.
  • a combination of Xaa 52, Xaa 133, and Xaa 154 can be, for example, when excited by the excitation light of the same intensity, since the emit stronger fluorescence, the following combinations (aa2) are preferred.
  • the combination of Xaa 52 , Xaa 133 , and Xaa 154 excludes the combination of H, S, and R, and the combination of D, S, and R, for example.
  • Xaa 205 is preferably substituted with I, for example, because it emits stronger fluorescence even with excitation light having a relatively low wavelength.
  • Xaa 198 is preferably substituted with L or H because, for example, Xaa 198 emits stronger fluorescence even with excitation light having a relatively low wavelength.
  • the 198th and 205th amino acids are presumed to each interact with the chromophore of the protein. For this reason, it is presumed that by performing the above-described substitution on at least one of the 198th and 205th amino acids, a protein that emits stronger fluorescence can be obtained even with excitation light having a relatively low wavelength.
  • Xaa 198 and Xaa 205 for example, emit more intense fluorescence even in the case of relatively low-wavelength excitation light, and are thus substituted with the combination of L and I or the combination of H and I, respectively. More preferably.
  • Examples of the protein (F1) include a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 47.
  • (F1) is a protein comprising the amino acid sequences of SEQ ID NOs: 2 to 47
  • (F1) is also referred to as (F1-1) to (F1-46)
  • (F2) is also referred to as (F2-1) to (F2-46)
  • the (F3) is also referred to as (F3-1) to (F3-46), respectively.
  • Examples of the protein (B1) include a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 7, 10, 11, 13, and 14 to 47.
  • the description of the protein consisting of the amino acid sequence of each SEQ ID NO in (B1) can be referred to the description of the protein consisting of the amino acid sequence of each SEQ ID NO in (F1).
  • the amino acid sequences of SEQ ID NOs: 2 to 47 in (F1) are as follows.
  • the amino acid sequences of SEQ ID NOs: 2 to 13 correspond to the combinations of Xaa 52 , Xaa 133 , and Xaa 154 in the amino acid sequence of SEQ ID NO: 1, respectively, of the combinations (aa1) to (aa12).
  • the amino acid sequences of SEQ ID NOs: 14 to 28 correspond to the case where the combination of Xaa 52 , Xaa 133 , and Xaa 154 is the combination of (aa1) in the amino acid sequence of SEQ ID NO: 1.
  • amino acid sequences of SEQ ID NOs: 29 to 47 correspond to the case where the combination of Xaa 52 , Xaa 133 , and Xaa 154 in the amino acid sequence of SEQ ID NO: 1 is the combination (aa9).
  • Xaa 52 , Xaa 133 and Xaa 154 of (F1) are conserved, and one or several amino acids are deleted, substituted or inserted in the amino acid sequence of (F1). It can also be referred to as a protein having a fluorescent activity and comprising an added amino acid sequence.
  • “1 or several” may be, for example, a range in which the (F2) has the fluorescence activity.
  • amino acid sequence of (F2) “1 or several” means, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, ⁇ 5, 1-3, 1 or 2.
  • the numerical range of numbers discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 5” means all disclosures of “1, 2, 3, 4, 5” (hereinafter the same).
  • the protein (F2) when Xaa 205 is substituted with I, it is preferable that the protein (F2) stores Xaa 205 of (F1).
  • the protein of (F2) for example, stores Xaa 52 , Xaa 133, Xaa 154, and Xaa 205 of (F1), and one or several amino acids in the amino acid sequence of (F1) are It is a protein having a fluorescent activity, consisting of an amino acid sequence deleted, substituted, inserted and / or added.
  • the protein (F2) when Xaa 198 is substituted with L or H, it is preferable that the protein (F2) contains Xaa 198 of (F1).
  • the protein of (F2) for example, contains Xaa 52 , Xaa 133, Xaa 154, and Xaa 198 of (F1), and one or several amino acids in the amino acid sequence of (F1) are It is a protein having a fluorescent activity, consisting of an amino acid sequence deleted, substituted, inserted and / or added.
  • the protein of (F2) when Xaa 198 and Xaa 205 are substituted with a combination of L and I, or a combination of H and I, respectively, the protein of (F2) becomes the Xaa 198 and of (F1).
  • Xaa 205 is preferably stored.
  • the protein (F2) contains, for example, Xaa 52 , Xaa 133, Xaa 154, Xaa 198, and Xaa 205 of (F1), and one or several proteins in the amino acid sequence of (F1) Is a protein having a fluorescent activity, consisting of an amino acid sequence in which the amino acids are deleted, substituted, inserted and / or added.
  • the “one or several”, for example, the above description can be used.
  • the protein of (F3) is an amino acid sequence in which Xaa 52 , Xaa 133, and Xaa 154 of (F1) are conserved and has 80% or more identity to the amino acid sequence of (F1), It can also be referred to as a protein having fluorescence activity.
  • the “identity” may be, for example, a range in which the (F3) has the fluorescence activity.
  • “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. .
  • the “identity” can be calculated from default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the protein (F3) when Xaa 205 is substituted with I, it is preferable that the protein (F3) stores Xaa 205 of (F1).
  • the protein (F3) contains, for example, Xaa 52 , Xaa 133, Xaa 154, and Xaa 205 of (F1), and is 80% or more identical to the amino acid sequence of (F1). It is a protein that has a fluorescent amino acid sequence and has an amino acid sequence.
  • the protein (F3) when Xaa 198 is substituted with L or H, it is preferable that the protein (F3) contains Xaa 198 of (F1).
  • the protein (F3) contains, for example, Xaa 52 , Xaa 133, Xaa 154, and Xaa 198 of (F1), and is 80% or more identical to the amino acid sequence of (F1). It is a protein that has a fluorescent amino acid sequence and has an amino acid sequence.
  • the protein of (F3) when Xaa 198 and Xaa 205 are substituted with a combination of L and I, or a combination of H and I, respectively, the protein of (F3) becomes the Xaa 198 of (F1) and Xaa 205 is preferably stored.
  • the protein of (F3) contains, for example, Xaa 52 , Xaa 133, Xaa 154, Xaa 198, and Xaa 205 of (F1), and 80% of the amino acid sequence of (F1). It is a protein having an amino acid sequence having the above identity and having fluorescence activity.
  • the fluorescent protein has the following chemical characteristics, for example.
  • the excitation wavelength, excitation maximum wavelength, fluorescence wavelength, and fluorescence maximum wavelength shown below are chemical characteristics in the wavelength range of 350 to 650 nm, for example.
  • the proteins (F1-1) to (F1-46) have, for example, the following chemical characteristics. When there are a plurality of maximum values of fluorescence intensity at the excitation wavelengths or fluorescence wavelengths of (F1-1) to (F1-46), a plurality of excitation maximum wavelengths and fluorescence maximum wavelengths are indicated.
  • the measurement method of the excitation wavelength, the excitation maximum wavelength, the fluorescence wavelength, and the fluorescence maximum wavelength is not particularly limited, and can be performed based on, for example, JIS K0120.
  • the fluorescent protein luminescence method of the present invention includes the step of irradiating the FRET fluorescent protein complex of the present invention with light having an excitation wavelength of the first fluorescent protein in the FRET fluorescent protein complex. Including By irradiating the first fluorescent protein with light having an excitation wavelength of the first fluorescent protein, the first fluorescent protein is caused to emit light, The second fluorescent protein in the FRET type fluorescent protein complex is excited by the light emission of the first fluorescent protein to cause the second fluorescent protein to emit light.
  • the luminescent method of the present invention uses the FRET fluorescent protein complex of the present invention, and the excitation light under the condition that the first fluorescent protein emits light with respect to the first fluorescent protein in the FRET fluorescent protein complex. And the second fluorescent protein is allowed to emit light by the light emission of the first fluorescent protein, and other configurations are not limited at all.
  • the wavelength of the light irradiated to the FRET fluorescent protein complex is not particularly limited, is the excitation wavelength of the first fluorescent protein, and the light emission of the first fluorescent protein by the excitation is Any condition including the excitation wavelength of the second fluorescent protein may be used.
  • the excitation wavelength and fluorescence wavelength of the first fluorescent protein and the excitation wavelength and fluorescence wavelength of the second fluorescent protein for example, the description of the FRET type fluorescent protein complex of the present invention can be used.
  • the luminescence method of the present invention may further include, for example, a detection step of detecting luminescence of the second fluorescent protein.
  • the light emission method of the present invention can also be referred to as, for example, the light emission detection method of the present invention.
  • the first fluorescent protein can be excited by irradiation light in the wavelength range of UV light, for example. And since the light emission of the said 1st fluorescence protein by excitation of the said irradiation light contains the excitation wavelength of a said 2nd fluorescence protein, this can make the said 2nd fluorescence protein light-emit, and can detect the said light emission.
  • the second fluorescent protein is used alone, for example, when the excitation wavelength of the exemplified second fluorescent protein is in the wavelength range of black light, for example, irradiation light of black light is irradiated.
  • the light emission of the second fluorescent protein overlaps with the wavelength of the black light, the light emission of the second fluorescent protein cannot be directly visually recognized, and an optical filter that cuts light other than the light emission (for example, a product) Through the name SC-52, manufactured by Fuji Film Co., Ltd.).
  • an optical filter that cuts light other than the light emission for example, a product
  • SC-52 manufactured by Fuji Film Co., Ltd.
  • external excitation light can be set according to the first fluorescent protein, and the second fluorescent protein is excited by light emission of the first fluorescent protein. Problems due to the use of the black light as described above can also be solved.
  • the FRET type fluorescent protein complex for example, it is possible to obtain luminescence with a stronger fluorescence intensity than when the second fluorescent protein is used alone.
  • the gene of the present invention is a gene encoding a FRET type fluorescent protein complex, and includes a first nucleic acid sequence encoding a first fluorescent protein and a second nucleic acid sequence encoding a second fluorescent protein.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked so as to be expressed as a complex in which the first fluorescent protein and the second fluorescent protein are linked, and the first fluorescence
  • the combination of the protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap.
  • the FRET fluorescent protein complex of the present invention can be easily produced by expressing it.
  • the first nucleic acid sequence and the second fluorescent protein are linked via a nucleic acid sequence encoding a linker.
  • the gene of the present invention may encode the FRET fluorescent protein complex of the present invention, and other configurations are not limited at all.
  • the gene of the present invention can be designed, for example, by appropriately replacing with a nucleic acid sequence consisting of codons encoding the first fluorescent protein, the second fluorescent protein, and any of the linker amino acid sequences.
  • the polynucleotide constituting the gene of the present invention can be produced by, for example, a known genetic engineering technique or a synthetic technique.
  • the expression vector of the present invention is characterized by including a gene encoding the FRET fluorescent protein complex of the present invention.
  • the FRET fluorescent protein complex of the present invention can be easily produced by introducing the expression vector into a host.
  • the description of the gene of the present invention can be used for the expression vector of the present invention.
  • the expression vector only needs to functionally include a polynucleotide constituting the gene so that the FRET fluorescent protein complex encoded by the gene of the present invention can be expressed. Is not particularly limited.
  • the expression vector can be prepared, for example, by inserting the polynucleotide into a backbone vector (hereinafter also referred to as “basic vector”).
  • the type of the vector is not particularly limited, and can be appropriately determined according to the type of the host. Examples of the vector include viral vectors and non-viral vectors.
  • the vector is preferably a binary vector, for example. When transforming a plant, the vector is preferably a T-DNA type binary vector.
  • the vectors include, for example, pBI121 vector, pPZP202 vector, pBINPLUS vector, pBIN19 vector, pBIG2113N, pBIG2113SF, pRI101 DNA vector (manufactured by TaKaRa), pRI201 DNA vector ( TaKaRa), pRI909 DNA vector (TaKaRa), pRI910 DNA vector (TaKaRa), and the like.
  • a microorganism such as E.
  • the vectors include, for example, pETDuet-1 vector (Novagen), pQE-80L vector (QIAGEN), pBluescript II SK vector, pET101 / D-TOPO vector (manufactured by Invitrogen). ), PGEX-6P-1 vector (Amersham Biosciences), pcDNA3.2 / V5-GW / D-TOPO vector (Invitrogen), pEGFP vector, pcDNA3.1-hygro (-) vector (Invitrogen) Etc.
  • pETDuet-1 vector Novagen
  • pQE-80L vector QIAGEN
  • pBluescript II SK vector pET101 / D-TOPO vector
  • PGEX-6P-1 vector Amersham Biosciences
  • pcDNA3.2 / V5-GW / D-TOPO vector Invitrogen
  • pEGFP vector pcDNA3.1-hygro (-) vector (Invitrogen) Etc.
  • the expression vector preferably has a regulatory sequence that regulates, for example, the expression of the polynucleotide and the expression of the FRET fluorescent protein complex of the present invention encoded by the polynucleotide.
  • the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ori) and the like.
  • the arrangement of the regulatory sequences is not particularly limited.
  • the regulatory sequence may be arranged so that, for example, the expression of the polynucleotide and the expression of the FRET fluorescent protein complex encoded by the polynucleotide can be functionally regulated. Can be arranged based on.
  • the regulatory sequence for example, a sequence provided in advance in the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, and the regulatory sequence provided in the basic vector It may be replaced with the regulatory sequence.
  • the expression vector may further include a selection marker coding sequence, for example.
  • a selection marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, and the like.
  • the method for producing the FRET fluorescent protein complex of the present invention is not particularly limited, and for example, it may be produced by a genetic engineering technique or may be produced by a known synthesis method. In the former case, the production method of the present invention includes an expression step of expressing the gene of the present invention. Thereby, the manufacturing method of the FRET type fluorescent protein complex of this invention can manufacture the said FRET type fluorescent protein complex of the said invention easily, for example.
  • the expression of the gene of the present invention is, for example, the expression of a polynucleotide constituting the gene, and may be performed using the expression vector of the present invention.
  • the method for expressing the polynucleotide is not particularly limited, and a known method can be adopted.
  • a cell-free protein synthesis system or a host may be used.
  • the polynucleotide is preferably expressed in a cell-free protein synthesis system.
  • an expression vector may be used for the expression of the polynucleotide.
  • the cell-free protein synthesis system can be performed by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which the target polynucleotide has been introduced. Reagent kits can be used.
  • the host in which the polynucleotide is introduced it is preferable to use the host in which the polynucleotide is introduced and to express the polynucleotide in the host by culturing the host.
  • a transformant that synthesizes the FRET fluorescent protein complex of the present invention can be produced by introducing the polynucleotide into a host, and the FRET of the present invention can be produced by culturing the transformant.
  • Type fluorescent protein complex can be synthesized.
  • Examples of the host include non-human hosts such as microorganisms, plants, animals, insects or cultured cells thereof, isolated human cells or cultured cells thereof, and preferably plants.
  • the plant When the host is a plant, the plant may be a plant body or a part thereof, for example.
  • Examples of the plant part include organs, tissues, cells, and vegetative propagation bodies.
  • Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like.
  • the tissue is, for example, a part of the organ.
  • Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus. The origin of the plant is not particularly limited. Department and so on.
  • Examples of the Brassicaceae include the Arabidopsis genus such as Arabidopsis thaliana .
  • the family Solanaceae for example, tobacco (Nicotiana tabacum) Nicotiana such as petunia (Petunia ⁇ hybrida) petunias genus such as (Petunia), Nierembergia (Nierembergia hippoamanica) Amamodoki genus such as, Calibrachoa (Calibrachoa hybrid Cultivar) such Examples include the genus Calibrachoa.
  • Gramineae examples include a genus of maize such as corn ( Zea mays ) and a genus of rice such as rice ( Oryza sativa ).
  • the legumes include soybean genus such as soybean ( Glycine max ).
  • the rose family for example, rose genus, such as roses (Rosa), and the like.
  • Examples of the Nadesico family include Nadesico genus such as carnation ( Dianthus caryophyllus ).
  • the Compositae family for example, chrysanthemum, such as cultivation chrysanthemum (Chrysantemum morifolium), Gerbera (Gerbera cvs.) Gerbera (Oosenbon'yari) genus, etc., and the like.
  • the Gentianaceae includes, for example, Eustoma genus such as Eustoma grandiflorum .
  • the Scrophulariaceae for example, Torenia sp such as Torenia (Torenia tenteri) and the like.
  • Examples of the family Oleaceae include Verbena genus such as Verbena ( Garden verbena ).
  • the Primulaceae for example, Cyclamen spp such as cyclamen (Cyclamen persicum) and the like.
  • the cactus family includes, for example, Austrokilondropuntia, Astrophytum, Echinocactus, Echinocereus, Echinopsis, Epiphylam, Opuntia, Crab Cactus, Kamaecereus, Kirindropuntia, Gymnocalycium Cactus cactus genus, Serenicerus genus, Teflocactus genus, Neobox baumia genus, Neoraimondia genus, Nopalea genus, Ferrocactus genus, Mamiglia genus, Melocatus genus, Lipsalis genus, Roseocactus genus, Lofophora genus and the like.
  • Phalaenopsis Phalaenopsis cvs.
  • Phalaenopsis Phalaenopsis
  • Cymbidium Cymbidium cvs.
  • Cymbidium Chunlan
  • Nobile system Dendrobium Dendrobium nobile hybrids
  • Denfare system Dendrobium D. phalaenopsis hybrids
  • Oncidium genus such as Oncidium cvs.
  • Cattleya genus such as Cattleya cvs .
  • the microorganism include eukaryotes and prokaryotes.
  • the prokaryotes for example, E.
  • Escherichia coli Escherichia genus such as Pseudomonas putida (Pseudomonas putida) Pseudomonas such as bacteria and the like.
  • the eukaryote include yeasts such as Saccharomyces cerevisiae .
  • the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.
  • a method for introducing the polynucleotide into the host that is, a method for transformation of the host is not particularly limited, and for example, a method using the expression vector may be used, or the expression vector is not used. It may be a known method introduced into. In the latter case, the introduction method can be appropriately set depending on, for example, the type of the host.
  • the introduction method examples include heat shock method, lithium acetate method, introduction method using gene gun such as particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposome, electroporation method, ultrasonic nucleic acid introduction method, DEAE -A dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method using Agrobacterium, and the like.
  • the liposome include lipofectamine and cationic liposome
  • the introduction aid include atelocollagen, nanoparticles, and polymers.
  • the introduction method is preferably an Agrobacterium-mediated method.
  • the polynucleotide constituting the gene of the present invention may be introduced into the host by the expression vector of the present invention, for example.
  • the method for culturing the host is not particularly limited, and can be appropriately set according to the type of the host.
  • the medium used for culturing the host is not particularly limited, and can be appropriately determined according to the type of the host.
  • the form of the medium used for culturing the host is not particularly limited, and a conventionally known medium such as a solid medium, a liquid medium, or an agar medium can be used as appropriate.
  • the components contained in the medium are not particularly limited.
  • the medium may include a commercially available medium, for example.
  • the commercial medium of the plant is not particularly limited, and examples thereof include Murashige-Skoog (MS) medium.
  • MS Murashige-Skoog
  • the commercially available medium for the plant cell is not particularly limited, and examples thereof include hyponex medium, MS medium, Gamborg B5 (B5) medium, White medium and the like.
  • the commercially available medium for the microorganism is not particularly limited, and examples thereof include LB medium, super broth medium, M9 medium and the like.
  • examples thereof include LB medium, super broth medium, M9 medium and the like.
  • one type of the medium may be used alone, or two or more types may be used in combination.
  • the pH of the medium is not particularly limited, and is, for example, in the range of pH 6 to 8, or in the range of pH 6.5 to 7.5.
  • the method for culturing the host is not particularly limited, and can be appropriately determined according to, for example, the type of the host.
  • examples of the culture method include a method of cultivating the plant in soil and water.
  • examples of the culture method include callus culture, root culture, ovule culture, embryo culture, and the like.
  • the culture temperature at the time of culturing the host is not particularly limited and can be appropriately determined according to, for example, the type of the host.
  • examples of the culture temperature include a plant-growing temperature and an optimum growth temperature.
  • the culture temperature can be, for example, in the range of 15 to 40 ° C., in the range of 30 to 37 ° C.
  • examples of the culture temperature include a plant cell growth temperature and a growth optimum temperature.
  • the culture temperature can be, for example, in the range of 15 to 40 ° C., in the range of 30 to 37 ° C.
  • the host may be cultured under an aerobic condition or an anaerobic condition, for example.
  • the aerobic condition or the anaerobic condition is not particularly limited, and can be set using a conventionally known method.
  • the transformant of the present invention includes the gene of the present invention or the expression vector.
  • the transformant of the present invention is characterized by including the gene of the present invention or the expression vector, and other configurations and conditions are not particularly limited. Since the transformant of the present invention contains the gene of the present invention or the expression vector, it has, for example, fluorescence activity.
  • the description of the FRET fluorescent protein complex of the present invention can be cited.
  • the transformant is not particularly limited, and examples thereof include animals and plants, but plants are preferred.
  • examples of the transformant include cancer cells such as human colon cancer cells.
  • the origin of the plant and the plant is not particularly limited, and for example, the above description can be used.
  • the plant may be a plant body or a part thereof, for example.
  • the plant part include organs, tissues, cells, and vegetative propagation bodies.
  • the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like.
  • the tissue is, for example, a part of the organ.
  • Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus.
  • the transformant of the present invention when the transformant is a plant, the transformant of the present invention can be further propagated, for example. At this time, the transformant of the present invention can be used as the propagation material.
  • the propagation material is not particularly limited, and may be the whole or a part of the transformant, for example.
  • examples of the propagation material include seeds, fruits, shoots, stems such as tubers, roots such as tuberous roots, strains, protoplasts, and callus.
  • the propagation method of the transformant of the present invention is not particularly limited, and a known method can be adopted.
  • the breeding method may be, for example, sexual reproduction or asexual reproduction, preferably asexual reproduction.
  • the reproduction by asexual reproduction includes, for example, vegetative propagation and is also called vegetative reproduction.
  • the method of vegetative propagation is not particularly limited, and in the case of the plant body or a part thereof, for example, bud propagation, propagation by cuttings, subdividing from an organ to a plant individual, growth by callus, and the like can be mentioned.
  • the organ for example, the leaves, stems, roots and the like as described above can be used.
  • the vegetative propagation material of the present invention preferably has the same properties as the transformant of the present invention.
  • the vegetative propagation material of the present invention is not particularly limited as in the case of the transformant, and examples thereof include a plant or a part thereof.
  • progeny such as growths grown from the seeds can be obtained. It is preferable that the progeny of the present invention obtains the same properties as the transformant of the present invention.
  • the progeny of the present invention may be, for example, a plant or a part thereof, for example, like the transformant.
  • the transformant of the present invention may be further processed, for example.
  • the kind of the transformant to be processed is not particularly limited, and examples thereof include flowers, leaves, branches and the like.
  • the processed product of the transformant is not particularly limited, and examples thereof include potpourri, dried flowers, dried flowers, preserved flowers, and resin-sealed products obtained by drying flowers, leaves, branches and the like.
  • the processed product of the present invention may be, for example, a processed product of a vegetative propagation body, organ, tissue or cell of the transformant.
  • the method for producing a transformant of the present invention includes an introducing step of introducing the gene of the present invention or the expression vector into a host.
  • the method for producing a transformant of the present invention includes an introduction step of introducing the gene of the present invention or the expression vector into a host, and other steps and conditions are not particularly limited. According to the method for producing a transformant of the present invention, for example, the transformant of the present invention can be easily produced.
  • the method for producing a transformant of the present invention may further include an expression step for causing the host to express the gene.
  • the method for introducing the gene of the present invention or the expression vector into the host is not particularly limited.
  • the polynucleotide in the method for producing the FRET fluorescent protein complex of the present invention is used as the host.
  • the explanation of the method to introduce can be used.
  • the host is preferably a plant.
  • the expression step is, for example, a step of expressing the FRET fluorescent protein complex of the present invention from the gene of the present invention in a host.
  • the host is preferably, for example, a transformant introduced with the gene of the present invention or the expression vector, and the FRET fluorescent protein of the present invention is cultured in the host by culturing the transformant. It is preferable to express the complex.
  • the method for producing a transformant of the present invention may further include a breeding step for breeding the transformant obtained by the introducing step.
  • the breeding method in the breeding process for example, the above description can be used.
  • the method for producing a transformant of the present invention may include a seeding process for seeding from the propagated transformant, and may further include a growth process for growing a growth body from the seed obtained in the seeding process. .
  • AAA in the linker region surrounded by a square is an amino acid sequence derived from a restriction enzyme site in the construction of an expression vector described later.
  • the combination 1 and 2 are FRET type fluorescent protein complexes, and the combination 3 is a non-FRET type fluorescent protein complex.
  • a polynucleotide encoding each region of the FRET type fluorescent protein complex is prepared, treated with a restriction enzyme, and between the multiple cloning sites of the pEGFP vector (clontech), instead of the EGFP region, each FRET type fluorescent protein complex A body-encoding polynucleotide was inserted. This was used as an expression vector for expressing each FRET type fluorescent protein complex.
  • Tables 10 to 12 below show the nucleotide sequences of the polynucleotides encoding the FRET fluorescent protein complexes of Tables 7 to 9 inserted into the respective expression vectors.
  • an expression vector into which only BK15 was introduced and an expression vector into which only BL was introduced were constructed in the same manner.
  • the following table shows the amino acid sequence of the protein expressed by the expression vector of BK15 alone or BL alone, and the base sequence of the polynucleotide encoding it. In the following amino acid sequence, the underlined portion is the same as in the FRET fluorescent protein complex.
  • the obtained suspension was subjected to ultrasonic treatment twice using an ultrasonic homogenizer (QSONICA, manufactured by Wakenbee Tech Co., Ltd.) under the conditions of Amp 25% and 1 minute. And the suspension after the said process was centrifuged for 10 minutes on the conditions of 1300 rpm and 4 degreeC, and the obtained supernatant was collect
  • the supernatant obtained from the transformant into which each expression vector was introduced was used as the FRET type fluorescent protein complex sample shown in Tables 7-9.
  • the protein concentration in each supernatant was determined by adding protein assay (manufactured by Bio Rad) to each supernatant and then using a microplate reader (Infinite (registered trademark) M1000Pro, TECAN) at 562 nm. Was determined by measuring.
  • the supernatant samples of BK15-gs1-BL and BK15-gs1-Wt prepared in Example 1 were diluted to 2 ⁇ mol / L. With respect to 50 ⁇ L of these diluted samples, fluorescence intensity (FI) was measured under the following measurement conditions using the microplate reader. Moreover, it measured similarly about the supernatant sample of BK15 which is a negative control.
  • the plate used was HTS 384 well C-Flat Fluorescence Black Plate® (# 781076, greiner® bio-one).
  • FIG. 1 shows the results of fixing the excitation wavelength (Ex) to 400 nm and the fluorescence wavelength (Em) to 516 nm.
  • FIG. 1 is a graph showing the fluorescence intensity (FI) under the above conditions for each FRET type fluorescent protein complex.
  • FI fluorescence intensity
  • the FRET type fluorescent protein complex in which BK15 and BL or Wt are linked showed emission of stronger fluorescence intensity.
  • BL and Wt corresponding to the second fluorescent protein in the FRET type fluorescent protein complex show stronger fluorescence intensity at the fluorescence wavelength 512 nm of BK15 in the complex than the excitation wavelength 400 nm from the outside.
  • the FRET fluorescent protein complex can be irradiated with light having an excitation wavelength (400 nm) that causes BK15 to emit light, instead of light having an excitation wavelength (507 to 508 nm) suitable for BL or Wt. By light emission, BL or Wt can be excited to emit light. For this reason, the optical film as described above is also unnecessary.
  • the supernatant samples of BK15-gs2-BL, BK15-gs2-Wt and BK15-gs2-BK15 prepared in Example 1 were diluted to 2 ⁇ mol / L. About 50 ⁇ L of these diluted samples, the fluorescence intensity (FI) was measured in the same manner as in Example 2 under the following measurement conditions using the microplate reader. Further, the same measurement was performed for the BK15 supernatant sample and the BL supernatant sample as negative controls.
  • FI fluorescence intensity
  • each FRET type fluorescent protein complex significantly increased the fluorescence intensity. Specifically, The fluorescence intensity at the fluorescence maximum wavelength increased significantly.
  • BK15 is used as the first fluorescent protein
  • BL or Wt which is a fluorescent protein different from the first fluorescent protein
  • the wavelength exhibiting the highest fluorescence intensity was shifted to the longer wavelength side than BK alone.
  • the measurement results of the entire range of excitation wavelengths and the entire range of fluorescence wavelengths are shown in FIG.
  • the X-axis direction indicates the excitation wavelength
  • the Y-axis direction indicates the fluorescence wavelength
  • the Z-axis direction indicates the fluorescence intensity.
  • Table 16 shows the emission intensity, maximum excitation wavelength, and emission maximum wavelength of the emission peak.
  • BK15 alone showed an emission peak in a low wavelength region such as 400 nm, but its intensity FI was about 15,000.
  • BL alone did not show an emission peak in the low wavelength region including 400 nm, but the intensity FI in the high wavelength region showed 150,000, which is one digit larger than that of BK15.
  • the FRET fluorescent protein complex in addition to the peak in the low wavelength region such as 400 nm, the peak was confirmed in the high wavelength region. Also from this result, as described in FIG. 2, when the FRET fluorescent protein complex is excited at 400 nm, the BK15 of the first fluorescent protein is excited and emits light. It can be seen that BL and Wt are excited to emit light.
  • the supernatant samples of BK15-gs3-BL, BK15-gs3-Wt and BK15-gs3-BK15 prepared in Example 1 were diluted to 2 ⁇ mol / L. About 50 ⁇ L of these diluted samples, the fluorescence intensity (FI) was measured in the same manner as in Example 2 under the following measurement conditions using the microplate reader. Further, the same measurement was performed for the BK15 supernatant sample and the BL supernatant sample as negative controls.
  • FI fluorescence intensity
  • the fluorescence intensity was significantly increased by each FRET type fluorescent protein complex as compared with the case of BK alone and BL alone, specifically, The fluorescence intensity at the fluorescence maximum wavelength increased significantly.
  • BK15 is used as the first fluorescent protein
  • BL or Wt which is a fluorescent protein different from the first fluorescent protein
  • the wavelengths of BK15-gs3-BL and BK15-gs3-Wt that showed the highest fluorescence intensity were shifted to the longer wavelength side than BK alone.
  • the measurement results of the entire excitation wavelength range and the entire fluorescence wavelength range are shown in FIG. 5, as in FIG. 4, the X-axis direction indicates the excitation wavelength, the Y-axis direction indicates the fluorescence wavelength, and the Z-axis direction indicates the fluorescence intensity.
  • Table 18 shows the maximum fluorescence intensity and the fluorescence detection wavelength indicating the fluorescence intensity.
  • BK15 alone showed a light emission peak in a low wavelength region such as 400 nm, but its intensity FI was about 15,000.
  • BL alone did not show an emission peak in the low wavelength region including 400 nm, but the intensity FI in the high wavelength region showed 150,000, which is one digit larger than that of BK15.
  • the FRET fluorescent protein complex was able to confirm a peak in a high wavelength region in addition to a peak in a low wavelength region such as 400 nm. Also from this result, as described in FIG. 4, when the FRET fluorescent protein complex is excited at 400 nm, the BK15 of the first fluorescent protein is excited and emits light. It can be seen that BL and Wt are excited to emit light.
  • Appendix 1 Including a first fluorescent protein and a second fluorescent protein, The first fluorescent protein and the second fluorescent protein are linked; The combination of the first fluorescent protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap.
  • a fluorescent protein complex characterized by the above. (Appendix 2) The second fluorescent protein is The second fluorescent protein was excited with the emission intensity (I2 E1 ) when the second fluorescent protein was excited with the excitation wavelength (E1) of the first fluorescent protein and the fluorescence wavelength (L1) of the first fluorescent protein. 2.
  • the fluorescent protein complex according to appendix 1 wherein the emission intensity (I2 L1 ) satisfies the relationship of I2 E1 ⁇ I2 L1 .
  • Appendix 3 The fluorescent protein complex according to appendix 1 or 2, wherein the first fluorescent protein and the second fluorescent protein are different proteins.
  • Appendix 4 The fluorescent protein complex according to appendix 3, wherein the maximum excitation wavelength of the first fluorescent protein and the maximum excitation wavelength of the second fluorescent protein are different.
  • Appendix 5 The fluorescent protein complex according to any one of appendices 1 to 4, wherein the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein include a wavelength of 350 to 650 nm. body.
  • (Appendix 13) Irradiating the fluorescent protein complex according to any one of appendices 1 to 12 with light having an excitation wavelength of the first fluorescent protein in the fluorescent protein complex, By irradiating the first fluorescent protein with light having an excitation wavelength of the first fluorescent protein, the first fluorescent protein is caused to emit light, A method for emitting a fluorescent protein, wherein the second fluorescent protein in the fluorescent protein complex is excited by light emission of the first fluorescent protein to cause the second fluorescent protein to emit light. (Appendix 14) Furthermore, the light-emission method of Additional remark 13 including the detection process of light emission of said 2nd fluorescent protein.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked so as to be expressed as a complex in which the first fluorescent protein and the second fluorescent protein are linked;
  • the combination of the first fluorescent protein and the second fluorescent protein is a combination in which the fluorescence wavelength (L1) of the first fluorescent protein and the excitation wavelength (E2) of the second fluorescent protein overlap.
  • a gene encoding a fluorescent protein complex characterized by (Appendix 16) The gene according to appendix 15, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked via a nucleic acid sequence encoding a linker.
  • Appendix 17 An expression vector comprising the gene encoding the fluorescent protein complex according to appendix 15 or 16.
  • Appendix 18 A transformant comprising the gene encoding the fluorescent protein complex according to appendix 15 or 16, or the expression vector according to appendix 17.
  • the present invention by combining the first fluorescent protein and the fluorescent protein under the above-described conditions, for example, when the excitation light from the outside is different from the excitation conditions of the second fluorescent protein or maximum excitation Even when the wavelength deviates from the wavelength, the second fluorescent protein can emit light by excitation due to light emission of the first fluorescent protein. For this reason, according to this invention, compared with the case where a fluorescent protein is used independently, the setting of a wider optical condition is attained, for example.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'objectif de la présente invention concerne une nouvelle protéine fluorescente. Ce complexe de protéine fluorescente de type FRET est caractérisé par les éléments suivants : il comprend une première protéine fluorescente et une deuxième protéine fluorescente; la première protéine fluorescente et la deuxième protéine fluorescente sont couplées; et la combinaison de la première protéine fluorescente et de la deuxième protéine fluorescente est telle que la longueur d'onde de fluorescence (L1) de la première protéine fluorescente chevauche la longueur d'onde d'excitation (E2) de la deuxième protéine fluorescente.
PCT/JP2017/028174 2016-11-30 2017-08-03 Complexe de protéine fluorescente et procédé électroluminescent de protéine fluorescente Ceased WO2018100803A1 (fr)

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JP2018553656A JPWO2018100803A1 (ja) 2016-11-30 2017-08-03 蛍光タンパク質複合体、および蛍光タンパク質の発光方法

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JP2016232746 2016-11-30
JP2016-232746 2016-11-30

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ARAI RYOICHI ET AL.: "Conformations of Variably Linked Chimeric Proteins Evaluated by Synchrotron X-ray Small-Angle Scattering", PROTEINS, vol. 57, no. 4, 2004, pages 829 - 838, XP002358153 *
LI GANG ET AL.: "Construction of a linker library with widely controllable flexibility for fusion protein design", APPL. MICROBIOL. BIOTECHNOL., vol. 100, no. 1, January 2016 (2016-01-01), pages 215 - 225, XP035870356 *

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