JP7031929B2 - New proteins and new genes - Google Patents

Description

The present invention relates to novel proteins and novel genes.

As the fluorescent protein, GFP (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein) and the like are generally used (Non-Patent Document 1). The fluorescent protein is used for various purposes such as research. However, fluorescent proteins suitable for various uses do not always exist. Therefore, a new fluorescent protein is required.

Chalfie M et al., “Green fluorescent protein as a marker for gene expression”, Science, February 1994, Vol.263, no.5148, p802-805.

Therefore, an object of the present invention is to provide a new fluorescent protein.

The novel protein of the present invention is characterized by being the following (F1) or (F2) protein.
(F1) In the amino acid sequence of SEQ ID NO: 1,
Protein (F2) consisting of an amino acid sequence in which the 52nd amino acid is an amino acid other than H. A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of the protein (F1) of ± 5 nm.

The novel gene of the present invention is characterized by being the following polynucleotide (f1) or (f2).
(F1) In the base sequence of SEQ ID NO: 12,
A polynucleotide in which the 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H (f2) The 154th base and the 155th base with respect to the polynucleotide of the above (f1). A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of (f1), and having a base sequence having an identity of 80% or more including the base of the above and the 156th base. The polynucleotide that encodes

The expression vector of the present invention is characterized by containing the novel gene of the present invention.

The transformant of the present invention is characterized by containing the novel gene of the present invention.

The method for producing a transformant of the present invention is characterized by comprising an introduction step of introducing the novel gene of the present invention into a host.

In the method for screening a novel fluorescent protein of the present invention, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H from the candidate protein in which a mutation is introduced into the novel protein of the present invention, and the above-mentioned (F1). It is characterized by including a selection step of selecting a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein.

According to the present invention, a novel fluorescent protein can be provided.

FIG. 1 is a graph showing fluorescence intensities at different excitation wavelengths and fluorescence wavelengths in Example 1. FIG. 2 is a graph showing fluorescence intensities at different excitation wavelengths and fluorescence wavelengths in Example 3. FIG. 3 is a graph showing the excitation wavelength at which the fluorescence intensity at the maximum value and the fluorescence intensity at the fluorescence wavelength in Example 3 are shown.

<New protein>
As described above, the novel protein of the present invention is characterized by being the following (F1) or (F2) protein.
(F1) In the amino acid sequence of SEQ ID NO: 1,
Protein (F2) consisting of an amino acid sequence in which the 52nd amino acid is an amino acid other than H. A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of the protein (F1) of ± 5 nm.

Further, in the novel protein of the present invention, for example, the protein of the above (F1) is the protein of the following (F1'), and the protein of the above (F2) is the protein of the following (F2').
(F1') In the amino acid sequence of SEQ ID NO: 1,
The 52nd amino acid is an amino acid other than H,
Protein consisting of an amino acid sequence in which the 136th amino acid is an amino acid other than T (F2') An amino acid sequence having 80% or more identity including the 52nd and 136th amino acids with respect to the amino acid sequence of the above (F1'). A protein consisting of and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).

Further, in the novel protein of the present invention, for example, the protein of the above (F1) is the protein of the following (F1 ″), and the protein of the above (F2) is the protein of the following (F2 ″).
(F1'') In the amino acid sequence of SEQ ID NO: 1.
The 52nd amino acid is an amino acid other than H,
The 56th amino acid is an amino acid other than Y,
The 136th amino acid is an amino acid other than T,
Protein consisting of an amino acid sequence in which the 192nd amino acid is an amino acid other than R (F2'') Contains the 52nd, 56th, 136th, and 192nd amino acids with respect to the amino acid sequence of (F1''). A protein consisting of an amino acid sequence having an identity of 80% or more and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).

The novel protein of the present invention is, for example, in the amino acid sequence of SEQ ID NO: 1.
The 52nd amino acid is A or S,
The 56th amino acid is R or Y,
The 136th amino acid is M, S, or T,
The 192nd amino acid is R, or I.

In the novel protein of the present invention, for example, the combination of the 52nd amino acid, the 56th amino acid, the 136th amino acid, and the 192nd amino acid is a combination of any one of the following (aa1) to (aa3). be.

In the novel protein of the present invention, for example, the fourth amino acid is replaced with Y.

In the novel protein of the present invention, for example, the 22nd amino acid is replaced with G.

In the novel protein of the present invention, for example, the 29th amino acid is replaced with V.

In the novel protein of the present invention, for example, the 37th amino acid is replaced with T.

In the novel protein of the present invention, for example, the 69th amino acid is replaced with M.

In the novel protein of the present invention, for example, the 153rd amino acid is replaced with V.

In the novel protein of the present invention, for example, the 172nd amino acid is replaced with M.

The novel protein of the present invention is, for example, a protein in which the protein of (F1) is composed of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11.

As a result of diligent research, the present inventors have found that in the protein having the amino acid sequence of SEQ ID NO: 1, the 52nd amino acid has a relationship with the fluorescence activity of the protein. Specifically, it was presumed that the 52nd amino acid of the protein interacts with, for example, the chromophore of the protein. Therefore, according to the novel protein of the present invention, the fluorescence activity of the protein can be adjusted by using an amino acid sequence in which the 52nd amino acid is mutated to an amino acid other than H, for example, the amino acid of SEQ ID NO: 1. A protein that emits fluorescence having a wavelength different from that of the protein having a sequence can be obtained. The above estimation does not limit the present invention in any way.

Hereinafter, the novel protein is also referred to as a novel fluorescent protein. The novel fluorescent protein is a protein newly prepared by the present inventors. In the present invention, the novel fluorescent protein may contain, for example, one kind of protein or two or more kinds of proteins (hereinafter, the same applies).

The amino acid sequence of SEQ ID NO: 1 in (F1) is as follows. In the following amino acid sequence, the amino acids (X) enclosed in four squares are the 52nd amino acid, the 56th amino acid, the 136th amino acid, and the 192nd amino acid, respectively. The seven underlined amino acids are the 4th amino acid, the 22nd amino acid, the 29th amino acid, the 37th amino acid, the 69th amino acid, the 153rd amino acid, and the 172nd amino acid, respectively. be. The protein having the amino acid sequence of SEQ ID NO: 1 is also referred to as, for example, CpYGFP.

In the protein of (F1), the 52nd amino acid in SEQ ID NO: 1 is an amino acid other than H. Amino acids other than H are A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably. A or S.

Examples of the protein of (F1) include a protein consisting of the amino acid sequence of SEQ ID NO: 2. When the (F1) is a protein consisting of the amino acid sequence of SEQ ID NO: 2, the (F1) is also referred to as the amino acid sequence of Y13.

The amino acid sequence of SEQ ID NO: 2 in (F1) is as follows. The amino acid sequence of SEQ ID NO: 2 corresponds to the case where the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is A.

The protein of (F2) is composed of, for example, an amino acid sequence in which the 52nd amino acid of (F1) is conserved and has 80% or more identity with respect to the amino acid sequence of (F1). ) Can be said to be a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm. In the (F2), the "identity" may be, for example, as long as the (F2) has the maximum fluorescence wavelength. In the amino acid sequence of (F2), the "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 the default parameters using, for example, analysis software such as BLAST or FASTA (hereinafter, the same applies).

Examples of the protein of (F2) include proteins consisting of the amino acid sequences of SEQ ID NOs: 3 and 4. The amino acid sequence of SEQ ID NO: 3 is an amino acid sequence in which the 22nd amino acid in the amino acid sequence of SEQ ID NO: 2 is substituted with G. The amino acid sequence of SEQ ID NO: 4 is an amino acid sequence in which the fourth amino acid is substituted with Y in the amino acid sequence of SEQ ID NO: 2. When the (F2) is a protein consisting of the amino acid sequences of SEQ ID NOs: 3 and 4, the (F2) is also referred to as the amino acid sequences of Y3 and Y6, respectively.

The novel protein of the present invention may be the following protein (F3).
(F3) In the amino acid sequence other than the 52nd amino acid of (F1), one or several amino acids are deleted, substituted, inserted and / or added, and the maximum of the protein of (F1) is described. Protein with maximum fluorescence wavelength in the range of fluorescence wavelength ± 5 nm

The protein of (F3) is an amino acid in which the 52nd amino acid of (F1) is conserved and one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of (F1). It can also be said that the protein is composed of an sequence and has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1). In the (F3), the "identity" may be, for example, a range in which the (F3) has the maximum fluorescence wavelength. In the amino acid sequence of (F3), "1 or several" means, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, 1 ~ 5, 1 ~ 3, 1 or 2. In the present invention, the numerical range of the number discloses, for example, all positive integers belonging to the range. That is, for example, the description of "1 to 5" means all disclosures of "1, 2, 3, 4, 5" (hereinafter, the same applies).

The protein of (F1) may be, for example, the protein of (F1') below.
(F1') In the amino acid sequence of SEQ ID NO: 1,
The 52nd amino acid is an amino acid other than H,
A protein consisting of an amino acid sequence in which the 136th amino acid is an amino acid other than T.

In the protein of (F1'), the 52nd amino acid in SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid is an amino acid other than T. Amino acids other than T are A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y, preferably. S, or M.

Examples of the protein of (F1') include a protein consisting of the amino acid sequence of SEQ ID NO: 5. When the (F1') is a protein consisting of the amino acid sequence of SEQ ID NO: 5, the (F1') is also referred to as an amino acid sequence of Y.

The amino acid sequence of SEQ ID NO: 5 in (F1') is as follows. The amino acid sequence of SEQ ID NO: 5 corresponds to the case where the 52nd amino acid is A and the 136th amino acid is S in the amino acid sequence of SEQ ID NO: 1.

In the protein of (F2'), for example, the 52nd and 136th amino acids of (F1') are conserved, and the amino acid sequence has 80% or more identity with respect to the amino acid sequence of (F1'). It can also be said that the protein has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1').

The novel protein of the present invention may be the following (F3') protein.
(F3') In the amino acid sequence other than the 52nd and 136th amino acids of the above (F1'), one or several amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of the above (F1'). ') Protein with maximum fluorescence wavelength in the range of ± 5 nm maximum fluorescence wavelength

The protein of (F3') contains the 52nd and 136th amino acids of (F1'), and one or several amino acids are deleted, substituted, inserted and / in the amino acid sequence of (F1). Alternatively, it can be said that the protein is composed of the added amino acid sequence and has the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).

The protein of (F1) may be, for example, the protein of (F1'') below.
(F1'') In the amino acid sequence of SEQ ID NO: 1.
The 52nd amino acid is an amino acid other than H,
The 136th amino acid is an amino acid other than T,
The 56th amino acid is an amino acid other than Y,
A protein consisting of an amino acid sequence in which the 192nd amino acid is an amino acid other than R.

In the protein of (F1''), the 52nd amino acid in SEQ ID NO: 1 is an amino acid other than H, the 136th amino acid is an amino acid other than T, and the 56th amino acid is an amino acid other than Y. The 192nd amino acid is an amino acid other than R. Amino acids other than Y are A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W, preferably. R, or I. Amino acids other than R are A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y, preferably. I.

Examples of the protein of (F1 ″) include a protein consisting of the amino acid sequence of SEQ ID NO: 6. When the (F1 ″) is a protein consisting of the amino acid sequence of SEQ ID NO: 6, the (F1 ″) is also referred to as an amino acid sequence of BG.

The amino acid sequence of SEQ ID NO: 6 in (F1 ″) is as follows. In the amino acid sequence of SEQ ID NO: 6, the 52nd amino acid is S, the 136th amino acid is M, the 56th amino acid is R, and the 192nd amino acid is S in the amino acid sequence of SEQ ID NO: 1. Corresponds to the case of I.

The protein of (F2'') contains, for example, the 52nd, 136th, 56th and 192nd amino acids of (F1''), and is 80 with respect to the amino acid sequence of (F1''). It can also be said that the protein has an amino acid sequence having an identity of% or more and has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1 ″).

Examples of the protein of (F2 ″) include proteins consisting of the amino acid sequences of SEQ ID NOs: 7 to 11. The amino acid sequence of SEQ ID NO: 7 is an amino acid sequence in which the 37th amino acid is substituted with T and the 153rd amino acid is substituted with V in the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of SEQ ID NO: 8 is an amino acid sequence in which the 29th amino acid is substituted with V and the 153rd amino acid is substituted with V in the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of SEQ ID NO: 9 is an amino acid sequence in which the 69th amino acid is substituted with M and the 153rd amino acid is substituted with V in the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of SEQ ID NO: 10 is an amino acid sequence in which the 153rd amino acid is substituted with V and the 172nd amino acid is substituted with M in the amino acid sequence of SEQ ID NO: 6. In the amino acid sequence of SEQ ID NO: 11, in the amino acid sequence of SEQ ID NO: 6, the 29th amino acid is replaced with V, the 37th amino acid is replaced with T, and the 153rd amino acid is replaced with V. It is an array. When the (F2 ″) is a protein consisting of the amino acid sequences of SEQ ID NOs: 7 to 11, the (F2 ″) is also referred to as the amino acid sequences of BG15, BG17, BG19, BG23, and BG29, respectively.

The novel protein of the present invention may be the following (F3') protein.
(F3'') In the amino acid sequence other than the 52nd, 136th, 56th and 192nd amino acids of (F1''), one or several amino acids were deleted, substituted, inserted and / or added. A protein consisting of an amino acid sequence and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1'').

In the protein of (F3''), the 52nd, 136th, 56th and 192nd amino acids of (F1'') are conserved, and one or several amino acids are contained in the amino acid sequence of (F1). It can also be said to be a protein consisting of an amino acid sequence deleted, substituted, inserted and / or added, and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).

Examples of the protein of (F3 ″) include a protein consisting of the amino acid sequences of SEQ ID NOs: 7 to 11.

The novel fluorescent protein of the present invention has, for example, the following chemical properties. The following excitation wavelength, excitation maximum wavelength, fluorescence wavelength and fluorescence maximum wavelength are chemical properties in the wavelength range of, for example, 350 to 650 nm.
Excitation wavelength: 350-650 nm
Maximum excitation wavelength: 386-514 nm
Fluorescence wavelength: 350-650 nm
Fluorescence maximum wavelength: 506 to 524 nm

When the novel fluorescent protein of the present invention is Y13, the excitation wavelength of Y13 is, for example, 485 to 495 nm, and the fluorescence wavelength is, for example, 505 to 515 nm.

When the novel fluorescent protein of the present invention is Y3, the excitation wavelength of Y3 is, for example, 485 to 495 nm, and the fluorescence wavelength is, for example, 505 to 515 nm.

When the novel fluorescent protein of the present invention is Y6, the excitation wavelength of Y6 is, for example, 485 to 495 nm, and the fluorescence wavelength is, for example, 505 to 515 nm.

When the novel fluorescent protein of the present invention is Y, the excitation wavelength of Y is, for example, 485 to 495 nm, and the fluorescence wavelength is, for example, 505 to 515 nm.

When the novel fluorescent protein of the present invention is the BG, the excitation wavelength of the BG is, for example, 445 to 455 nm, and the fluorescence wavelength is, for example, 480 to 490 nm.

When the novel fluorescent protein of the present invention is the BG15, the excitation wavelength of the BG15 is, for example, 435 to 445 nm, and the fluorescence wavelength is, for example, 485 to 495 nm.

When the novel fluorescent protein of the present invention is the BG17, the excitation wavelength of the BG17 is, for example, 440 to 450 nm, and the fluorescence wavelength is, for example, 485 to 495 nm.

When the novel fluorescent protein of the present invention is the BG19, the excitation wavelength of the BG19 is, for example, 435 to 445 nm, and the fluorescence wavelength is, for example, 485 to 495 nm.

When the novel fluorescent protein of the present invention is the BG23, the excitation wavelength of the BG23 is, for example, 435 to 445 nm, and the fluorescence wavelength is, for example, 485 to 495 nm.

When the novel fluorescent protein of the present invention is the BG29, the excitation wavelength of the BG29 is, for example, 440 to 450 nm, and the fluorescence wavelength is, for example, 485 to 495 nm.

The method for measuring the excitation wavelength, the fluorescence wavelength, and the fluorescence maximum wavelength is not particularly limited, and can be performed based on, for example, JIS K0120. The method for measuring the excitation wavelength, the excitation maximum wavelength, the fluorescence wavelength, and the fluorescence maximum wavelength may be, for example, according to the measurement method of Example 1 described later.

The novel fluorescent protein emits fluorescence of a different wavelength as compared with, for example, the CpYGFP. When the novel fluorescent protein and the CpYGFP emit fluorescence having different wavelengths, for example, the maximum fluorescence wavelength is measured for the novel fluorescent protein and the CpYGFP, and the difference between the maximum fluorescence wavelengths is, for example, 20 nm or more and 10 nm. It can be judged by whether or not it is the above. Further, for example, for the novel fluorescent protein and the CpYGFP, a gene encoding each amino acid sequence is inserted into an Escherichia coli plasmid, the color of the colony of the Escherichia coli is visually observed, and the novel fluorescent protein and the CpYGFP are described above. It can be judged by whether or not the color of the colony is different.

The novel fluorescent protein may have a fluorescence activity and may have a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1), and may further have other activities, for example. good.

<New gene>
As described above, the novel gene of the present invention is characterized by being the following polynucleotide (f1) or (f2).
(F1) In the base sequence of SEQ ID NO: 12,
A polynucleotide in which the 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H (f2) The 154th base and the 155th base with respect to the polynucleotide of the above (f1). A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of (f1), and having a base sequence having an identity of 80% or more including the base of the above and the 156th base. The polynucleotide that encodes

Further, in the novel gene of the present invention, for example, the polynucleotide of (f1) is the polynucleotide of the following (f1'), and the polynucleotide of (f2) is the polynucleotide of the following (f2'). ..
(F1') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
The 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T polynucleotide (f2') The 154th base with respect to the above-mentioned (f1') polynucleotide, The polynucleotide consisting of a base sequence having 80% or more identity including the 155th base, the 156th base, the 406th base, the 407th base, and the 408th base, and the polynucleotide of (f1) is said. A polynucleotide encoding a protein having a maximum fluorescence wavelength in the range of ± 5 nm maximum fluorescence wavelength of the encoding protein.

Further, in the novel gene of the present invention, for example, the polynucleotide of (f1) is the polynucleotide of the following (f1''), and the polynucleotide of (f2) is the polynucleotide of the following (f2''). Is.
(F1'') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
The 166th base, the 167th base, and the 168th base are codons encoding amino acids other than Y.
The 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T.
The 574th base, the 575th base, and the 576th base are polynucleotides (f2'') which are codons encoding amino acids other than R. The 154th base with respect to the above-mentioned (f1'') polynucleotide. Base, 155th base, 156th base, 166th base, 167th base, 168th base, 406th base, 407th base, 408th base, 574th base, 575th base A protein having a base sequence having an identity of 80% or more including a base and the 576th base and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) above. Polynucleotide to encode

The novel gene of the present invention is, for example, in the base sequence of SEQ ID NO: 12.
The 154th base, the 155th base, and the 156th base are codons encoding A or S.
The 166th base, the 167th base, and the 168th base are codons encoding R or Y.
The 406th base, the 407th base, and the 408th base are codons encoding M, S, or T.
The 574th base, the 575th base, and the 576th base are codons encoding R or I.

The novel genes of the present invention include, for example, the 154th base, the 155th base, and the 156th base coordinator, the 166th base, the 167th base, and the 168th base coordinator, the 406th base, and the like. The combination of the 407th base and the 408th base codon, and the 574th base, the 575th base, and the 576th base codon is one of the following (aa1') to (aa3'). A combination of codons encoding a combination of amino acids.

The novel genes of the present invention include, for example, the 154th base, the 155th base, and the 156th base coordinator, the 166th base, the 167th base, and the 168th base coordinator, the 406th base, and the like. The 407th base, the 408th base coordinator, and the 574th base, the 575th base, and the 576th base coordinator are a combination of any one of the following (n1) to (n4).

In the novel gene of the present invention, for example, the 10th base, the 11th base, and the 12th base are replaced with codons encoding Y.

In the novel gene of the present invention, for example, the 64th base, the 65th base, and the 66th base are replaced with codons encoding G.

In the novel gene of the present invention, for example, the 85th base, the 86th base, and the 87th base are replaced with codons encoding V.

In the novel gene of the present invention, for example, the 109th base, the 110th base, and the 111th base are replaced with codons encoding T.

In the novel gene of the present invention, for example, the 205th base, the 206th base, and the 207th base are replaced with codons encoding M.

In the novel gene of the present invention, for example, the 457th base, the 458th base, and the 459th base are replaced with codons encoding V.

In the novel gene of the present invention, for example, the 514th base, the 515th base, and the 516th base are replaced with codons encoding M.

The novel gene of the present invention is, for example, a polynucleotide in which the polynucleotide of (f1) is composed of at least one base sequence selected from the group consisting of SEQ ID NOs: 13 to 22.

Since the novel gene is a gene encoding the novel fluorescent protein of the present invention, it is also hereinafter referred to as a novel fluorescent protein gene. The novel fluorescent protein gene may contain, for example, one kind of polynucleotide or two or more kinds of polynucleotides (hereinafter, the same applies).

The base sequence of SEQ ID NO: 12 in (f1) is as follows. In the following base sequence, the base sequence surrounded by three squares is that the first base sequence is the 154th base, the 155th base, and the 156th base, and the second base sequence is 166. The second base, the 167th base, and the 168th base, the third base sequence is the 406th base, the 407th base, and the 408th base, and the fourth base sequence is the 574th base. It is a base sequence corresponding to the base, the 575th base, and the 576th base. The seven underlined codons are the first base, the tenth base, the eleventh base, and the twelfth base, and the second codon is the 64th base, the 65th base, and so on. And the 66th base, the 3rd base is the 85th base, the 86th base, and the 87th base, and the 4th base is the 109th base, the 110th base, and the 111th base. Base, 5th base is 205th base, 206th base, and 207th base, 6th base is 457th base, 458th base, and 459th base, 7th place The codon of the order is the codon corresponding to the 514th base, the 515th base, and the 516th base. The protein encoded by the base sequence of SEQ ID NO: 12 is also referred to as, for example, CpYGFP. Further, the polynucleotide consisting of the base sequence of SEQ ID NO: 12 is also referred to as a polynucleotide of CpYGFP.

In the polynucleotide (f1), the 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H. Codons encoding amino acids other than H encode A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. A codon, preferably a codon encoding A, or S.

Examples of the polynucleotide (f1) include a polynucleotide consisting of the base sequence of SEQ ID NO: 13. When the (f1) is a polynucleotide consisting of the base sequence of SEQ ID NO: 13, the above (f1) is also referred to as a polynucleotide of Y13. The polynucleotide of Y13 is a polynucleotide encoding the amino acid sequence of Y13.

The base sequence of SEQ ID NO: 13 is as follows. The base sequence of SEQ ID NO: 13 corresponds to the case where the 154th base, the 155th base, and the 156th base are codons encoding A in the base sequence of SEQ ID NO: 12.

In the polynucleotide (f2), for example, the 154th base, the 155th base, and the 156th base of the (f1) are conserved, and 80% or more of the base sequence of the (f1) is stored. It can also be said to be a polynucleotide having a base sequence having the same identity and encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1). In the above (f2), "identity" may be, for example, as long as the protein encoded by the polynucleotide of the above (f2) has the maximum fluorescence wavelength. The identity of (f2) 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. In the above (f1), when the 154th base, the 155th base, and the 156th base are replaced with the codon encoding A, the polynucleotide of the (f2) is the 154 of the above (f1). It is preferable that the 155th base and the 156th base are conserved. In this case, in the polynucleotide of (f2), for example, the 154th base, the 155th base, and the 156th base of the above (f1) are conserved, and 80 with respect to the base sequence of the above (f1). A polynucleotide comprising a base sequence having% or more identity and encoding the protein having the maximum fluorescence wavelength.

Examples of the polynucleotide (f2) include proteins consisting of the nucleotide sequences of SEQ ID NOs: 14 and 15. The base sequence of SEQ ID NO: 14 is a base sequence in which the 64th base, the 65th base, and the 66th base are replaced with codons encoding G in the base sequence of SEQ ID NO: 13. The base sequence of SEQ ID NO: 15 is a base sequence in which the 10th base, the 11th base, and the 12th base are replaced with codons encoding Y in the base sequence of SEQ ID NO: 13. When the (f2) is a polynucleotide consisting of the nucleotide sequences of SEQ ID NOs: 14 and 15, the said (f2) is also referred to as a polynucleotide of Y3 and Y6, respectively. The polynucleotides of Y3 and Y6 are polynucleotides encoding the amino acid sequences of Y3 and Y6.

The novel gene of the present invention may be the following polynucleotides (f3) to (f7).
(F3) One or several bases are deleted, substituted, inserted and / in the base sequence other than the 154th base, the 155th base, and the 156th base with respect to the above-mentioned polynucleotide (f1). Alternatively, a polynucleotide (f4) that comprises the added base sequence and encodes a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) above (f1). It consists of a base sequence complementary to a polynucleotide that hybridizes to a polynucleotide under stringent conditions, and in the complementary sequence, the 154th base of (f1), the 155th base, and the 156th base. Conservation of the base corresponding to the base of (f5), the polynucleotide encoding the protein having the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) (f5). Nucleotide encoding a protein in which the 52nd amino acid is an amino acid other than H in the amino acid sequence (f6) In the amino acid sequence other than the 52nd amino acid in (f5) above, one or several amino acids are deleted. A polynucleotide (f7) consisting of a substituted, inserted and / or added amino acid sequence and encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) above. It consists of an amino acid sequence having 80% or more identity with respect to the amino acid sequence other than the 52nd amino acid of (f5), and has a maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of (f1). A polynucleotide encoding a protein with the maximum fluorescence wavelength in the range

In the polynucleotide of (f3), for example, the 154th base, the 155th base, and the 156th base of the above (f1) are conserved, and one or several bases in the polynucleotide of (f1). A poly comprising a base sequence in which is deleted, substituted, inserted and / or added, and encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) above. It can also be called a nucleotide. In the above (f2), "1 or several" may be, for example, as long as the protein encoded by the polynucleotide of the above (f2) has the maximum fluorescence wavelength. The "1 or several" in (f2) is, for example, 1 to 132, 1 to 99, 1 to 66, 1 to 33, 1 to 26, 1 to 20, 1 to 13. 1 to 7, 1, 2, 3, or 4. In the above (f1), when the 154th base, the 155th base, and the 156th base are replaced with the codon encoding I, the polynucleotide of the (f2) is the 154 of the above (f1). It is preferable that the 155th base and the 156th base are conserved. In this case, the polynucleotide of (f2) is stored, for example, the 154th base, the 155th base, and the 156th base of the (f1), and is 1 or a number in the base sequence of the (f1). A polynucleotide in which a base sequence is deleted, substituted, inserted and / or added, and encodes the protein having the maximum fluorescence wavelength.

In the above (f4), the "hybridizable polynucleotide" is, for example, a polynucleotide completely or partially complementary to the polynucleotide of the above (f1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and is not particularly limited, for example, "Molecular Cloning: A Laboratory Manual 2nd ^Ed ." [(Cold Spring Harbor Labor)) edited by Sambrook et al. The method described in Press (1989)] and the like can also be adopted.

In the above (f4), the "stringent condition" may be, for example, any of a low stringent condition, a medium stringent condition, and a high stringent condition. “Low stringent conditions” are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 32 ° C. conditions. The "medium stringent condition" is, for example, a condition of 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 42 ° C. "High stringent conditions" are, for example, 5 × SSC, 5 × Denhardt solution, 0.5% SDS, 50% formamide, 50 ° C. conditions. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent condition" is, for example, "Molecular Cloning: A Laboratory Manual 2nd ^Ed ." Edited by Sambrook et al. [(Cold Spring Harbor L)). (1989)] and the like can also be adopted.

The polynucleotide of (f5) has, for example, a protein encoded by the polynucleotide of (f5) having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1). It may be a base sequence. The base sequence of the polynucleotide of (f5) can be designed, for example, by substituting the corresponding codon based on the amino acid sequence of SEQ ID NO: 1 and the 52nd amino acid. The protein encoded by the polynucleotide of (f5) can be, for example, the protein of (F1) in the novel protein of the present invention, and the description thereof can be incorporated.

In the above (f6), "one or several" regarding the amino acid sequence means, for example, that the protein encoded by the polynucleotide of the (f6) is the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of the (f1). Any range may be used as long as it has the maximum fluorescence wavelength in the range of. The "1 or several" in (f6) is, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, 1 to 5, etc. 1 to 3 pieces, 1 or 2 pieces. The protein of (f6) can also be said to be the protein of (F2) in the novel protein of the present invention, and the description thereof can be incorporated.

In the above (f7), the "identity" regarding the amino acid sequence means, for example, that the protein encoded by the polynucleotide of the (f7) is in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of the (f1). It may be in the range having the maximum fluorescence wavelength. The identity of (f7) 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 protein of (f7) can also be said to be the protein of (F3) in the novel protein of the present invention, and the description thereof can be incorporated.

The polynucleotide of (f1) may be, for example, the polynucleotide of (f1') below.
(F1') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
A polynucleotide in which the 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T.

In the polynucleotide (f1'), the 154th base, the 155th base, and the 156th base in SEQ ID NO: 12 are codons encoding amino acids other than H, and the 406th base and the 407th base are The base and the 408th base are codons encoding amino acids other than T. Codons encoding amino acids other than T encode A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y. Codon, preferably a codon encoding S, or M.

Examples of the polynucleotide (f1') include a polynucleotide consisting of the base sequence of SEQ ID NO: 16. When the (f1') is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16, the (f1') is also referred to as a Y polynucleotide. The polynucleotide of Y is a polynucleotide encoding the amino acid sequence of Y.

The base sequence of SEQ ID NO: 16 is as follows. In the base sequence of SEQ ID NO: 16, in the base sequence of SEQ ID NO: 13, the 154th base, the 155th base, and the 156th base are codons encoding A, and the 406th base and the 407th base. Corresponds to the case where the base of and the 408th base are codons encoding S.

The polynucleotide of (f2') includes, for example, the 154th base, the 155th base, and the 156th base, the 166th base, the 167th base, and the 168th base of the (f1'). Consisting of a base sequence that is conserved and has 80% or more identity with respect to the base sequence of (f1'), the maximum fluorescence wavelength of the protein encoded by the polynucleotide of (f1') is within ± 5 nm. It can also be said to be a polynucleotide encoding a protein having a fluorescence wavelength. In the above (f1'), the 154th base, the 155th base, and the 156th base, and the 166th base, the 167th base, and the 168th base are replaced with codons encoding A and S. If so, the polynucleotide of (f2') is the 154th base, the 155th base, and the 156th base of the (f1'), and the 166th base, the 167th base, and 168th. It is preferred that the second base is preserved. In this case, the polynucleotide of (f2') is, for example, the 154th base, the 155th base, and the 156th base of the (f1'), and the 166th base, the 167th base, and 168th. A polynucleotide in which the second base is conserved, comprises a base sequence having 80% or more identity with respect to the base sequence of (f1'), and encodes the protein having the maximum fluorescence wavelength.

The novel gene of the present invention may be the following polynucleotides (f3') to (f7').
(F3') Base sequences other than the 154th base, the 155th base, the 156th base, the 166th base, the 167th base, and the 168th base with respect to the above-mentioned (f1') nucleotide. In, one or several bases consist of a deleted, substituted, inserted and / or added base sequence, and the maximum fluorescence wavelength of the protein encoded by the above-mentioned (f1') nucleotide is within ± 5 nm. Nucleotide encoding a protein having a fluorescence wavelength (f4') The above-mentioned complementary sequence consists of a base sequence complementary to a polynucleotide that hybridizes to the above-mentioned (f1') polynucleotide under stringent conditions. In (f1'), the 154th base, the 155th base, the 156th base, the 166th base, the 167th base, and the base corresponding to the 168th base are stored, and the above-mentioned ( A polynucleotide encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of f1') (f5') In the amino acid sequence of SEQ ID NO: 1, the 52nd amino acid is other than H. A polynucleotide encoding a protein in which the 136th amino acid is an amino acid other than T (f6') In the amino acid sequence other than the 52nd and 136th amino acids of the above (f5'), one or several. A protein consisting of an amino acid sequence in which the amino acid of (f1') is deleted, substituted, inserted and / or added, and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of (f1'). The encoding polynucleotide (f7') consists of an amino acid sequence having 80% or more identity with respect to the amino acid sequences other than the 52nd and 136th amino acids of the above (f5'), and of the above (f1'). A polynucleotide encoding a protein having a maximum fluorescence wavelength in the range of ± 5 nm maximum fluorescence wavelength of the protein encoded by the polynucleotide.

The polynucleotide of (f1) may be, for example, the polynucleotide of (f1 ″) below.
(F1'') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
The 166th base, the 167th base, and the 168th base are codons encoding amino acids other than Y.
The 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T.
A polynucleotide in which the 574th base, the 575th base, and the 576th base are codons encoding amino acids other than R.

In the above (f1'') polynucleotide, the 154th base, the 155th base, and the 156th base in SEQ ID NO: 12 are codons encoding amino acids other than H, and the 166th base and the 167th base are used. The base and the 168th base are codons encoding amino acids other than Y, and the 406th base, 407th base, and 408th base are codons encoding amino acids other than T, and the 574th base. The base of the 575th base and the 576th base are codons encoding amino acids other than R. Codons encoding amino acids other than Y encode A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W. Codon, preferably a codon encoding R, or I. Codons encoding amino acids other than R encode A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y. A codon that encodes I, preferably a codon that encodes I.

Examples of the polynucleotide (f1 ″) include a polynucleotide consisting of the base sequence of SEQ ID NO: 17. When the (f1 ″) is a polynucleotide consisting of the base sequence of SEQ ID NO: 17, the (f1 ″) is also referred to as a BG polynucleotide. A BG polynucleotide is a polynucleotide encoding the amino acid sequence of BG.

The base sequence of SEQ ID NO: 17 is as follows. In the base sequence of SEQ ID NO: 17, in the base sequence of SEQ ID NO: 12, the 154th base, the 155th base, and the 156th base are codons encoding S, and the 166th base and the 167th base are used. The base of, and the 168th base are the codons encoding R, the 406th base, the 407th base, and the 408th base are the codons encoding M, and the 574th base and the 575th base. Corresponds to the case where the base and the 576th base are codons encoding I.

The polynucleotide of (f2'') is, for example, the 154th base of the above (f1''), the 155th base, the 156th base, the 166th base, the 167th base, the 168th base, and the like. The 406th base, the 407th base, the 408th base, the 574th base, the 575th base, and the 576th base are conserved, and 80% or more of the base sequence of the above (f1 ″). It can also be said that it is a polynucleotide encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1 ″). In the above (f1''), the 154th base, the 155th base, the 156th base, the 166th base, the 167th base, and the 168th base, the 406th base, the 407th base, And when the 408th base, the 574th base, the 575th base, and the 576th base are replaced with codons encoding S, R, M, and I, respectively, the above (f2''). The polynucleotide of (f1'') is the 154th base, the 155th base, the 156th base, the 166th base, the 167th base, and the 168th base, the 406th base, and the 407th base. , The 408th base, the 574th base, the 575th base, and the 576th base are preferably stored. In this case, the polynucleotide of (f2'') is, for example, the 154th base of (f1''), the 155th base, the 156th base, the 166th base, the 167th base, and the 168th base. , The 406th base, the 407th base, the 408th base, the 574th base, the 575th base, and the 576th base are conserved, and the base sequence of the above (f1'') is conserved. A polynucleotide comprising a base sequence having 80% or more identity and encoding the protein having the maximum fluorescence wavelength.

Examples of the polynucleotide of (f2 ″) include a protein consisting of the base sequences of SEQ ID NOs: 18 to 22. In the base sequence of SEQ ID NO: 18, in the base sequence of SEQ ID NO: 17, the 109th base, the 110th base, and the 111th base are replaced with codons encoding T, and the 457th base and the 458th base are replaced. The base and the 459th base are base sequences substituted with codons encoding V. In the base sequence of SEQ ID NO: 19, in the base sequence of SEQ ID NO: 17, the 85th base, the 86th base, and the 87th base are replaced with codons encoding V, and the 457th base and the 458th base are replaced. The base and the 459th base are base sequences substituted with codons encoding V. In the base sequence of SEQ ID NO: 20, in the base sequence of SEQ ID NO: 17, the 205th base, the 206th base, and the 207th base are replaced with codons encoding M, and the 457th base and the 458th base are replaced. The base and the 459th base are base sequences substituted with codons encoding V. In the base sequence of SEQ ID NO: 21, in the base sequence of SEQ ID NO: 17, the 457th base, the 458th base, and the 459th base are replaced with codons encoding V, and the 514th base and the 515th base are replaced. The base and the 516th base are base sequences substituted with codons encoding M. In the base sequence of SEQ ID NO: 22, in the base sequence of SEQ ID NO: 17, the 85th base, the 86th base, and the 87th base are replaced with the codon encoding V, and the 109th base and the 110th base are replaced. The base and the 111th base are substituted with the codon encoding T, and the 457th base and the 458th base and the 459th base are substituted with the codon encoding V. When the (f2 ″) is a polynucleotide consisting of the nucleotide sequences of SEQ ID NOs: 18 to 22, the (f2 ″) is also referred to as a polynucleotide of BG15, BG17, BG19, BG23, and BG29, respectively. The polynucleotides of BG15, BG17, BG19, BG23, and BG29 are polynucleotides encoding the amino acid sequences of BG15, BG17, BG19, BG23, and BG29, respectively.

The novel gene of the present invention may be the following polynucleotides (f3'') to (f7'').
(F3'') The 154th base, the 155th base, the 156th base, the 166th base, the 167th base, the 168th base, and the 406th base with respect to the above-mentioned (f1'') polynucleotide. 1 or several bases are deleted, substituted, inserted and / or in the base sequence other than the base of, the base of the 407th, the base of the 408th, the base of the 574th, the base of the 575th, and the base of the 576th. A polynucleotide (f4'') that comprises an added base sequence and encodes a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of the above (f1 ″). It consists of a base sequence complementary to a polynucleotide that hybridizes to the polynucleotide of f1'') under stringent conditions, and in the complementary sequence, the 154th base of the above (f1''), 155. The second base, the 156th base, the 166th base, the 167th base, the 168th base, the 406th base, the 407th base, the 408th base, the 574th base, the 754th base, and the base. The nucleotide corresponding to the 576th base is conserved, and the nucleotide (f5') encoding the protein having the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the above-mentioned (f1'') polynucleotide. ') In the amino acid sequence of SEQ ID NO: 1, the 52nd amino acid is an amino acid other than H, the 136th amino acid is an amino acid other than T, and the 56th amino acid is an amino acid other than Y, 192. Nucleotide encoding a protein in which the second amino acid is an amino acid other than R (f6'') In the amino acid sequence other than the 52nd, 136th, 136th and 192nd amino acids of the above (f5''), 1 or It consists of an amino acid sequence in which several amino acids are deleted, substituted, inserted and / or added, and the maximum fluorescence wavelength is within the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide encoded by the above (f1 ″). Nucleotide encoding a protein having (f7'') An amino acid sequence having 80% or more identity with respect to the amino acid sequences other than the 52nd, 136th, 136th and 192nd amino acids of the above (f5''). The maximum fluorescence wavelength of the protein encoded by the above-mentioned (f1'') polynucleotide is ± 5 nm. A polynucleotide encoding a protein with the maximum fluorescence wavelength in

In the present invention, the various polynucleotides can be produced, for example, by a known genetic engineering method or synthetic method.

<Expression vector of new protein>
As described above, the expression vector of the present invention is characterized by containing the novel gene of the present invention. According to the expression vector of the present invention, for example, by introducing the expression vector into the host, the novel protein of the present invention can be easily produced. For the expression vector of the present invention, for example, the description of the novel gene of the present invention can be incorporated.

The expression vector may functionally contain, for example, the polynucleotide so that it can express a novel protein encoded by the polynucleotide, which is the novel gene of the present invention, and other configurations are particularly limited. Not limited.

The expression vector can be prepared, for example, by inserting the polynucleotide into a vector that serves as a skeleton (hereinafter, also referred to as a "basic vector"). The type of the vector is not particularly limited and can be appropriately determined, for example, depending on the type of the host. Examples of the vector include a viral vector and a non-viral vector. The vector is preferably, for example, a binary vector. When transforming a plant, the vector is preferably a T-DNA type binary vector. When transforming a plant by a method using Agrobacterium, the vector may be, for example, pBI121 vector, pPZP202 vector, pBINPLUS vector, pBIN19 vector, pBIG2113N, pBIG2113SF, pRI101DNA vector (manufactured by TaKaRa), pRI201DNA vector ( (TaKaRa), pRI909DNA vector (TaKaRa), pRI910DNA vector (TaKaRa) and the like can be mentioned. When transforming a microorganism such as Escherichia coli, the vector is, for example, pETDuet-1 vector (Novagen), pQE-80L vector (QIAGEN), pBluescript II SK vector, pET101 / D-TOPO vector (Invitrogen). ), PGEX-6P-1 vector (manufactured by Amersham Biosciences), pcDNA3.2 / V5-GW / D-TOPO vector (manufactured by Invitrogen), pEGFP vector, pcDNA3.1-hygro ^(-) vector (manufactured by Invitrogen). And so on.

The expression vector preferably has, for example, a regulatory sequence that regulates the expression of the polynucleotide and the novel protein of the invention encoded by the polynucleotide. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, an origin of replication sequence (ori), and the like. In the expression vector, the arrangement of the regulatory sequence is not particularly limited. In the expression vector, the regulatory sequence may be arranged, for example, so as to be able to functionally regulate the expression of the polynucleotide and the expression of the novel protein encoded by the polynucleotide, and can be arranged based on a known method. .. As the regulatory sequence, for example, the sequence provided in advance by the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence provided by the basic vector may be used. It may be replaced with the regulatory sequence of.

The expression vector may further have, for example, a coding sequence for a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, a cell surface receptor marker and the like.

<Manufacturing method of new protein>
The method for producing the novel protein of the present invention is not particularly limited, and may be produced by, for example, a genetic engineering method or a known synthetic method. In the former case, the production method of the present invention comprises an expression step of expressing the novel gene of the present invention. Thereby, the method for producing a novel protein of the present invention can easily produce, for example, the novel fluorescent protein of the present invention.

The expression of the polynucleotide which is the novel gene of the present invention may be carried out using, for example, the expression vector of the present invention.

The method for expressing the polynucleotide is not particularly limited, and a known method can be adopted. For example, a cell-free protein synthesis system may be used, or a host may be used.

In the former case, it is preferable to express the polynucleotide in a cell-free protein synthesis system. In this case, an expression vector may be used for the expression of the polynucleotide. The cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which a target polynucleotide has been introduced, and is, for example, commercially available. Reagent kits can be used.

In the latter case, for example, it is preferable to use the host into which the polynucleotide has been introduced and to express the polynucleotide in the host by culturing the host. As described above, for example, by introducing the polynucleotide into a host, a transformant for synthesizing the novel protein of the present invention can be produced, and by culturing the transformant, the novel protein of the present invention 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 the like, and plants are preferable. When the host is a plant, the plant may be, for example, a plant or a portion thereof. Examples of the plant portion include organs, tissues, cells, vegetative propagules and the like. Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like. The tissue is, for example, a portion of the organ. Examples of the cells include cells collected from the plant body or its tissue, cultured cells of the cells, protoplasts, callus and the like. The origin of the plant is not particularly limited, and is, for example, Abranaceae, Nathaceae, Rice family, Bean family, Rose family, Dianthus family, Kiku family, Gentianaceae, Figwort family, Verbenaceae, Sakurasou family, Cactus family, Orchid. Family etc. can be mentioned. Examples of the Brassicaceae include Arabidopsis thaliana and the genus Arabidopsis thaliana . The Solanaceae includes, for example, the genus Tobacco such as tobacco ( Nicotiana tabacum ), the genus Petunia such as Petunia ( Petunia × hybrida ), the genus Petunia , the genus Nierembergia ( Nierembergia cybali ) Calibrachoa , etc. Examples include Calibrachoa. Examples of the Gramineae include the genus Oryza such as corn ( Zea mays ) and the genus Oryza such as rice ( Oryza sativa ). Examples of the legume family include the genus Glycine such as soybean ( Glycine max ). The Rosaceae includes, for example, the genus Rose such as rose ( Rosa ). Examples of the dianthus family include the genus Dianthus such as carnation ( Dianthus caryophyllus ). Examples of the Asteraceae include the genus Chrysanthemum such as cultivated chrysanthemum ( Chrysanthemum morifolia ) and the genus Gerbera ( Osenbonyari ) such as Gerbera cvs . Examples of the Gentianaceae include the genus Eustoma such as Eustoma grandiflorum . Examples of the figwort family include Torenia ( Torenia furnieri ) and the genus Torenia. Examples of the Verbenaceae include the genus Verbena such as Garden verbena. Examples of the Primrose family include the genus Cyclamen such as Cyclamen persicum . The cactaceae are, for example, the genus Austrokilindropuntia, the genus Astrofitum, the genus Echinocactus, the genus Equinoceleus, the genus Echinopsis, the genus Epiphyllum, the genus Opuntia, the genus Caniva cactus, the genus Kamaeseleus, the genus Kirindropuntia, the genus Gymnocaricium. , Shakobasaboten, Serenicereus, Teflocactus, Neobractus baumia, Neolymondia, Nopalea, Ferrocactus, Mamilaria, Melocacus, Lipsalis, Roseocactus, Lofophora and the like. The Orchidaceae includes, for example, Phalaenopsis ( Phalaenopsis cvs. ) And other Phalaenopsis (Moth orchids), Cymbidium ( Cymbidium cvs. ) And other Cymbidium ( Shunlan ) genus, Dendrobium nobil ( Dendrobium ) dendrobium (Dendrobium) and Dendrobium (Dendrobium). Examples thereof include the genus Dendrobium (Sekoku) such as D. phalaenopsis hybrids , the genus Oncidium such as Oncidium ( Oncidium cvs. ), And the genus Moth orchid such as Cattleya cvs . Examples of the microorganism include eukaryotes and prokaryotes. Examples of the prokaryote include bacteria of the genus Escherichia such as Escherichia coli and the genus Pseudomonas such as Pseudomonas putida . Examples of the eukaryote include yeasts such as Saccharomyces cerevisiae . Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.

The method for introducing the polynucleotide into the host, that is, the method for transforming the host is not particularly limited, and for example, a method for introducing the polynucleotide using the expression vector may be used, or the expression vector may not be used. A known method to be introduced into the above may be used. In the latter case, the introduction method can be appropriately set, for example, according to the type of the host. The introduction method is, for example, a heat shock method, a lithium acetate method, an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, DEAE. -The dextran method, the direct injection method using a microglass tube or the like, the hydrodynamic method, the cationic liposome method, the method using an introduction aid, the method via Agrobacterium, etc. can be mentioned. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles and polymers. When the host is a plant, the introduction method is preferably an Agrobacterium-mediated method. The polynucleotide which is the novel gene of the present invention may be introduced into the host by, for example, the expression vector of the present invention.

The method of 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 conventionally known media such as solid medium, liquid medium, and agar medium can be appropriately used. The components contained in the medium are not particularly limited. The medium may contain, for example, a commercially available medium. When the host is a plant, the commercially available medium of the plant is not particularly limited, and examples thereof include Murashige-Skoog (MS) medium and the like. When the host is a plant cell, the commercially available medium of the plant cell is not particularly limited, and examples thereof include Hyponex medium, MS medium, Gamborg B5 (B5) medium, and White medium. When the host is a microorganism, the commercially available medium of the microorganism is not particularly limited, and examples thereof include LB medium, super broth medium, and M9 medium. As the medium, for example, one type 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 and 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, for example, according to the type of the host. When the host is a plant, the culturing method includes, for example, a method of cultivating the plant in soil or in water. When the plant is a plant cell, the culture method includes, for example, callus culture, root culture, embryo pearl 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, for example, according to the type of the host. When the host is a plant, the culture temperature may be, for example, a temperature at which the plant can grow, an optimum temperature for growth, or the like. Specifically, the culture temperature can be, for example, in the range of 15 to 40 ° C, in the range of 30 to 37 ° C, or the like. When the host is a plant cell, the culture temperature may be, for example, a temperature at which the plant cell can grow, an optimum temperature for growth, or the like. Specifically, the culture temperature can be, for example, in the range of 15 to 40 ° C, in the range of 30 to 37 ° C, or the like.

The host may be cultured under aerobic conditions or anaerobic conditions, for example. The aerobic condition or the anaerobic condition is not particularly limited and can be set by using a conventionally known method.

<Transformant>
As described above, the transformant of the present invention is characterized by containing the novel gene of the present invention. The transformant of the present invention is characterized by containing the novel gene of the present invention, and other configurations and conditions are not particularly limited. Since the transformant of the present invention contains the novel gene of the present invention, for example, it has fluorescent activity. The transformant of the present invention can be referred to, for example, the description of the novel protein of the present invention.

In the present invention, the transformant is not particularly limited, and examples thereof include animals and plants, but plants are preferable. When the transformant is an animal, examples of the transformant include cancer cells such as human colon cancer cells. When the transformant is a plant, the plant and the origin of the plant are not particularly limited, and for example, the above-mentioned description can be incorporated. Specifically, the plant may be, for example, a plant body or a part thereof. Examples of the plant portion include organs, tissues, cells, vegetative propagules and the like. Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like. The tissue is, for example, a portion of the organ. Examples of the cells include cells collected from the plant body or its tissue, cultured cells of the cells, protoplasts, callus and the like.

In 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 breeding material. The breeding material is not particularly limited, and may be, for example, the whole or a part of the transformant. When the transformant is the plant body or a portion thereof, the breeding material includes, for example, seeds, fruits, shoots, stems such as tubers, roots such as tubers, strains, protoplasts, callus and the like.

The method for breeding the transformant of the present invention is not particularly limited, and a known method can be adopted. The reproduction method may be, for example, either sexual reproduction or asexual reproduction, and is preferably asexual reproduction. The reproduction by asexual reproduction includes, for example, vegetative propagation, and is also referred to as 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, breeding by cuttings, propagation by cuttings, subdivision from organs into individual plants, proliferation by callus and the like can be mentioned. As the organ, for example, leaves, stems, roots and the like as described above can be used.

The propagule obtained by vegetatively propagating the transformant is hereinafter referred to as the vegetative propagule of the present invention. The vegetative propagule of the present invention preferably has the same properties as the transformant of the present invention. The vegetative propagule of the present invention is not particularly limited as in the transformant, and examples thereof include a plant body or a portion thereof.

In addition, when the transformant is sexually reproduced, for example, seeds, progeny of the grown body and the like 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, either a plant or a portion thereof, as in the transformant.

The transformant of the present invention may be further processed, for example. The type of the transformant to be processed is not particularly limited, and examples thereof include flowers, leaves, and branches. The processed product of the transformant is not particularly limited, and examples thereof include potpourri obtained by drying flowers, leaves, branches and the like, pressed flowers, dried flowers, preserved flowers, resin-sealed products and the like. Further, the processed product of the present invention may be, for example, a processed product of a vegetative propagule, an organ, a tissue or a cell of the transformant.

<Manufacturing method of transformant>
As described above, the method for producing a transformant of the present invention is characterized by comprising an introduction step of introducing a novel gene of the present invention into a host. The method for producing a transformant of the present invention is characterized by comprising an introduction step of introducing a novel gene of the present invention 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 of expressing the novel gene in the host.

In the introduction step, the method for introducing the novel gene of the present invention into the host is not particularly limited, and for example, the description of the method for introducing the polynucleotide in the method for producing the novel protein of the present invention into the host is incorporated. can. In the production method of the present invention, the host is preferably a plant.

The expression step is, for example, a step of expressing a novel protein of the present invention from a novel gene of the present invention in a host. In the expression step, the host is preferably, for example, a transformant into which the novel gene of the present invention or the expression vector has been introduced, and by culturing the transformant, the novel protein of the present invention is introduced into the host. It is preferable to express it.

When the host is a plant, the method for producing a transformant of the present invention may further include a breeding step of propagating the transformant obtained by the introduction step. As the breeding method in the breeding step, for example, the above-mentioned description can be incorporated. In addition, the method for producing a transformant of the present invention may include a seeding step of seeding from a propagated transformant, and may further include a growth step of growing a grower from the seeds obtained in the seeding step. ..

<Screening method for new fluorescent proteins>
The method for screening a novel fluorescent protein of the present invention (hereinafter, also referred to as “screening method of the present invention”) is the amino acid of SEQ ID NO: 1 from a candidate protein in which a mutation is introduced into the novel protein of the present invention, as described above. The 52nd amino acid in the sequence is an amino acid other than H, and is characterized by comprising a selection step of selecting a protein having a maximum fluorescence wavelength within the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1). The process and conditions are not particularly limited. According to the screening method of the present invention, for example, a protein having fluorescent activity can be easily screened. As the screening method of the present invention, for example, the description of the novel protein, the novel gene, the expression vector, the production method and the like of the present invention can be incorporated.

The candidate protein is a protein in which a mutation is introduced into the novel protein of the present invention. The type of mutation introduced into the novel protein is not particularly limited and is, for example, amino acid deletion, substitution, insertion and / or addition. As a specific example, the candidate protein is, for example, a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of the novel protein, or an amino acid of the novel protein. Examples thereof include proteins consisting of amino acid sequences having 80% or more identity with respect to the sequence.

In the amino acid sequence of the candidate protein, "1 or several" is, for example, 1 to 43, 1 to 33, 1 to 22, 1 to 11, 1 to 9, 1 to 7, 1 to 1. 5 pieces, 1 to 3 pieces, 1 or 2 pieces.

In the amino acid sequence of the candidate protein, "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 candidate protein may be produced by a known protein synthesis method or a genetic engineering method based on the amino acid sequence of the candidate protein. In the latter case, the screening method of the present invention is, for example, a mutation step of introducing a mutation into a base sequence encoding the novel protein, that is, a new gene of the present invention to prepare a mutant polynucleotide, from the mutant polynucleotide. It includes an expression step for expressing a candidate protein.

In the mutation step, the mutation introduced into the novel gene is not particularly limited, and is, for example, deletion, substitution, insertion and / or addition of a base. The method for introducing a mutation into the novel gene is not particularly limited, and can be carried out by, for example, a method for introducing a mutation into a known polynucleotide, and as a specific example, random fragmentation of the polynucleotide of the novel gene of the present invention. Examples thereof include a method of reconstructing a gene by recombination using PCR (so-called DNA shuffling method).

In the expression step, the method for expressing the mutant polynucleotide is not particularly limited, and for example, the above-mentioned description of the method for producing a novel protein of the present invention can be referred to, and a cell-free protein synthesis system may be used. , Hosts may be used. As the host, for example, it is preferable to use a prokaryote, particularly Escherichia coli .

The screening method of the present invention may include, for example, a step of purifying the candidate protein obtained in the expression step. The purification method is not particularly limited, and examples thereof include salting out, various column chromatography, and the like. The solvent used in the various purification steps is not particularly limited, and for example, a buffer solution can be used. In the screening method of the present invention, for example, the candidate protein obtained in the expression step may be used as it is in the selection step described later, or a partially purified candidate protein for partial purification may be used. A single purified candidate protein may be used.

In the selection step, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H from the candidate protein, and has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1). This is the process of selecting proteins.

In the selection step, the 52nd amino acid of the selected candidate protein is an amino acid other than H. Amino acids other than H are A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, preferably. A or S.

In the screening method of the present invention, for example, from the candidate protein in which a mutation is introduced into the novel protein of the present invention in the selection step, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid. The amino acid of is an amino acid other than T, and a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) may be selected.

In the selection step, the 52nd amino acid of the selected candidate protein is an amino acid other than H, and the 136th amino acid is an amino acid other than T. The amino acids other than T are S or M.

In the screening method of the present invention, for example, from the candidate protein in which a mutation is introduced into the novel protein of the present invention in the selection step, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid. The amino acid of (F1) is an amino acid other than T, the 56th amino acid is an amino acid other than Y, the 192nd amino acid is an amino acid other than R, and the maximum fluorescence wavelength of the protein (F1) is within ± 5 nm. A protein having the maximum fluorescence wavelength may be selected.

In the selection step, the 52nd amino acid of the selected candidate protein is an amino acid other than H, the 136th amino acid is an amino acid other than T, and the 56th amino acid is an amino acid other than Y. The 192nd amino acid is an amino acid other than R. The amino acids other than Y are A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W, and are preferable. , R, or I. The amino acids other than R are A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y, and are preferable. , I.

In the selection step, for the selected candidate protein, for example, the combination of the 52nd amino acid, the 56th amino acid, the 136th amino acid, and the 192nd amino acid in the amino acid sequence of SEQ ID NO: 1 is as follows (aa1). ) To (aa3), and the protein has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).

In the selection step, the method for identifying the amino acid sequence of the candidate protein is not particularly limited, and can be carried out, for example, by a known amino acid identification method. When the candidate protein is expressed from the mutant polynucleotide, the amino acid sequence of the candidate protein may be identified, for example, by replacing the codon of the base sequence of the mutant polynucleotide with the corresponding amino acid sequence.

In the selection step, the method for confirming the fluorescence activity of the candidate protein is not particularly limited, and a known method for confirming the fluorescence activity can be used. For example, it is generated by excitation light of each excitation wavelength using a known optical measuring instrument. It can be confirmed by measuring the fluorescence spectrum. When the candidate protein is expressed in the Escherichia coli , the method for confirming the fluorescence activity is not particularly limited, and confirmation is performed using, for example, an LEDUV (Light Emitting DIode Ultra Violet) light source and a UV transmission filter. can.

In the screening method of the present invention, it is preferable to select a protein having an amino acid sequence having 80% or more identity with respect to the amino acid sequence of SEQ ID NO: 1 and having fluorescent activity in the selection step. The "identity" may be, for example, as long as the selected candidate protein has the fluorescent activity. The "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.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
It was confirmed that the novel protein of the present invention has fluorescent activity.

(1) Construction of expression vector Vector insertion sequences (SEQ ID NOs: 23 and 24) containing novel genes (SEQ ID NOs: 16 and 17) encoding new fluorescent proteins (SEQ ID NOs: 5 and 6) were obtained by chemical synthesis. In the vector insertion sequence, the base sequence in the underlined portion enclosed in parentheses is the base sequence corresponding to the novel gene encoding the novel fluorescent protein. These were designated as Y and BG vector insertion sequences, respectively.

The Y and BG vector insertion sequences were ligated into the pUC19 vector using restriction enzymes HindIII and EcoRI sites, respectively. The vector into which the Y and BG vector insertion sequences were inserted was used as a Y and BG expression vector, respectively. The expression vector was constructed by Gene Script.

(2) Introduction to Escherichia coli The Y and BG expression vectors were independently transduced into the Escherichia coli DH5α strain. The obtained transformant was cultured overnight at 37 ° C. in LB medium containing 100 μg / mL of an antibiotic (carbenicillin). Then, the cultured cells were centrifuged at 6000 rpm and 4 ° C. for 10 minutes to collect the cells. The resulting pellet was washed once with 40 mL of PBS solution. The washed pellet was suspended in 1.6 mL of PBS solution. Next, the obtained suspension was ultrasonically treated with an ultrasonic homogenizer (QSONICA, manufactured by Waken Yakuhin Co., Ltd.) under the conditions of Amp 30% for 1 minute. Then, the suspension after the treatment was centrifuged at 15,000 rpm and 4 ° C. for 10 minutes, and the obtained supernatant was recovered. To the supernatant, 0.2 ml (50% slurry) of TALON® Metal Infinity Resin (Z5502N, manufactured by TAKARA) was added, and the mixture was reacted at room temperature for 1 hour. The reaction solution was washed with a PBS solution, and then 0.5 ml of a PBS solution containing 300 mmol / L imidazole was added to elute the protein. Then, using Amicon Ultra-0.5 (UFC5010, manufactured by Merck Millipore), the reaction solution was buffer-exchanged and replaced with a PBS solution. Purified samples obtained from the transformants into which the Y and BG expression vectors were introduced were used as Y and BG proteins, respectively, and used in the following experiments. The protein concentration in each purified sample is determined by diluting each sample with a 10% SDS solution and then developing a color using a Pierce BCA Protein Assay Kit (# 23225, manufactured by Pierce) and using a microplate reader (Infinite (registered)). It was determined by measuring the absorbance at 562 nm using (trademark) M1000Pro (manufactured by TECAN).

(3) Measurement of fluorescence activity The purified sample of (2) was diluted to a protein concentration of 1 mg / mL in each sample based on the protein concentration of each sample obtained in (2). .. For each 70 μL of the diluted sample, the fluorescence intensity (FI) was measured under the following measurement conditions using the microplate reader.

(Measurement condition)
Excitation wavelength: 350-650 nm (bandwidth: 5 nm)
Fluorescence (detection) wavelength: 350 to 650 nm (bandwidth: 5 nm)
Laser intensity (Gain): 105

FIG. 1 shows the measurement results of the entire range of the excitation wavelength and the entire range of the fluorescence wavelength. In FIG. 1, (A) shows the result of Y protein, and (B) shows the result of BG protein. Further, in FIG. 1, 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. As shown in FIG. 1, the Y and BG proteins showed fluorescent activity.

Table 5 below shows the combination of the excitation wavelength (Ex) and the fluorescence wavelength (Em) at which the fluorescence intensity (FI) becomes the maximum value in each sample. As shown in Table 5, the Y protein had a maximum fluorescence intensity at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and the BG protein had a maximum fluorescence intensity at an excitation wavelength of 450 nm and a fluorescence wavelength of 485 nm. .. Since the excitation and fluorescence wavelengths of CpYGFP are known to be 508 nm and 518 nm, respectively (references: PLoS One. 2017 Jul 11; 12 (7): e0181186.), The Y and BG proteins are It was found that the fluorescence wavelength was changed as compared with CpYGFP.

From these results, it was found that the novel fluorescent protein of the present invention has fluorescent activity and the fluorescence wavelength is changed as compared with CpYGFP.

[Example 2]
It was confirmed that a novel fluorescent protein can be screened by the screening method of the present invention.

(1) Construction of expression vector A mutant polynucleotide was prepared by using the DNA shuffling method, and the mutant polynucleotide was ligated to the vector to construct a mutant polynucleotide expression vector. Specifically, it was carried out as follows. First, the Y polynucleotide (SEQ ID NO: 16) and the BG polynucleotide (SEQ ID NO: 17) were amplified by 1st PCR (Polymerase Chain Reaction) using the following primer set to obtain a 1st PCR product. The PCR reaction solution was prepared according to the instructions using a kit of TaKaRa EX Taq (registered trademark) (manufactured by TAKARA).

Primer set Primer sequence 1 (SEQ ID NO: 25)
5'-AATAAGCTTGATGCATCATCATCATCATC-3'
Primer sequence 2 (SEQ ID NO: 26)
5'-GTACGGCCGACTAGTAGGCC-3'

1.9 μg of the 1st PCR product was suspended in a buffer solution containing deoxyribonuclease (DNase) having the following composition, reacted at 25 ° C for 20 minutes, and further reacted at 90 ° C for 10 minutes to obtain DNA. Fragmentation was performed.

[Buffer solution composition]
Total amount 40 μL
DNaseI (manufactured by TaKaRa) 0.05U
(PCR product of Y polynucleotide)
0.025U
(PCR product of BG polynucleotide)
Tris-Hcl 50 mmol / L
MnCl ₂ 10 mmol / L
pH 7.4

Each fragmented DNA was purified by agarose gel electrophoresis and gel extraction. Then, the obtained purified product was added to the PCR reaction solution so that the concentration in the liquid was 10 to 30 μg / μL, and amplified by 2nd PCR to obtain a large number of 2nd PCR products. The primer set was not used in the 2nd PCR. Then, 1/50 of each of the large number of 2nd PCR products was added to the PCR reaction solution and amplified by 3rd PCR using the primer set to obtain a mutant polynucleotide. The mutant polynucleotide was cleaved with the restriction enzyme and ligated with a pEGFP vector (manufactured by clonetec) cleaved with the restriction enzyme. The vector into which the mutant polynucleotide was inserted was used as a mutant polynucleotide expression vector. In this way, a large number of mutant polynucleotide expression vectors were obtained.

(2) Introduction to Escherichia coli The mutant polynucleotide expression vector was transduced into the Escherichia coli DH5α strain independently. The obtained transformants were cultured overnight (18 hours) at 37 ° C. using LB medium containing 100 μg / mL of an antibiotic (carbenicillin) until about 5,000 to 10,000 colonies grew.

(3) Confirmation of fluorescence activity Then, the cultured cells are collected and used as a light source, a 420 nm LED handy light for fluorescence excitation (LED420-3W, manufactured by Optcode Co., Ltd.), and a 470 nm LED handy light for fluorescence excitation (LED470-3W, Optcode Co., Ltd.), SC-48 (4902520067654, manufactured by Fuji Film Co., Ltd.) as an ultraviolet absorption filter was used to confirm the fluorescence activity of the cells. For the cells whose fluorescent activity was confirmed, a plasmid was purified, the operations of Examples 2 (1) and (2) were repeated three times, and then the fluorescent activity of the cells was confirmed in the same manner.

(4) Sequence of Escherichia coli The colonies of the cells whose fluorescent activity was confirmed in (3) above were collected, the plasmid was purified, and subjected to the direct sequence under the following conditions. As a result, it was confirmed that the cells whose fluorescent activity was confirmed had the novel genes of the present invention (SEQ ID NOs: 13 to 15 and 18 to 22). From these results, it was found that the novel fluorescent protein of the present invention can be screened by the screening method of the present invention.

[Direct sequence conditions]
Sequencer ABI 3130xl (manufactured by Applied Biosystems)
Sequencing Reagent BigDye Terminator V1-1 cycle sequencing kit (manufactured by Applied Biosystems)
Sequence primer Primer sequence 1 (SEQ ID NO: 27)
5'-ATGCTTCCGGCTCGTATGTTG-3'
Primer sequence 2 (SEQ ID NO: 28)
5'-GTACGGCCGACTAGTAGGCC-3'

[Example 3]
It was confirmed that the novel protein of the present invention has fluorescent activity. The description of the experimental conditions, the experimental method, and the like was the same as in Example 1 unless otherwise specified.

(1) Construction of expression vector A sequence in which a His tag sequence is added to the N-terminal of a novel gene (SEQ ID NOs: 13 to 15 and 18 to 22) encoding a new fluorescent protein (SEQ ID NOs: 2 to 4 and 7 to 11) is added. The vector insertion sequences (SEQ ID NOs: 29 to 36, respectively) containing each were obtained by chemical synthesis. In the vector insertion sequence, the base sequence enclosed in parentheses is the base sequence corresponding to the novel gene encoding the novel fluorescent protein, and the underlined base sequence outside the parentheses is the His tag sequence. .. The vector insertion sequences were Y13, Y3, Y6, BG15, BG17, BG19, BG23, and BG29 vector insertion sequences, respectively.

Each of the vector insertion sequences was cleaved with the restriction enzyme and ligated to the pEGFP vector cleaved with the restriction enzyme. The vector into which the vector insertion sequence was inserted was used as a Y13, Y3, Y6, BG15, BG17, BG19, BG23, and BG29 expression vector, respectively.

(2) Introduction into Escherichia coli A purified sample obtained from the transformant by introducing the expression vector into the Escherichia coli DH5α strain in the same manner as in Example 1 (2) except for the experimental conditions shown below. Was designated as Y13, Y3, Y6, BG15, BG17, BG19, BG23, and BG29 proteins, respectively.

(3) Measurement of Fluorescence Activity The fluorescence intensity (FI) of the purified sample was measured in the same manner as in Example 1.

FIG. 2 shows the measurement results of the entire range of the excitation wavelength and the entire range of the fluorescence wavelength. In FIG. 2, (A) to (H) show the results of Y3, Y6, Y13, BG15, BG17, BG19, BG23, and BG29 proteins, respectively. Further, in FIG. 2, 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. The numbers in the figure indicate the combination of the excitation wavelength (Ex) and the fluorescence wavelength (Em) at which the fluorescence intensity (FI) becomes the maximum value. As shown in FIG. 2, the Y3, Y6, Y13, BG15, BG17, BG19, BG23, and BG29 proteins showed fluorescent activity.

FIG. 3 shows the fluorescence intensity (FI) at the maximum value for each protein. In FIG. 3, the vertical axis indicates the relative value of fluorescence intensity (FI), the horizontal axis indicates each protein, and the error bar indicates the standard deviation (SD). In FIG. 3, (A) shows the relative value of the fluorescence intensities of the Y3, Y6, and Y13 proteins when the result of the Y6 protein is 1, and (B) shows the relative value of the fluorescence intensity of the BG23 protein when the result is 1. The relative values of the fluorescence intensities of the BG15, BG17, BG19, BG23, and BG29 proteins in BG15, BG17, BG19, BG23, and BG29 proteins are shown. As shown in FIGS. 2 and 3, the Y3 protein has a maximum fluorescence intensity at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and the Y6 protein has a maximum fluorescence intensity at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm. , Y13 protein has a maximum fluorescence intensity at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, BG15 protein has a maximum fluorescence intensity at an excitation wavelength of 440 nm and a fluorescence wavelength of 490 nm, and BG17 protein has a maximum fluorescence intensity. The fluorescence intensity is maximum at 445 nm and the fluorescence wavelength is 490 nm, the fluorescence intensity of the BG19 protein is maximum at the excitation wavelength of 440 nm and the fluorescence wavelength is 490 nm, and the fluorescence intensity of the BG23 protein is maximum at the excitation wavelength of 440 nm and the fluorescence wavelength of 490 nm. The intensity became the maximum value, and the BG29 protein showed the maximum fluorescence intensity at the excitation wavelength of 445 nm and the fluorescence wavelength of 490 nm, and showed strong fluorescence activity at the maximum value. Since the excitation wavelength and the fluorescence wavelength of CpYGFP are known to be 508 nm and 518 nm, respectively, the Y and BG proteins have different fluorescence wavelengths and exhibit strong fluorescence activity as compared with CpYGFP. all right.

Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of the present invention.

According to the present invention, a novel fluorescent protein can be provided. Therefore, it can be said that the present invention is an extremely useful technique in, for example, fields such as life science field, medical field, and agricultural field.

（付記６）
４番目のアミノ酸が、Ｙに置換された、付記１から５のいずれかに記載の新規タンパク質。
（付記７）
２２番目のアミノ酸が、Ｇに置換された、付記１から６のいずれかに記載の新規タンパク質。
（付記８）
２９番目のアミノ酸が、Ｖに置換された、付記１から７のいずれかに記載の新規タンパク質。
（付記９）
３７番目のアミノ酸が、Ｔに置換された、付記１から８のいずれかに記載の新規タンパク質。
（付記１０）
６９番目のアミノ酸が、Ｍに置換された、付記１から９のいずれかに記載の新規タンパク質。
（付記１１）
１５３番目のアミノ酸が、Ｖに置換された、付記１から１０のいずれかに記載の新規タンパク質。
（付記１２）
１７２番目のアミノ酸が、Ｍに置換された、付記１から１１のいずれかに記載の新規タンパク質。
（付記１３）
前記（Ｆ１）のタンパク質が、配列番号２～１１からなる群から選択された少なくとも１つのアミノ酸配列からなるタンパク質である、付記１から１２のいずれかに記載の新規タンパク質。
（付記１４）
下記（ｆ１）または（ｆ２）のポリヌクレオチドであることを特徴とする、新規遺伝子。
（ｆ１） 配列番号１２の塩基配列において、
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基が、Ｈ以外のアミノ酸をコードするコドンであるポリヌクレオチド
（ｆ２） 前記（ｆ１）のポリヌクレオチドに対して、１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基を含む同一性が８０％以上の塩基配列からなり、且つ、前記（ｆ１）のポリヌクレオチドがコードするタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質をコードするポリヌクレオチド
（付記１５）
前記（ｆ１）のポリヌクレオチドが、下記（ｆ１’）のポリヌクレオチドであり、前記（ｆ２）のポリヌクレオチドが、下記（ｆ２’）のポリヌクレオチドである、付記１４記載の新規遺伝子。
（ｆ１’） 配列番号１２の塩基配列において、
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基が、Ｈ以外のアミノ酸をコードするコドンであり、
４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基が、Ｔ以外のアミノ酸をコードするコドンであるポリヌクレオチド
（ｆ２’） 前記（ｆ１’）のポリヌクレオチドに対して、１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基、４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基を含む同一性が８０％以上の塩基配列からなり、且つ、前記（ｆ１）のポリヌクレオチドがコードするタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質をコードするポリヌクレオチド
（付記１６）
前記（ｆ１）のポリヌクレオチドが、下記（ｆ１’’）のポリヌクレオチドであり、前記（ｆ２）のポリヌクレオチドが、下記（ｆ２’’）のポリヌクレオチドである、付記１４または１５記載の新規遺伝子。
（ｆ１’’） 配列番号１２の塩基配列において、
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基が、Ｈ以外のアミノ酸をコードするコドンであり、
１６６番目の塩基、１６７番目の塩基、および１６８番目の塩基が、Ｙ以外のアミノ酸をコードするコドンであり、
４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基が、Ｔ以外のアミノ酸をコードするコドンであり、
５７４番目の塩基、５７５番目の塩基、および５７６番目の塩基が、Ｒ以外のアミノ酸をコードするコドンであるポリヌクレオチド
（ｆ２’’） 前記（ｆ１’’）のポリヌクレオチドに対して、１５４番目の塩基、１５５番目の塩基、１５６番目の塩基、１６６番目の塩基、１６７番目の塩基、１６８番目の塩基、４０６番目の塩基、４０７番目の塩基、４０８番目の塩基、５７４番目の塩基、５７５番目の塩基、および５７６番目の塩基を含む同一性が８０％以上の塩基配列からなり、且つ、前記（ｆ１）のポリヌクレオチドがコードするタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質をコードするポリヌクレオチド
（付記１７）
前記配列番号１２の塩基配列において、
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基が、Ｓ、またはＡをコードするコドンであり、
１６６番目の塩基、１６７番目の塩基、および１６８番目の塩基が、Ｒ、またはＹをコードするコドンであり、
４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基が、Ｍ、Ｓ、またはＴをコードするコドンであり、
５７４番目の塩基、５７５番目の塩基、および５７６番目の塩基が、Ｉ、またはＲをコードするコドンである、付記１４から１６のいずれかに記載の新規遺伝子。
（付記１８）
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基のコドン、１６６番目の塩基、１６７番目の塩基、および１６８番目の塩基のコドン、４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基のコドン、ならびに５７４番目の塩基、５７５番目の塩基、および５７６番目の塩基のコドンの組合せが、下記（ａａ１’）～（ａａ３’）のいずれか１つのアミノ酸の組合せをコードするコドンの組合せである、付記１４から１７のいずれかに記載の新規遺伝子。

（付記１９）
１５４番目の塩基、１５５番目の塩基、および１５６番目の塩基のコドン、１６６番目の塩基、１６７番目の塩基、および１６８番目の塩基のコドン、４０６番目の塩基、４０７番目の塩基、および４０８番目の塩基のコドン、ならびに５７４番目の塩基、５７５番目の塩基、および５７６番目の塩基のコドンが、下記（ｎ１）～（ｎ４）のいずれか１つの組合せである、付記１４から１８のいずれかに記載の新規遺伝子。

（付記２０）
１０番目の塩基、１１番目の塩基、および１２番目の塩基が、Ｙをコードするコドンに置換された、付記１４から１９のいずれかに記載の新規遺伝子。
（付記２１）
６４番目の塩基、６５番目の塩基、および６６番目の塩基が、Ｇをコードするコドンに置換された、付記１４から２０のいずれかに記載の新規遺伝子。
（付記２２）
８５番目の塩基、８６番目の塩基、および８７番目の塩基が、Ｖをコードするコドンに置換された、付記１４から２１のいずれかに記載の新規遺伝子。
（付記２３）
１０９番目の塩基、１１０番目の塩基、および１１１番目の塩基が、Ｔをコードするコドンに置換された、付記１４から２２のいずれかに記載の新規遺伝子。
（付記２４）
２０５番目の塩基、２０６番目の塩基、および２０７番目の塩基が、Ｍをコードするコドンに置換された、付記１４から２３のいずれかに記載の新規遺伝子。
（付記２５）
４５７番目の塩基、４５８番目の塩基、および４５９番目の塩基が、Ｖをコードするコドンに置換された、付記１４から２４のいずれかに記載の新規遺伝子。
（付記２６）
５１４番目の塩基、５１５番目の塩基、および５１６番目の塩基が、Ｍをコードするコドンに置換された、付記１４から２５のいずれかに記載の新規遺伝子。
（付記２７）
前記（ｆ１）のポリヌクレオチドが、配列番号１３～２２からなる群から選択された少なくとも１つの塩基配列からなるポリヌクレオチドである、付記１４から２６のいずれかに記載の新規遺伝子。
（付記２８）
付記１４から２７のいずれかに記載の新規遺伝子を含むことを特徴とする、発現ベクター。
（付記２９）
付記１４から２７のいずれかに記載の新規遺伝子を含むことを特徴とする、形質転換体。
（付記３０）
前記形質転換体が、植物である、付記２９記載の形質転換体。
（付記３１）
宿主に、付記１４から２７のいずれかに記載の新規遺伝子を導入する導入工程を含むことを特徴とする、形質転換体の製造方法。
（付記３２）
さらに、前記宿主に、前記新規遺伝子を発現させる発現工程を含む、付記３１記載の形質転換体の製造方法。
（付記３３）
前記宿主が、植物である、付記３１または３２記載の形質転換体の製造方法。
（付記３４）
付記１から１２のいずれかに記載の新規タンパク質に変異を導入した候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する選抜工程を含むことを特徴とする、新規蛍光タンパク質のスクリーニング方法。
（付記３５）
付記１から１２のいずれかに記載の新規タンパク質に変異を導入した候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、１３６番目のアミノ酸がＴ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４記載のスクリーニング方法。
（付記３６）
付記１から１２のいずれかに記載の新規タンパク質に変異を導入した候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、１３６番目のアミノ酸がＴ以外のアミノ酸であり、５６番目のアミノ酸がＹ以外のアミノ酸であり、１９２番目のアミノ酸がＲ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４または３５記載のスクリーニング方法。
（付記３７）
前記選抜工程において、
前記配列番号１のアミノ酸配列における５２番目のアミノ酸が、Ｓ、またはＡであり、
５６番目のアミノ酸が、Ｒ、またはＹであり、
１３６番目のアミノ酸が、Ｍ、Ｓ、またはＴであり、
１９２番目のアミノ酸が、Ｉ、またはＲであり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４から３６のいずれかに記載のスクリーニング方法。
（付記３８）
前記選抜工程において、
前記配列番号１のアミノ酸配列における、５２番目のアミノ酸、５６番目のアミノ酸、１３６番目のアミノ酸、および１９２番目のアミノ酸の組合せが、下記（ａａ１）～（ａａ３）のいずれか１つの組合せであり、且つ前記（Ｆ１）のタンパク質のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４から３７のいずれかに記載のスクリーニング方法。

（付記３９）
付記１４から２７のいずれかに記載の新規遺伝子に変異を導入した変異ポリヌクレオチドがコードする候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４から３８のいずれかに記載のスクリーニング方法。
（付記４０）
付記１４から２７のいずれかに記載の新規遺伝子に変異を導入した変異ポリヌクレオチドがコードする候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、１３６番目のアミノ酸がＴ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４から３９のいずれかに記載のスクリーニング方法。
（付記４１）
付記１４から２７のいずれかに記載の新規遺伝子に変異を導入した変異ポリヌクレオチドがコードする候補タンパク質から、配列番号１のアミノ酸配列における５２番目のアミノ酸がＨ以外のアミノ酸であり、１３６番目のアミノ酸がＴ以外のアミノ酸であり、５６番目のアミノ酸がＹ以外のアミノ酸であり、１９２番目のアミノ酸がＲ以外のアミノ酸であり、且つ前記（Ｆ１）のタンパク質の最大蛍光波長±５ｎｍの範囲に最大蛍光波長を有するタンパク質を選抜する、付記３４から４０のいずれかに記載のスクリーニング方法。 Some or all of the above embodiments may also be described as in the appendix below, but are not limited to:
(Appendix 1)
A novel protein characterized by being the following (F1) or (F2) protein.
(F1) In the amino acid sequence of SEQ ID NO: 1,
Protein (F2) consisting of an amino acid sequence in which the 52nd amino acid is an amino acid other than H. A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) (Appendix 2).
The novel protein according to Appendix 1, wherein the protein of (F1) is the protein of the following (F1'), and the protein of the (F2) is the protein of the following (F2').
(F1') In the amino acid sequence of SEQ ID NO: 1,
The 52nd amino acid is an amino acid other than H,
Protein consisting of an amino acid sequence in which the 136th amino acid is an amino acid other than T (F2') An amino acid sequence having 80% or more identity including the 52nd and 136th amino acids with respect to the amino acid sequence of the above (F1'). A protein consisting of the above (F1) and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm (Appendix 3).
The novel protein according to Supplementary note 1 or 2, wherein the protein of (F1) is the protein of the following (F1''), and the protein of the (F2) is the protein of the following (F2'').
(F1'') In the amino acid sequence of SEQ ID NO: 1.
The 52nd amino acid is an amino acid other than H,
The 56th amino acid is an amino acid other than Y,
The 136th amino acid is an amino acid other than T,
Protein consisting of an amino acid sequence in which the 192nd amino acid is an amino acid other than R (F2'') Contains the 52nd, 56th, 136th, and 192nd amino acids with respect to the amino acid sequence of (F1''). A protein having an amino acid sequence having an identity of 80% or more and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) (Appendix 4).
In the amino acid sequence of SEQ ID NO: 1,
The 52nd amino acid is A or S,
The 56th amino acid is R or Y,
The 136th amino acid is M, S, or T,
The novel protein according to any one of Supplementary note 1 to 3, wherein the 192nd amino acid is R or I.
(Appendix 5)
Any one of Supplementary note 1 to 4, wherein the combination of the 52nd amino acid, the 56th amino acid, the 136th amino acid, and the 192nd amino acid is a combination of any one of the following (aa1) to (aa3). The novel protein described in.

(Appendix 6)
The novel protein according to any one of Supplementary note 1 to 5, wherein the fourth amino acid is substituted with Y.
(Appendix 7)
The novel protein according to any one of Supplementary note 1 to 6, wherein the 22nd amino acid is replaced with G.
(Appendix 8)
The novel protein according to any one of Supplementary note 1 to 7, wherein the 29th amino acid is replaced with V.
(Appendix 9)
The novel protein according to any one of Supplementary note 1 to 8, wherein the 37th amino acid is replaced with T.
(Appendix 10)
The novel protein according to any one of Supplementary note 1 to 9, wherein the 69th amino acid is replaced with M.
(Appendix 11)
The novel protein according to any one of Supplementary note 1 to 10, wherein the 153rd amino acid is replaced with V.
(Appendix 12)
The novel protein according to any one of Supplementary note 1 to 11, wherein the 172nd amino acid is replaced with M.
(Appendix 13)
The novel protein according to any one of Supplementary note 1 to 12, wherein the protein of (F1) is a protein consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11.
(Appendix 14)
A novel gene characterized by being the following (f1) or (f2) polynucleotide.
(F1) In the base sequence of SEQ ID NO: 12,
A polynucleotide in which the 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H (f2) The 154th base and the 155th base with respect to the polynucleotide of the above (f1). A protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of (f1), and having a base sequence having an identity of 80% or more including the base of the above and the 156th base. (Appendix 15)
The novel gene according to Appendix 14, wherein the polynucleotide (f1) is the polynucleotide of the following (f1'), and the polynucleotide of the (f2) is the polynucleotide of the following (f2').
(F1') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
A polynucleotide (f2') in which the 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T. The polynucleotide consisting of a base sequence having 80% or more identity including the 155th base, the 156th base, the 406th base, the 407th base, and the 408th base, and the polynucleotide of (f1) is said. A polynucleotide encoding a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the encoding protein (Appendix 16).
The novel gene according to Supplementary note 14 or 15, wherein the polynucleotide of (f1) is the polynucleotide of the following (f1''), and the polynucleotide of the (f2) is the polynucleotide of the following (f2''). ..
(F1'') In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding amino acids other than H.
The 166th base, the 167th base, and the 168th base are codons encoding amino acids other than Y.
The 406th base, the 407th base, and the 408th base are codons encoding amino acids other than T.
The 574th base, the 575th base, and the 576th base are polynucleotides (f2'') which are codons encoding amino acids other than R. The 154th base with respect to the above-mentioned (f1'') polynucleotide. Base, 155th base, 156th base, 166th base, 167th base, 168th base, 406th base, 407th base, 408th base, 574th base, 575th base A protein having a base sequence having an identity of 80% or more including a base and the 576th base and having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein encoded by the polynucleotide of (f1) above. Polynucleotide to be encoded (Appendix 17)
In the base sequence of SEQ ID NO: 12,
The 154th base, the 155th base, and the 156th base are codons encoding S or A.
The 166th base, the 167th base, and the 168th base are codons encoding R or Y.
The 406th base, the 407th base, and the 408th base are codons encoding M, S, or T.
The novel gene according to any of Supplementary note 14 to 16, wherein the 574th base, the 575th base, and the 576th base are codons encoding I or R.
(Appendix 18)
The 154th base, the 155th base, and the 156th base codon, the 166th base, the 167th base, and the 168th base codon, the 406th base, the 407th base, and the 408th base. The combination of the base codon and the codon of the 574th base, the 575th base, and the 576th base encodes the combination of any one of the following amino acids (aa1') to (aa3'). The novel gene according to any one of Supplementary note 14 to 17.

(Appendix 19)
Base 154, base 155, and base 156, base 166, base 167, and base 168, base 406, base 407, and base 408. It is described in any one of Supplementary note 14 to 18, wherein the base coordinator, the 574th base, the 575th base, and the 576th base coordinator are a combination of any one of the following (n1) to (n4). New gene.

(Appendix 20)
The novel gene according to any of Supplementary note 14 to 19, wherein the 10th base, the 11th base, and the 12th base are replaced with codons encoding Y.
(Appendix 21)
The novel gene according to any of Supplementary note 14 to 20, wherein the 64th base, the 65th base, and the 66th base are replaced with codons encoding G.
(Appendix 22)
The novel gene according to any of Supplementary note 14 to 21, wherein the 85th base, the 86th base, and the 87th base are replaced with codons encoding V.
(Appendix 23)
The novel gene according to any of Supplementary note 14 to 22, wherein the 109th base, the 110th base, and the 111th base are replaced with codons encoding T.
(Appendix 24)
The novel gene according to any of Supplementary note 14 to 23, wherein the 205th base, the 206th base, and the 207th base are replaced with codons encoding M.
(Appendix 25)
The novel gene according to any of Supplementary note 14 to 24, wherein the 457th base, the 458th base, and the 459th base are replaced with codons encoding V.
(Appendix 26)
The novel gene according to any of Supplementary note 14 to 25, wherein the 514th base, the 515th base, and the 516th base are replaced with codons encoding M.
(Appendix 27)
The novel gene according to any one of Supplementary note 14 to 26, wherein the polynucleotide of (f1) is a polynucleotide consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 13 to 22.
(Appendix 28)
An expression vector comprising the novel gene according to any one of Supplementary note 14 to 27.
(Appendix 29)
A transformant comprising the novel gene according to any one of Supplementary note 14 to 27.
(Appendix 30)
The transformant according to Appendix 29, wherein the transformant is a plant.
(Appendix 31)
A method for producing a transformant, which comprises an introduction step of introducing the novel gene according to any one of Supplementary note 14 to 27 into a host.
(Appendix 32)
The method for producing a transformant according to Appendix 31, further comprising an expression step of expressing the novel gene in the host.
(Appendix 33)
The method for producing a transformant according to Supplementary note 31 or 32, wherein the host is a plant.
(Appendix 34)
From the candidate protein in which the mutation was introduced into the novel protein according to any one of Supplementary note 1 to 12, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the maximum fluorescence of the protein of (F1) above. A method for screening a novel fluorescent protein, which comprises a selection step of selecting a protein having a maximum fluorescence wavelength in the range of a wavelength of ± 5 nm.
(Appendix 35)
From the candidate proteins in which a mutation was introduced into the novel protein according to any one of Supplementary note 1 to 12, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid is an amino acid other than T. The screening method according to Appendix 34, wherein a protein having a maximum fluorescence wavelength within the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) is selected.
(Appendix 36)
From the candidate proteins in which a mutation was introduced into the novel protein according to any one of Supplementary note 1 to 12, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid is an amino acid other than T. Yes, the 56th amino acid is an amino acid other than Y, the 192nd amino acid is an amino acid other than R, and a protein having a maximum fluorescence wavelength within the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) is selected. The screening method according to Supplementary note 34 or 35.
(Appendix 37)
In the selection process,
The 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is S or A, and is
The 56th amino acid is R or Y,
The 136th amino acid is M, S, or T,
The screening according to any one of Supplementary note 34 to 36, wherein the 192nd amino acid is I or R, and the protein having the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1) is selected. Method.
(Appendix 38)
In the selection process,
The combination of the 52nd amino acid, the 56th amino acid, the 136th amino acid, and the 192nd amino acid in the amino acid sequence of SEQ ID NO: 1 is any one of the following combinations (aa1) to (aa3). The screening method according to any one of Supplementary note 34 to 37, wherein a protein having a maximum fluorescence wavelength is selected in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the protein of the above (F1).

(Appendix 39)
From the candidate protein encoded by the mutant polynucleotide in which the mutation was introduced into the novel gene according to any one of Supplementary note 14 to 27, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the above-mentioned (F1). ), The screening method according to any one of Supplementary note 34 to 38, wherein the protein having the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein is selected.
(Appendix 40)
From the candidate proteins encoded by the mutant polynucleotide in which the mutation was introduced into the novel gene according to any one of Supplementary note 14 to 27, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid. The screening method according to any one of Supplementary note 34 to 39, wherein the protein is an amino acid other than T and has the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1).
(Appendix 41)
From the candidate proteins encoded by the mutant polynucleotide in which the mutation was introduced into the novel gene according to any one of Supplementary note 14 to 27, the 52nd amino acid in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than H, and the 136th amino acid. Is an amino acid other than T, the 56th amino acid is an amino acid other than Y, the 192nd amino acid is an amino acid other than R, and the maximum fluorescence is within the range of the maximum fluorescence wavelength ± 5 nm of the protein of the above (F1). The screening method according to any one of Supplementary note 34 to 40, which selects a protein having a wavelength.

Claims (10)

A novel protein characterized by being the following (F1), (F2), or (F3) protein.
(F1) Protein consisting of any of the amino acid sequences of SEQ ID NOs: 5 to 11 (F2) The amino acid sequence of SEQ ID NO: 5 consists of an amino acid sequence having 90% or more identity with respect to the amino acid sequence of SEQ ID NO: 5. The 52nd and 136th amino acids of the above are conserved, and the protein having the maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein consisting of the amino acid sequence of SEQ ID NO: 5 (F3) Any of the above SEQ ID NOs: 6 to 11. It consists of an amino acid sequence having 90% or more identity with respect to the amino acid sequence, and the 52nd, 56th, 136th, and 192nd amino acids of the corresponding amino acid sequences in the amino acid sequences of SEQ ID NOs: 6 to 11 are. A protein that is conserved and has a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength ± 5 nm of the protein of the corresponding amino acid sequence in the amino acid sequences of SEQ ID NOs: 6 to 11. The novel protein according to claim 1, wherein the novel protein is a protein consisting of any of the amino acid sequences of SEQ ID NOs: 5 to 11. The novel protein according to claim 1 or 2, wherein the novel protein is a protein consisting of any of the amino acid sequences of SEQ ID NOs: 6 to 11. A novel gene characterized by being the following polynucleotide (f1), (f2) or (f3).
(F1) Nucleotide sequence consisting of any of SEQ ID NOs: 16 to 22 (f2) Nucleotide sequence of SEQ ID NO: 16 having 90% or more identity with respect to the nucleotide of SEQ ID NO: 16. The maximum fluorescence wavelength of the protein encoded by the polynucleotide having the 154th, 155th, 156th, 406th, 407th, 407th, and 408th base sequences of the sequence and consisting of the base sequence of SEQ ID NO: 16 is ± 5 nm. Polynucleotide encoding a protein having the maximum fluorescence wavelength in the range of (f3) The nucleotide sequence having 90% or more identity with respect to any of the nucleotide sequences of SEQ ID NOs: 17 to 22 is composed of the nucleotide sequences of 90% or more. The 154th, 155th, 156th, 166th, 167th, 168th, 406th, 407th, 408th, 574th, 575th, and 576th base sequences of the corresponding base sequences in the 22nd base sequence are A polynucleotide that is conserved and encodes a protein having a maximum fluorescence wavelength in the range of the maximum fluorescence wavelength of ± 5 nm of the protein encoded by the polynucleotide of the corresponding base sequence in the base sequences of SEQ ID NOs: 17 to 22. The novel gene according to claim 4, wherein the novel gene is a polynucleotide consisting of any of the nucleotide sequences of SEQ ID NOs: 16 to 22. The novel gene according to claim 4 or 5, wherein the novel gene is a polynucleotide consisting of any of the nucleotide sequences of SEQ ID NOs: 17 to 22. An expression vector comprising the novel gene according to any one of claims 4 to 6. A transformant comprising the novel gene according to any one of claims 4 to 6 or the expression vector according to claim 7. The transformant according to claim 8, wherein the transformant is a plant. A method for producing a transformant, which comprises an introduction step of introducing the novel gene according to any one of claims 4 to 6 or the expression vector according to claim 7 into a host.

Images