JP6425483B2 - Fluorescent proteins, DNA, vectors, transformants, and methods for monitoring the redox state in cells - Google Patents

Description

  The present invention relates to fluorescent proteins, DNA, vectors, transformants, and methods for monitoring the redox state in cells.

  In cells, various events involving redox reactions occur. For example, in the electron transfer system located at the final stage of the metabolic system of cellular respiration, the electrons generated by the oxidation of NADH are finally transferred to oxygen to generate water. ATP is generated by oxidative phosphorylation that occurs in conjunction with the electron transfer system.

Redox reactions also occur in plant photosynthesis. In chloroplast thylakoid membranes, chlorophyll (photosynthetic pigments) use light energy to break down water, producing protons, molecular oxygen and electrons. The electrons generated at this time reduce NADP ⁺ to generate NADPH. Furthermore, ATP is generated by ATP synthase using a proton concentration gradient across the thylakoid membrane.

  In addition, reactive oxygen species generated during aerobic consumption of aerobic organisms have a very strong oxidizing action, and when present in excess, they constitute many of the cells, such as intracellular proteins, enzymes, cell membranes, and DNA. Molecules can be oxidatively denatured causing cellular dysfunction.

  In order to understand the details of such redox events in cells, development of molecular tools capable of monitoring the redox state in cells is desired.

  As a molecular tool that can monitor the redox state in cells, roGFP (Redox-sensitive green fluorescent protein) is known, in which S147 and Q204 of the green fluorescent protein (GFP), which is a fluorescent protein, is replaced with a cysteine residue (Non-patent documents 1 and 2). roGFP is present in an oxidized form in which the thiol group in the two cysteine residues is linked by a disulfide bond, or in a reduced form in which the thiol group in the cysteine residue is in the free state. The abundance ratio of the oxidized form to the reduced form varies depending on the redox state of the surrounding environment of roGFP. The change is detected as a change in shape of the excitation spectrum of roGFP. There are two excitation maximum wavelengths (400 nm and 490 nm) in the excitation spectrum of roGFP, and it is possible to monitor the redox state of the surrounding environment of roGFP by measuring the ratio of the fluorescence intensity at the excitation wavelength .

Hanson G. T. et al., J. Biol. Chem., 2004, 279: 13044-13053. Lohman J. R. et. Al., Biochemistry, 2008, 47, 8678-8688

However, when roGFP is used, in order to monitor the redox state in cells, it is necessary to measure the excitation spectrum and analyze the shape of the spectrum, resulting in a problem that it takes time and effort. For this reason, a simpler method has been required which can directly evaluate changes in the redox state in cells by observation with a fluorescent microscope or the like.
Therefore, an object of the present invention is to provide a molecular tool capable of more easily evaluating changes in redox state in cells without analyzing the excitation spectrum or the like.

  As a result of intensive investigations in view of the above problems, the present inventors surprisingly fixed the excitation light wavelength when a fluorescent protein in which a specific amino acid mutation was applied to a GFP mutant was exposed to oxidation conditions or reduction conditions. It was found that the fluorescence intensity of the fluorescent protein measured was significantly changed. For example, a fluorescent protein which has been subjected to a specific amino acid mutation in Sirius (SEQ ID NO: 1) (Nagai, T. et al., Nature Methods, 2009, 6, 351-353) known as a GFP mutant is oxidized It was found that when exposed to the conditions, the fluorescence intensity of the fluorescent protein measured by fixing the excitation light wavelength is significantly reduced. Furthermore, it has been found that use of the fluorescent protein makes it possible to measure the change in the redox state in cells based on the fluorescence intensity, and it has been found that it can be used as a simpler molecular tool, and the present invention has been achieved.

Therefore, the present invention has the following aspects.
[1] 90% or more of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61, or the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61 In the amino acid sequence having identity, the 147th and 204th amino acid residues are substituted with cysteine (C), and between the 147th and 148th amino acid residues, glycine (G), aspartic acid (D) 2.) A fluorescent protein consisting of an amino acid sequence into which any one amino acid selected from the group consisting of glutamic acid (E), leucine (L) and arginine (R) is inserted.
[2] The amino acid residue at position 146 in the amino acid sequence is selected from the group consisting of leucine (L), tryptophan (W), asparagine (N), phenylalanine (F), serine (S) and glycine (G) The fluorescent protein according to the above [1], which is further substituted with any one amino acid.
[3] In the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, the 146th amino acid residue is substituted with asparagine (N), 147 and 204 The fluorescence according to the above [2], which consists of an amino acid sequence in which the th amino acid residue is substituted with cysteine (C) and aspartic acid (D) is inserted between the 147th and 148th amino acid residues. protein.
[4] In the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, the 65th amino acid residue is substituted with serine (S), and the 66th amino acid The residue is substituted with tryptophan (W), the 146th amino acid residue is substituted with glycine (G), the 147th and 204th amino acid residues are substituted with cysteine (C), and the 148th amino acid residue The fluorescent protein according to the above-mentioned [2], which comprises an amino acid sequence in which is substituted with aspartic acid (D) and glutamic acid (E) is inserted between the 147th and 148th amino acid residues.
[5] In the amino acid sequence of SEQ ID NO: 59, or the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59, the 146th amino acid residue is substituted with glycine (G), 147 and 204 The fluorescent protein according to the above-mentioned [2], which consists of an amino acid sequence in which the th amino acid residue is substituted with cysteine (C) and glutamic acid (E) is inserted between the 147th and 148th amino acid residues. .
[6] In the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 60, the 146th amino acid residue is substituted with leucine (L), and 147 and 204 The fluorescence according to the above [2], which consists of an amino acid sequence in which the th amino acid residue is substituted with cysteine (C) and aspartic acid (D) is inserted between the 147th and 148th amino acid residues. protein.
[7] In the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59, the 146th amino acid residue is substituted with tryptophan (W), 147 and 204 The fluorescent protein according to the above-mentioned [2], which consists of an amino acid sequence in which the th amino acid residue is substituted with cysteine (C) and glycine (G) is inserted between the 147th and 148th amino acid residues. .
[8] In the amino acid sequence of SEQ ID NO: 61, or the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 61, the 146th amino acid residue is substituted with tryptophan (W), 147 and 204 The fluorescent protein according to the above-mentioned [2], which consists of an amino acid sequence in which the th amino acid residue is substituted with cysteine (C) and arginine (R) is inserted between the 147th and 148th amino acid residues. .
[9] A fusion protein in which the fluorescent protein according to any one of the above [1] to [8] and a second protein are fused.
[10] A DNA encoding the fluorescent protein according to any one of the above [1] to [8] or the fusion protein according to the above [5].
[11] A recombinant vector having the DNA of the above-mentioned [9].
[12] A host cell transformed with the recombinant vector of [11] above.
[13] A method of monitoring the redox state in a cell, which comprises introducing the fluorescent protein according to any one of the above [1] to [8] or the fusion protein according to the above [9] into a cell Detecting the fluorescence of the fluorescent protein or the fusion protein, and monitoring the redox state in the cell based on the fluorescence intensity of the detected fluorescence.

  According to the fluorescent protein of the present invention, the change in redox state in cells can be conveniently monitored based on the fluorescence intensity of the fluorescent protein without measuring the excitation spectrum.

FIG. 1A is a graph showing the fluorescence intensity in each of the oxidized and reduced forms of the fluorescent protein of the present invention (Examples 1 to 10). FIG. 1B is a graph showing the fluorescence intensity in each of the oxidized and reduced forms of the fluorescent protein of the present invention (Examples 11 to 14). FIG. 2A is a graph showing the ratio of the fluorescence intensity of the fluorescent protein of the present invention (Examples 1 to 10) to the higher one of the oxidized and reduced fluorescence intensities. FIG. 2B is a graph showing the ratio of the fluorescence intensity of the fluorescent protein of the present invention (Examples 11 to 14) to the higher one of the oxidized and reduced fluorescence intensities. FIG. 3A is an excitation spectrum and a fluorescence spectrum of each of oxidized and reduced forms of Oba-Qs (oxidation balanced sensed protein derived from serius) (SEQ ID NO: 5). In addition, after conversion from the reduced form to the oxidized form, one converted again to the reduced form is shown as a rereduced form. The excitation spectrum was at a fluorescence wavelength of 425 nm and was measured in the range of 250 nm to 400 nm. The fluorescence spectrum was measured at an excitation light wavelength of 375 nm. FIG. 3B is an excitation spectrum and a fluorescence spectrum of Re-Qy (reduction sensed quenching protein derived from YFP) (SEQ ID NO: 65) in an oxidized form and a reduced form, respectively. The excitation spectrum was at a fluorescence wavelength of 525 nm and was measured in the range of 250 nm to 518 nm. The fluorescence spectrum was measured with an excitation light wavelength of 510 nm. FIG. 4 is a graph showing how the fluorescence intensity of Oba-Qs decreases with time by adding H ₂ O ₂ . FIG. 5 is a graph showing how the fluorescence intensity of the fusion protein of Oba-Qs and AtGpx1 decreases with time by adding H ₂ O ₂ .

  In the present specification, amino acids are represented by one-letter notation unless otherwise specified. The notation of the amino acid sequence represents, for example, serine, which is the amino acid residue at position 147 from the N-terminus of the amino acid sequence of SEQ ID NO: 1, as S147 unless otherwise specified. The same applies to other amino acid residues. Further, the notation of substitution in the amino acid sequence is represented as S147C unless otherwise specified, for example, when S147 of SEQ ID NO: 1 is substituted by C. The same applies to other amino acid substitutions. Also, the notation of insertion in the amino acid sequence is, for example, C147_S148insD unless otherwise specified when aspartic acid (D) is inserted between C147 and S148 of SEQ ID NO: 1 in which substitution of S147C is performed. The same applies for other amino acid insertions. In addition, a variant in which a plurality of amino acid residues are simultaneously substituted and / or inserted in the amino acid sequence (Sirius) shown in SEQ ID NO: 1 is described as “Sirius” at the beginning, and each substitution and / or insertion is hyphenated. We will tie by (-). For example, amino acid substitution of S147C and Q204C is made to the amino acid sequence (Sirius) shown in SEQ ID NO: 1, and amino acid sequence in which aspartate (D) is inserted between C147 and S148 is “Sirius-S147C-Q204C It represents as "C147_S148insD." The other variants to SEQ ID NO: 1 are also denoted similarly. In addition, a mutant in which a plurality of amino acid residues are simultaneously substituted and / or inserted into the amino acid sequence shown in SEQ ID NO: 59, 60 or 61 also has a fluorescent protein name (“mTurquoise”, “ In the same manner as in SEQ ID NO: 1 except that “EBFP 2” or “Venus” is described.

  As used herein, "sequence identity" refers to sequence identity between two DNAs or two proteins. The "sequence identity" is determined by comparing two sequences aligned in the optimal state over the region of the sequences to be compared. Here, the DNA or protein to be compared may have additions or deletions (such as gaps) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using Vector NTI and the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)). Can. Sequence identity is measured using sequence analysis software, specifically analysis tools provided by Vector NTI, GENETYX and public databases. The public database is generally available, for example, at a homepage address http://www.ddbj.nig.ac.jp. In the present specification, the numbering of the amino acid sequences in variants of the amino acid sequences of SEQ ID NOs: 1, 59, 60 and 61 is carried out based on the amino acid sequences of SEQ ID NOs: 1, 59, 60 and 61, respectively. The numbering of the nucleotide sequences of variants of the nucleotide sequences of 44, 80, 81 and 82 is carried out based on the nucleotide sequences of SEQ ID NO: 44, 80, 81 and 82, respectively.

  In the present specification, the “excitation spectrum” is a fluorescence wavelength to be detected is fixed, the wavelength of excitation light is continuously changed, and the obtained fluorescence intensity is plotted for each wavelength. In the present specification, the “excitation maximum wavelength” is a wavelength that gives the strongest fluorescence intensity in the excitation spectrum. The excitation spectrum is measured by a spectrofluorimeter.

  In the present specification, the “fluorescence spectrum” refers to a plot of fluorescence intensity obtained for each wavelength while fixing the wavelength of excitation light. The difference in excitation wavelength does not change the shape of the fluorescence spectrum. Usually, the excitation wavelength to be fixed uses the excitation maximum wavelength. In the present specification, the "fluorescent maximum wavelength" is a wavelength that gives the strongest fluorescence intensity in the fluorescence spectrum. The peak of the fluorescence spectrum appears on the longer wavelength side than the peak of the excitation spectrum. The fluorescence spectrum is measured by a spectrofluorimeter.

  The fluorescence intensity of the fluorescence emitted from the oxidized and reduced fluorescent proteins excited by the excitation light can be measured by a spectrofluorimeter, a fluorescence microscope, or the like.

  As used herein, a "GFP variant" is a variant of GFP that has high identity (for example, 90% or more identity) with the amino acid sequence of GFP and has unique fluorescence characteristics. Although not particularly limited, for example, Sirius (SEQ ID NO: 1), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP) and the like. In addition, mTurquoise (SEQ ID NO: 59) prepared by adding a mutation to CFP; EBFP2 (SEQ ID NO: 60) prepared by adding a mutation to BFP; Venus (SEQ ID NO: 61) prepared by adding a mutation to YFP And the like are also included in the GFP mutant.

  One aspect of the invention relates to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61, or 90 for the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61 % Or more, preferably an amino acid sequence having a sequence identity of 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more In which the 147th and 204th amino acid residues are substituted with cysteine (C), and between the 147th and 148th amino acid residues, glycine (G), aspartic acid (D), glutamic acid (E) It is a fluorescent protein consisting of an amino acid sequence into which any one amino acid selected from the group consisting of leucine (L) and arginine (R) is inserted.

  The fluorescent protein of the present invention is either an oxidized form in which a thiol group in the introduced two cysteine residues is linked by a disulfide bond, or a reduced form in which the thiol group in the cysteine residue is in a free state. Exist in the form. The half reaction formula is as follows.

  In the cell, an electron acceptor (oxidizing agent) such as active oxygen containing hydrogen peroxide, and an electron donor (reducing agent) such as glutathione or thioredoxin are present. These amounts vary depending on the environment in which the cells are present. Here, in the present invention, "monitoring the redox state in cells" refers to monitoring changes in the amounts of oxidizing agent and reducing agent in the cells. In the case where the fluorescent protein of the present invention is present in cells, the balance of the above semi-reactive formula is biased to the oxidized form when the amount of oxidizing agent present in the cells is increased. Conversely, when the amount of reducing agent present in the cell increases, the equilibrium of the above semi-reaction formula is biased to the reduced form. And, the change of the redox state in the cell is observed as the change of the fluorescence intensity of the fluorescent protein of the present invention.

  When the excitation light wavelength is fixed and the fluorescence intensity at a specific wavelength (for example, fluorescence maximum wavelength) is measured, the fluorescence intensity of the oxidized type of the fluorescent protein of the present invention is different from the fluorescence intensity of the reduced type. Therefore, by measuring the fluorescence intensity of the fluorescent protein, it is possible to distinguish whether the fluorescent protein is present in an oxidized form or in a reduced form, and oxidation in the surrounding environment in which the fluorescent protein is present The reduction status can be monitored.

  The ratio of the other fluorescence intensity to the higher one of the fluorescence intensities of the oxidized and reduced forms of the fluorescent protein of the present invention is usually 80% or less, preferably 70% or less, more preferably 60% or less, further It is preferably at most 50%, particularly preferably at most 40%, particularly preferably at most 30%, most preferably at most 20%.

  In the amino acid sequence of the fluorescent protein of the present invention, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61 The amino acids inserted between the 147th and 148th amino acid residues of the amino acid sequence having 90% or more of the sequence identity are glycine (G), aspartic acid (D), glutamic acid (E), leucine It is any one selected from the group consisting of (L) and arginine (R). When the excitation light wavelength is fixed and the fluorescence intensity at a specific wavelength (for example, fluorescence maximum wavelength) is measured, the difference between the fluorescence intensity of the reduced type and the fluorescence intensity of the oxidized type of the fluorescent protein of the present invention Will occur. In order to expand the difference in fluorescence intensity between the reduced form and the oxidized form, the above amino acids to be inserted include glycine (G), aspartic acid (D), glutamic acid (E) or arginine (R) preferable.

  In order to further increase the difference in fluorescence intensity between the reduced and oxidized forms of the fluorescent protein of the present invention, the fluorescent protein of the present invention is an amino acid selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61 Or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 59, 60 and 61, wherein the 146th amino acid residue is leucine (L), Asparagine (N), preferably further substituted with any one amino acid selected from the group consisting of tryptophan (W), asparagine (N), phenylalanine (F), serine (S) and glycine (G), More preferably, it is further substituted with any one amino acid selected from the group consisting of serine (S) and glycine (G).

  In a preferred embodiment of the fluorescent protein of the present invention, the amino acid residue at position 146 of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 90% or more sequence identity to SEQ ID NO: Consisting of an amino acid sequence in which the 147th and 204th amino acid residues are substituted with cysteine (C) and an aspartic acid (D) is inserted between the 147th and 148th amino acid residues. It is a fluorescent protein.

  A plurality of redox responsive fluorescent proteins having different color emissions (ie, different maximum fluorescence wavelengths) can be distinguished simultaneously even in the same cell depending on the color differences. In addition, a combination of redox-responsive fluorescent proteins with greatly different maximum excitation wavelengths can be used to observe the fluorescence of only the other fluorescent protein by irradiating excitation light of a wavelength at which one fluorescent protein hardly reacts. Do.

  One of the fluorescent proteins of the present invention is Sirius (excitation maximum wavelength 355 nm: fluorescence maximum wavelength 424 nm) in which Y66 involved in fluorescence emission in GFP (excitation maximum wavelength 395 nm: fluorescence maximum wavelength 510 nm) is substituted with phenylalanine (F) Can be made on the basis of Therefore, the fluorescent protein of the present invention having F66 has different fluorescence characteristics from fluorescent proteins (for example, roGFP) for monitoring the redox state in cells, which are made on the basis of GFP, so the combination of both is Even when used simultaneously in the same cell, they can be distinguished.

  In addition, one of the fluorescent proteins of the present invention has tryptophan (W) as the 66th amino acid involved in fluorescence emission, and has fluorescence characteristics like CFP (excitation maximum wavelength 452 nm: fluorescence maximum wavelength 505 nm). In addition, one of the fluorescent proteins of the present invention has tyrosine (Y) as the 66th and 203rd amino acids involved in fluorescence emission, and exhibits fluorescence characteristics like YFP (excitation maximum wavelength 513 nm: fluorescence maximum wavelength 527 nm). Have. Further, one of the fluorescent proteins of the present invention has histidine (H) as the 66th amino acid involved in fluorescence emission, and has fluorescence characteristics like BFP (excitation maximum wavelength 380 nm: fluorescence maximum wavelength 440 nm). A plurality of fluorescent proteins of the present invention having different fluorescent properties can be distinguished even when used simultaneously in the same cell.

  In the fluorescent protein of the present invention having F66 having Sirius-like fluorescence properties, for example, the excitation light wavelength is fixed at around 365-385 nm, preferably around 375 nm, and around 415-435 nm, preferably around 425 nm The fluorescence intensity at the wavelength is measured. In the fluorescent protein of the present invention having W66 having CFP-like fluorescence properties, for example, the excitation light wavelength is fixed at around 420 to 440 nm, preferably around 430 nm, and around 470 to 490 nm, preferably 480 nm The fluorescence intensity at nearby wavelengths is measured. In the fluorescent protein of the present invention having Y66 and Y203 having YFP-like fluorescence properties, for example, the excitation light wavelength is fixed at around 490 to 515 nm, preferably around 515 nm, preferably around 520 to 540 nm The fluorescence intensity at a wavelength around 528 nm is measured. In the fluorescent protein of the present invention having H66 having BFP-like fluorescence properties, for example, the excitation light wavelength is fixed at around 370 to 390 nm, preferably at around 380 nm, and preferably at around 430 to 450 nm, preferably 440 nm The fluorescence intensity at nearby wavelengths is measured.

  In a preferred embodiment of the fluorescent protein of the present invention, the amino acid residue at position 65 is a serine (S (S) in the amino acid sequence of SEQ ID NO: 1 or in the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1 Amino acid residue is substituted with tryptophan (W), amino acid residue 146 is substituted with glycine (G), and amino acid residues 147 and 204 are cysteine (C). It is a fluorescent protein consisting of an amino acid sequence which is substituted, and wherein the 148th amino acid residue is substituted with aspartic acid (D), and glutamic acid (E) is inserted between the 147th and 148th amino acid residues.

  In a preferred embodiment of the fluorescent protein of the present invention, the amino acid residue at position 146 is glycine (G (G) in the amino acid sequence of SEQ ID NO: 59 or amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59). Consisting of an amino acid sequence in which the 147th and 204th amino acid residues are substituted with cysteine (C), and a glutamic acid (E) is inserted between the 147th and 148th amino acid residues, It is a fluorescent protein.

  In a preferred embodiment of the fluorescent protein according to the present invention, the amino acid residue at position 146 is leucine (L) in the amino acid sequence of SEQ ID NO: 60 or in the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 60 Consisting of an amino acid sequence in which the 147th and 204th amino acid residues are substituted with cysteine (C) and an aspartic acid (D) is inserted between the 147th and 148th amino acid residues. , Fluorescent protein.

  In a preferred embodiment of the fluorescent protein of the present invention, the amino acid residue at position 146 is tryptophan (W) in the amino acid sequence of SEQ ID NO: 59 or in the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59 Consisting of an amino acid sequence in which the 147th and 204th amino acid residues are substituted with cysteine (C), and a glycine (G) is inserted between the 147th and 148th amino acid residues, It is a fluorescent protein.

  In a preferred embodiment of the fluorescent protein of the present invention, in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence having 90% or more sequence identity to SEQ ID NO: Consisting of an amino acid sequence in which the 147th and 204th amino acid residues are substituted with cysteine (C), and arginine (R) is inserted between the 147th and 148th amino acid residues, It is a fluorescent protein.

  One of the fluorescent proteins of the invention can be made on the basis of Sirius. The fluorescence intensity of conventional fluorescent proteins, including wild-type GFP, fluctuates depending on the pH change of the surrounding environment. However, Sirius is known to have its fluorescence intensity independent of pH (Nagai, T. et al., Nature Methods, 2009, 6, 351-353). Therefore, the fluorescent protein of the present invention produced using Sirius as a parent protein preferably has low pH sensitivity of the fluorescent property. More preferably, the fluorescent protein of the present invention has low pH sensitivity of the fluorescent property in the range of pH 6.5 to 8.0. Here, "low pH sensitivity" means changing the pH, fixing the excitation light wavelength and measuring the fluorescence intensity at the fluorescence maximum wavelength, the oxidized fluorescence to the reduced fluorescence intensity of the fluorescent protein The variation ratio of intensity ratio is usually within 70%.

  The fluorescent protein of the present invention can be produced by introducing an amino acid mutation to a parent protein (Sirius, mTurquoise, Venus or EBFP2). The method of introducing mutations is described in detail below. In addition, the fluorescent protein of the present invention can be synthesized by an organic chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method) or appropriate peptide synthesis commercially available. It can also be manufactured using a machine.

  The fluorescent protein of the present invention is not limited to only its single form, and can be used in an embodiment to which any modification, modification or the like is added. For example, fusion with a second protein, addition of a peptide tag (eg His tag, HQ tag, HN tag, HAT tag) to facilitate protein purification, various chemical modifications to proteins, macromolecules such as polyethylene glycol Processing by a variety of techniques known to those skilled in the art, such as binding to an insoluble carrier, binding to an insoluble carrier, and encapsulation in a liposome.

One aspect of the present invention is a fusion protein in which a fluorescent protein of the present invention and a second protein are fused. The fluorescent protein of the present invention and the second protein are linked via a linker having an amino acid sequence composed of a predetermined number of amino acid residues. The amino acid sequence constituting the linker and the number of amino acid residues thereof can be arbitrarily selected as long as the function of the fluorescent protein of the present invention is not inhibited. For example, (GGSGG) _6, which is a flexible linker, can be mentioned. The second protein is not particularly limited, and examples thereof include peroxidase and glutaredoxin. In particular, a protein in which the fluorescent protein of the invention is fused with peroxidase or glutaredoxin can improve redox sensitivity as compared to the fluorescent protein of the present invention before fusion.

  The fusion protein of the present invention is obtained by inserting the DNA encoding the fluorescent protein of the present invention and the DNA encoding the second protein into a recombinant vector in a form linked to the DNA encoding the linker, and using the vector for the host It can be produced by transforming cells. Alternatively, the fluorescent protein of the present invention and the second protein can be separately produced and then linked to each other. There is no particular limitation on the time of linking the fluorescent protein of the present invention and the second protein, and they may be linked individually immediately after preparation, or any one of the proteins may be prepared and used for a specific use. After the reaction, it may be linked to the other protein.

  One aspect of the present invention is DNA encoding the fluorescent protein of the present invention or the fusion protein of the present invention. The DNA encoding the fluorescent protein of the present invention has a base sequence selected from the group consisting of SEQ ID NOs: 44, 80, 81 and 82, or a base selected from the group consisting of SEQ ID NOs: 44, 80, 81 and 82 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the sequence identity to the sequence The codon corresponding to the amino acid at the substitution site characterizing the fluorescent protein of the present invention is replaced with the codon corresponding to each amino acid substitution, and the codon corresponding to the 147th and 148th amino acid residues The base sequence consists of the inserted codon corresponding to the amino acid to be inserted.

  The method for producing the DNA encoding the fluorescent protein of the present invention is not particularly limited, but is selected from the group consisting of SEQ ID NOs: 44, 80, 81 and 82 encoding the parent protein (Sirius, mTurquoise, Venus and EBFP2) Based on a DNA comprising a nucleotide sequence having a sequence identity of 90% or more to a nucleotide sequence or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 44, 80, 81 and 82, PCR, site-directed mutagenesis It can be produced by methods or other common genetic engineering techniques. In addition, genetic engineering techniques such as site-directed mutagenesis are widely used by those skilled in the art, for example, Maniatis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring harbor Laboratory, New York, 1982) and other such techniques. It is described in the experimental operation manual.

  One aspect of the present invention is a recombinant vector having a fluorescent protein of the present invention or a DNA encoding a fusion protein of the present invention. The recombinant vector of the present invention may be in any form such as circular, linear, etc., and may have other base sequences, if necessary, in addition to the DNA encoding the fluorescent protein or fusion protein of the present invention. May be Other nucleotide sequences include an enhancer sequence, a promoter sequence, a ribosome binding sequence, a nucleotide sequence used for the purpose of amplification of copy number, a nucleotide sequence encoding a signal peptide, a polyA addition sequence, a splicing sequence, an origin of replication, The base sequence of the gene used as a selection marker etc. are mentioned.

  In genetic recombination, a translation initiation codon or translation termination codon is added to the fluorescent protein of the present invention or the DNA encoding the fusion protein of the present invention using a suitable synthetic DNA adapter, or a suitable restriction enzyme within the nucleotide sequence It is also possible to newly generate or eliminate the cleavage sequence. These are within the scope of work routinely performed by those skilled in the art, and can be arbitrarily and easily processed based on the fluorescent protein of the present invention or the DNA encoding the fusion protein of the present invention.

  Moreover, the vector carrying the fluorescent protein of the present invention or the DNA encoding the fusion protein of the present invention may be selected from appropriate vectors according to the host to be used. The vector may be an autonomously replicating vector, ie a vector capable of autonomously replicating independently of chromosomal replication of the host cell. It may also be integrated into the host cell's genome and replicated together with the integrated chromosome. For example, in addition to plasmids, various viruses such as bacteriophage, baculovirus, retrovirus, vaccinia virus can be used.

  Commercially available expression vectors that can be used include pcDM8 (Funakoshi), pcDNAI (Funakoshi), pcDNAI / AmP (Invitrogen), EGFP-C1 (Clontech), pREP4 (Invitrogen), pGBT -9 (made by Clontech), pet23a (made by Novagen) etc. can be illustrated. The expression of the fluorescent protein of the present invention or the fusion protein of the present invention can be expressed under the control of a gene-specific promoter sequence encoding the protein. Alternatively, another appropriate expression promoter can be linked upstream of the nucleotide sequence encoding the fluorescent protein of the present invention or the fusion protein of the present invention. Such expression promoter may be appropriately selected according to the host and the purpose of expression, for example, when the host is E. coli, T7 promoter, lac promoter, trp promoter, λPL promoter, etc., and the host is yeast PHO5 promoter, GAP promoter, ADH promoter etc., SV40-derived promoter when the host is an animal cell, retrovirus promoter, IE (immediate early) gene promoter of cytomegalovirus (human CMV) gene, metallothionein promoter, A heat shock promoter, SR alpha promoter etc. can be mentioned. The procedure for linking the DNA encoding the fluorescent protein or fusion protein of the present invention to the promoter exemplified above or incorporating it into an expression vector is also described in Maniatis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring It can carry out based on the description of harbor laboratory, New York, 1982) and other experimental operation manual.

  One aspect of the invention is a host cell transformed with a recombinant vector of the invention. Examples of host cells include Escherichia bacteria, Corynebacterium bacteria, Brevibacterium bacteria, Bacillus bacteria, Serratia bacteria, Pseudomonas. Genus bacteria, Arthrobacter bacteria, Erwinia bacteria, Methylobacterium (Methylobacterium) bacteria, Rhodobacter bacteria, Streptomyces bacteria, Zymomonas bacteria , Microorganisms such as yeast belonging to the genus Saccharomyces, insect cells such as silkworm, HEK 293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI 38 cells, BHK cells, COS-7 cells, MDCK cells, C 127 cells, HKG Cells, animal cells such as human kidney cell lines It can gel.

  As a method for introducing a recombinant vector into a host cell, it is described in the experimental operation manual including Mananitis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring harbor Laboratory, New York, 1982) described above. For example, it can be carried out by an electroporation method, a protoplast method, an alkali metal method, a calcium phosphate precipitation method, a DEAE dextran method, a microinjection method, a particle gun method or the like. The use of insect cells such as Sf9 and Sf21 is described in Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), and BIO / TECHNOLOGY vol. 6, 1988, page 47. .

  The fluorescent protein of the present invention or the fusion protein of the present invention can be obtained by expressing the recombinant vector of the present invention in the above-mentioned host cell, recovering the target protein from the host cell or culture medium, and purifying it. As a method for purifying a protein, an appropriate method can be appropriately selected from the methods usually used for protein purification and can be carried out. Namely, salting out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, various affinity chromatography such as hydrophobic chromatography or antibody chromatography, chromatofocusing method, adsorption Appropriate methods may be appropriately selected from methods that can be usually used, such as chromatography and reverse phase chromatography, and purification may be performed in an appropriate order using an HPLC system or the like as necessary.

  One aspect of the present invention is a method of monitoring the redox state in a cell, wherein the fluorescent protein or fusion protein of the present invention is present in the cell, and the fluorescence of the fluorescent protein or fusion protein is detected. It is a method comprising monitoring the redox state in cells based on the fluorescence intensity of the detected fluorescence. In the method, in order to cause the fluorescent protein or fusion protein of the present invention to be present in cells, a recombinant vector having a DNA encoding the fluorescent protein or fusion protein of the present invention is transformed into cells, and the cells are Or express the fluorescent protein or fusion protein of the present invention directly into cells. The method for directly introducing the fluorescent protein or fusion protein of the present invention into cells is not particularly limited, and for example, it can be carried out using a cationic lipid-based protein transfer reagent. It is also performed by electroporation or microinjection.

  The detection of the fluorescence of the fluorescent protein or the fusion protein in the above is not particularly limited, but is performed using, for example, a fluorescence microscope or using a spectrofluorimeter. For example, the fluorescence intensity of the detected fluorescence is measured, and the fluorescence intensity of the protein when all is oxidized and / or the fluorescence intensity of the protein when all is reduced, The redox state in the cell can be monitored by comparing it with the fluorescence intensity.

  EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is by no means limited to the following examples, and can be implemented with appropriate modifications.

  In the following comparative examples and examples, generation of variants from parent proteins (Sirius, mTurquoise, Venus and EBFP2) was performed by site-directed mutagenesis. The sequences of the primers used are shown in Table 1 below.

  Here, the primer name in Tables 1 and 2 is written as "Primer_primer number (F / R)-introduced mutation (in order of introduction)". "F" indicates a forward primer and "R" indicates a reverse primer. For example, Primer_4 (F) -C147_S148insD (SEQ ID NO: 20) is a forward primer and is shown to be a primer for introducing C147_S148insD. Moreover, Primer_5 (F) -C147_S148insD-I146N (sequence number 22) is a forward primer, and shows that it is a primer for introduce | transducing I146N, after C147_S148insD is introduce | transduced. Moreover, Primer_13 (F) -Q65S + F66W is a forward primer and shows that it is a primer for introduce | transducing Q65S and F66W simultaneously. Moreover, Primer_15 (F) -C147_S148insE-S148D-I146G (sequence number 42) is a forward primer, It shows that it is a primer for introduce | transducing I146G, after C147_S148insE is introduce | transduced and S148D is introduce | transduced. Other primers are similarly described.

Comparative Example 1: Preparation of Sirius-S147C-Q204C (SEQ ID NO: 2) The DNA encoding Sirius (SEQ ID NO: 44) prepared by the method described in WO 2009/020197 was converted to pet 23a (manufactured by Novagen). Amino acids S147C and Q204C of Sirius (SEQ ID NO: 1) by site-directed mutagenesis using PrimStar® Mutagenesis Basal Kit (Takara Bio Inc.) with the incorporated vector (pet 23a / Sirius) as a template A mutation of the nucleotide sequence corresponding to the mutation was introduced into SEQ ID NO: 44.

  As a primer for introducing a mutation, the combination of SEQ ID NOS: 14 and 15 and the combination of SEQ ID NOS: 16 and 17 were used.

  PCR (50 μl reaction system) was performed using pet23a / Sirius 10 pg as a template, the above primers, and PrimeSTAR (registered trademark) Max Premix. The PCR was carried out 30 cycles of one cycle of 98 ° C., 10 seconds of DNA denaturation, 55 ° C., 15 seconds of annealing reaction and 72 ° C., 20 seconds of extension and ligation reaction.

  The prepared mutant was transformed into E. coli BL21 (DE3) and cultured overnight in LB + 50 μM ampicillin plate medium, then inoculated into 20 ml of 2 × YT medium and precultured at 37 ° C. for about 3 hours. This is inoculated in 1 L of 2 × YT medium, and main culture is performed at 37 ° C. When the absorbance is 0.4 to 0.6, IPTG with a final concentration of 1 mM is added, and the culture temperature is lowered to 25 ° C. It was cultured overnight. The cultured cells are collected by centrifugation, suspended in 20 mM Tris-HCl (pH 8.0), crushed using a French press, centrifuged at 20000 × G for 30 minutes, and containing Sirius-S147C-Q204C. I got Qing.

  The above supernatant was heated at 70 ° C. for 30 minutes to denature proteins from E. coli. Then, it centrifuged at 20000xG for 15 minutes, and removed the denatured protein. The supernatant after centrifugation was applied to a TOYOPEARL Butyl-650 column (manufactured by Tosoh Corporation), and the ammonium sulfate concentration in the eluate was continuously reduced between 20% and 0% to separate fractions. In the fractionated fraction, the less contaminated portion is dialyzed overnight in 20 mM Tris-HCl (pH 8.0) solution, and then concentrated using Amicon Ultra 10K (manufactured by Merck Millipore) to obtain Sirius-S147C-Q204C. I got The resulting protein was frozen in liquid nitrogen with glycerol added to a final concentration of 20% and stored. In addition, the DNA encoding the obtained protein is referred to as SEQ ID NO: 45.

Comparative Example 2: Preparation of Sirius-S147C-Q204C-C147_S148insA (SEQ ID NO: 3) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, and a combination of SEQ ID NOs: 18 and 19 Except using it, it carried out similarly to the comparative example 1, and produced. In addition, the DNA encoding the obtained protein is referred to as SEQ ID NO: 46.

Example 1: Preparation of Sirius-S147C-Q204C-C147_S148insD (SEQ ID NO: 4) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, and a combination of SEQ ID NOs: 20 and 21 Except using it, it carried out similarly to the comparative example 1, and produced. The DNA encoding the obtained protein is referred to as SEQ ID NO: 47.

Example 2: Preparation of Oba-Qs (Sirius-I146N-S147C-Q204C-C147_S148insD) (SEQ ID NO: 5) A combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NO: as primers for introducing mutations The same procedure as in Comparative Example 1 was repeated except that the combination of 20 and 21 and SEQ ID NO: 22 and 23 was used. However, after introducing a mutation corresponding to C147_S148insD using SEQ ID NOS: 20 and 21, a mutation corresponding to I146N was introduced using a combination of SEQ ID NOS: 22 and 23. The obtained DNA encoding the protein is referred to as SEQ ID NO: 48.

Example 3: Preparation of Sirius-S147C-Q204C-C147_S148insE (SEQ ID NO: 6) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, and a combination of SEQ ID NOs: 24 and 25 Except using it, it carried out similarly to the comparative example 1, and produced. The obtained DNA encoding the protein is referred to as SEQ ID NO: 49.

Example 4: Preparation of Sirius-I146N-S147C-Q204C-C147_S148insE (SEQ ID NO: 7) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOS: 16 and 17, SEQ ID NOs: 24 and 25, and The procedure of Comparative Example 1 was repeated except that the combination of SEQ ID NOS: 26 and 27 was used. However, after introducing a mutation corresponding to C147_S148insE using SEQ ID NO: 24 and 25, a mutation corresponding to I146N was introduced using a combination of SEQ ID NOs: 26 and 27. The DNA encoding the obtained protein is referred to as SEQ ID NO: 50.

Example 5 Preparation of Sirius-S147C-Q204C-C147_S148insL (SEQ ID NO: 8) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, and a combination of SEQ ID NOs: 28 and 29 Except using it, it carried out similarly to the comparative example 1, and produced. The obtained DNA encoding the protein is referred to as SEQ ID NO: 51.

Example 6: Preparation of Sirius-S147C-Q204C-C147_S148insR (SEQ ID NO: 9) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, and a combination of SEQ ID NOs: 30 and 31 Except using it, it carried out similarly to the comparative example 1, and produced. The DNA encoding the obtained protein is referred to as SEQ ID NO: 52.

Example 7 Preparation of Sirius-I146F-S147C-Q204C-C147_S148insR (SEQ ID NO: 10) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOS: 30 and 31, and Comparative Example 1 was prepared in the same manner as Comparative Example 1 except that the combination of SEQ ID NOS: 32 and 33 was used. However, after introducing a mutation corresponding to C147_S148insR using SEQ ID NOS: 30 and 31, a mutation corresponding to I146F was introduced using a combination of SEQ ID NOS: 32 and 33. The DNA encoding the obtained protein is referred to as SEQ ID NO: 53.

Example 8 Preparation of Sirius-I146N-S147C-Q204C-C147_S148insR (SEQ ID NO: 11) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 30 and 31, and Except using the combination of SEQ ID NO: 34 and 35, it was prepared in the same manner as Comparative Example 1. However, after introducing a mutation corresponding to C147_S148insR using SEQ ID NOS: 30 and 31, a mutation corresponding to I146N was introduced using a combination of SEQ ID NOS: 34 and 35. The DNA encoding the obtained protein is referred to as SEQ ID NO: 54.

Example 9 Preparation of Sirius-I146S-S147C-Q204C-C147_S148insR (SEQ ID NO: 12) As a primer for introducing a mutation, a combination of SEQ ID NOs: 14 and 15, SEQ ID NOS: 16 and 17, SEQ ID NOs: 30 and 31, and The procedure of Comparative Example 1 was repeated except that the combination of SEQ ID NOS: 36 and 37 was used. However, after introducing a mutation corresponding to C147_S148insR using SEQ ID NOS: 30 and 31, a mutation corresponding to I146S was introduced using a combination of SEQ ID NOS: 36 and 37. The obtained DNA encoding the protein is referred to as SEQ ID NO: 55.

Example 10 Preparation of roCFP (Sirius-Q65S-F66W-I146G-S147C-S148D-Q204C-C147_D148insE) (SEQ ID NO: 13) (SEQ ID NO: 13) as a primer for introducing a mutation, SEQ ID NO: 16 and SEQ ID NO: 16 and Comparative Example 1 was prepared in the same manner as Comparative Example 1 except that the combination of SEQ ID NO: 24 and SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, and SEQ ID NO: 42 and 43 was used. However, after a mutation corresponding to C147_S148insE is introduced using the combination of SEQ ID NO: 24 and 25 and a mutation corresponding to S148D is introduced using the combination of SEQ ID NO: 40 and 41, the combination of SEQ ID NO: 42 and 43 is The I146G corresponding mutation was introduced. The obtained DNA encoding the protein is referred to as SEQ ID NO: 56.

Example 11 Preparation of Oba-Qc (mTurquoise-I146G-S147C-Q204C-C147_D148insE) (SEQ ID NO: 62) A vector (pet 23a /) obtained by incorporating mTurquoise-encoding DNA (SEQ ID NO: 80) into pet23a (Novagen). Using mTurquoise) as a template, a combination of SEQ ID NOS: 73 and 74, and a combination of SEQ ID NOS: 78 and 79 were used as primers for introducing a mutation, and they were prepared in the same manner as Comparative Example 1.

Example 12 Preparation of Oba-Qb (EBFP2-N146L-S147C-Q204C-C147_H148insD) (SEQ ID NO: 63) A vector (pet 23a / Novagen) in which a DNA (SEQ ID NO: 81) encoding EBFP2 was incorporated into pet23a (Novagen). Comparative Example 1 was prepared in the same manner as Comparative Example 1 except that the combination of SEQ ID NOS: 66 and 67 and the combination of SEQ ID NOS: 68 and 69 were used as primers for introducing a mutation using EBFP2) as a template. The obtained Oba-Qb was used for Example 15 in the state which contains the sequence (LEHHHHHH) containing the histidine tag from pet23a in a C terminal. In addition, 11 amino acids at the C-terminal side of GFP and its variants are generally not involved in the fluorescence characteristics, and it is known that the fluorescence characteristics do not change even if other proteins are linked ahead of that. .

Example 13 Preparation of Re-Qc (mTurquoise-I146W-S147C-Q204C-C147_D148insG) (SEQ ID NO: 64) A vector (pet 23a /) obtained by incorporating mTurquoise-encoding DNA (SEQ ID NO: 80) into pet23a (Novagen). Using mTurquoise) as a template, a combination of SEQ ID NOS: 70 and 71, and a combination of SEQ ID NOS: 72 and 73 were used as primers for introducing a mutation, in the same manner as in Comparative Example 1. The obtained Re-Qc was used for Example 15 in the state which contains the sequence (LEHHHHHH) containing the histidine tag from pet23a in a C terminal.

Example 14 Preparation of Re-Qy (Venus-N146W-S147C-Q204C-C147_H148insR) (SEQ ID NO: 65) A vector (pet 23a / Novagen) in which DNA encoding Venus (SEQ ID NO: 82) was incorporated into pet23a (Novagen). The procedure of Comparative Example 1 was repeated except that the combination of SEQ ID NOS: 74 and 75 and the combination of SEQ ID NOS: 76 and 77 were used as primers for introducing a mutation using Venus) as a template. The obtained Re-Qy was used for Example 15 in the state which contains the sequence (LEHHHHHH) containing the histidine tag from pet23a in a C terminal.

Example 15 Measurement of Fluorescent Intensity of Fluorescent Protein A 1 μM solution of the fluorescent protein prepared in Examples 1 to 14 and Comparative Examples 1 and 2 is prepared, and a spectrofluorimeter FP-8500 (JASCO Corporation) is used. The fluorescence intensity of each fluorescent protein was measured. In the case of measuring the fluorescence intensity of the reduced form of the fluorescent protein, the fluorescent protein was kept at room temperature for 15 minutes in the presence of 1 mM dithiothreitol (DTT) prior to the measurement of the fluorescent intensity to make it in advance in the reduced form. In the case of measuring the fluorescence intensity of the oxidized form of the fluorescent protein, the fluorescent protein was kept at room temperature for 15 minutes in the presence of 50 μM copper chloride prior to the measurement of the fluorescence intensity to make the oxidized form in advance.
In the fluorescent proteins produced in Comparative Examples 1 and 2 and Examples 1 to 9, the sensitivity of the spectrofluorimeter is set to medium, the fluorescence wavelength is 425 nm, and the excitation and fluorescence bandwidths are both set to 5 nm. The excitation spectrum was measured in the range of 250 nm to 400 nm. The fluorescence intensity of each fluorescent protein at the maximum fluorescence wavelength (around 375 nm) was recorded.
The fluorescence intensity of the fluorescent protein produced in Example 10, 11 and 13 measured the excitation spectrum in the range of 250-460 nm in the fluorescence wavelength 480 nm, and recorded the fluorescence intensity of the fluorescent protein in maximum excitation wavelength (around 430 nm).
The fluorescence intensity of the fluorescent protein produced in Example 12 measured the excitation spectrum in the range of 250 to 500 nm at a fluorescence wavelength of 440 nm, and recorded the fluorescence intensity of the fluorescent protein at the maximum excitation wavelength (about 380 nm).
The fluorescence intensity of the fluorescent protein produced in Example 14 measured the excitation spectrum in the range of 250 to 518 nm at a fluorescence wavelength of 525 nm, and recorded the fluorescence intensity of the fluorescent protein at the maximum excitation wavelength (around 515 nm).
The fluorescence intensities of the oxidized and reduced forms of each fluorescent protein are shown in FIGS. 1A and B.
Further, the ratio of the fluorescence intensity of the reduced type and the oxidized type of each fluorescent protein to the other one of the fluorescence intensities of the higher one is shown in FIGS. 2A and 2B.

  In the case of Comparative Example 1 (Sirius-S147C-Q204C), there was no difference in the fluorescence intensity between the oxidized form and the reduced form. However, by inserting a specific amino acid (aspartic acid (D), glutamic acid (E), leucine (L) or arginine (R)) between the 147th and 148th amino acids of the fluorescent protein of Comparative Example 1, The fluorescence intensity of the oxidized form was lower than that of the reduced form (Examples 1, 3, 5 and 6). In particular, in the fluorescent protein of Example 1, the fluorescence intensity ratio of the oxidized form to the reduced form was about 40%. However, when alanine (A) was inserted between the 147th and 148th amino acids, there was no difference in fluorescence intensity between the reduced and oxidized forms (Comparative Example 2).

  The amino acids 146 of the fluorescent proteins of Examples 1, 3, 5 and 6 were further substituted with specific amino acids (asparagine (N), phenylalanine (F), serine (S)). As a result, the fluorescence intensity of the oxidized form was further reduced compared to the reduced form (Examples 2, 4 and 7 to 9). In particular, in the fluorescent protein of Example 2, the fluorescence intensity ratio of the oxidized form to the reduced form was 20% or less. Based on such a very remarkable quenching effect which is shown when the fluorescent protein of Example 2 is oxidized, the protein is also referred to herein as Oba-Qs (oxidation balanced sensed quenching derived from serius).

  The fluorescent protein (roCFP) produced in Example 10 is the fluorescent protein of Example 3 further mutated. Specifically, by introducing a mutation into an amino acid involved in fluorescence emission with reference to the structure of CFP, CFP-like fluorescence characteristics are exhibited (excitation maximum wavelength 435 nm, emission maximum wavelength 480 nm). This is largely different from the fluorescence characteristics (excitation maximum wavelength: about 375 nm, emission maximum wavelength: about 425 nm) of the fluorescent proteins of Examples 1 to 9 which are Sirius mutants. In addition, the feature of the fluorescent protein of the present invention in that the fluorescence intensity of the oxidized form is lower than that of the reduced form is not lost (FIGS. 1A and 2A). Therefore, if the fluorescent protein of Example 10 and any one of the fluorescent proteins of Examples 1 to 9 are used at the same time, it is possible to make an analysis with better differentialness than before.

  The fluorescent proteins produced in Example 11 and Example 12 are variants in which mTurquoise and EBFP2 are employed as parent proteins, respectively. The characteristics of the fluorescent protein of the present invention, that the fluorescence intensity of the oxidized form is lower compared to the reduced form, while retaining the fluorescence properties of mTurquoise and EBFP2, respectively, are not lost (FIGS. 1B and 2B). Based on the very pronounced quenching effect shown when the fluorescent proteins of Examples 11 and 12 are oxidized, the fluorescent proteins of Examples 11 and 12 herein will be referred to as Oba-Qc (oxidation balance sensed quenching protein derived from respectively). It is also called CFP) and Oba-Qb (oxidation balanced sensed quenching protein derived from BFP).

  The fluorescent proteins produced in Example 13 and Example 14 are variants in which mTurquoise and Venus are employed as parent proteins, respectively. The fluorescence properties of mTurquoise and Venus were retained, respectively. And, surprisingly, it became clear that the fluorescence intensity of the reduced form was remarkably reduced compared with the oxidized form, unlike the fluorescent proteins of Examples 1 to 12 (FIGS. 1B and 2B). Based on such a very remarkable quenching effect which is shown when the fluorescent proteins of Examples 13 and 14 are reduced, the fluorescent proteins of Examples 13 and 14 will be referred to herein as Re-Qc (reduction sensed quenching protein, respectively). Also known as derived from CFP and Re-Qy (reduction sensed quenching protein derived from YFP).

  FIG. 3A is an excitation spectrum and a fluorescence spectrum of each of the oxidized form and the reduced form of Oba-Qs (SEQ ID NO: 5). In addition, after conversion from the reduced form to the oxidized form, one converted again to the reduced form is shown as a rereduced form. The excitation spectrum was at a fluorescence wavelength of 425 nm and was measured in the range of 250 nm to 400 nm. The fluorescence spectrum was measured at an excitation light wavelength of 375 nm. As shown in FIG. 3A, the fluorescence intensity at the maximum excitation wavelength of the excitation spectrum of the fluorescent protein of the present invention is almost the same as the fluorescence intensity at the maximum fluorescence wavelength of the fluorescence spectrum. Therefore, the value of the fluorescence intensity at the maximum fluorescence wavelength (about 430 nm) obtained by the excitation spectrum measurement can be regarded as equivalent to the value of the fluorescence intensity at the maximum excitation wavelength of the fluorescence spectrum.

  FIG. 3B is an excitation spectrum and a fluorescence spectrum of Re-Qy (SEQ ID NO: 65) in an oxidized form and a reduced form, respectively. The excitation spectrum was at a fluorescence wavelength of 525 nm and was measured in the range of 250 nm to 518 nm. The fluorescence spectrum was measured with an excitation light wavelength of 510 nm. Unlike the case of Oba-Qs in FIG. 3A, the fluorescence intensity in the excitation spectrum and fluorescence spectrum of the reduced form of Re-Qy was significantly reduced compared to the fluorescence intensity in the excitation spectrum and fluorescence spectrum of the oxidized form.

Example 16 Preparation of Fusion Protein of Oba-Qs and AtGpx1 (Oba-Qs-AtGpx1) (SEQ ID NO: 57) A vector prepared in Example 2 in which the nucleotide sequence corresponding to Oba-Qs was inserted into pet 23a. The nucleotide sequence corresponding to glutathione peroxidase 1 (AtGpx1) of P. vulgaris was inserted into (referred to as oba-Qs / pet 23a). Furthermore, the base sequence corresponding to (GGSGG) ₆ which is a flexible linker was inserted between the base sequence corresponding to Oba-Q and the base sequence corresponding to AtGpx1. The obtained vector is called Oba-Qs-AtGpx1 / pet23a.

  After Oba-Qs-AtGpx1 / pet23a was transformed into E. coli BL21 (DE3) and cultured overnight in LB + 50 μM ampicillin plate medium, it was inoculated into 20 ml 2 × YT medium and precultured at 37 ° C. for about 3 hours . This is inoculated in 1 L of 2 × YT medium, and main culture is performed at 37 ° C. When the absorbance is 0.4 to 0.6, IPTG with a final concentration of 1 mM is added, and the culture temperature is lowered to 25 ° C. It was cultured overnight. The cultured cells are collected by centrifugation, suspended in 20 mM Tris-HCl (pH 8.0), crushed using a French press, centrifuged at 20000 × G for 30 minutes, and the supernatant containing Oba-Qs-AtGpx1. I got

  The obtained supernatant is applied to a Ni-NTA column (manufactured by QIAGEN) and washed with a washing buffer containing 20 mM imidazole-20 mM Tris-HCl (pH 8.0), 100 mM imidazole, 20 mM Tris-HCl (pH 8.0) Elution was carried out using. In the fractionated fraction, the less contaminated portion is dialyzed overnight in 20 mM Tris-HCl (pH 8.0) solution and then concentrated using Amicon Ultra 10K (manufactured by Merck Millipore) to obtain Oba-Qs-AtGpx1. I got The resulting fusion protein was frozen in liquid nitrogen with glycerol added to a final concentration of 20% and stored. The obtained DNA encoding the protein is referred to as SEQ ID NO: 58.

Example 17: Measurement of the time-dependent change of the fluorescence intensity of Oba-Qs and Oba-Qs-AtGpx1 by addition of H ₂ O ₂ 10 μM solution of Oba-Qs and Oba-Qs-AtGpx1 solution previously reduced with 500 μM DTT Each sample (all degassed) was diluted 100-fold with 50 mM tricine buffer (final concentration of protein 100 nM) was used as a sample. The fluorescence intensity of each sample was measured using a spectrofluorimeter FP-8500 (JASCO Corporation). The sensitivity of the spectrofluorimeter was set to medium, the excitation wavelength was 375 nm, the excitation / fluorescence bandwidth was set to 5 nm, and the fluorescence intensity at a fluorescence wavelength of 425 nm was measured every 10 seconds. One minute after the start of the measurement, H ₂ O ₂ having a predetermined concentration was added, and the temporal change of the fluorescence intensity was observed (FIGS. 4 and 5).

In the case of Oba-Qs alone (FIG. 4), the fluorescence intensity decreased by about 30% 100 seconds after the start of measurement (40 seconds after the addition of 10 mM H ₂ O ₂ ). On the other hand, in the case of Oba-Qs-AtGpx1 (FIG. 5), the fluorescence intensity decreased by as much as 60% 100 seconds after the start of the measurement (40 seconds after the addition of 10 mM H ₂ O ₂ ). By fusing the fluorescent protein of the present invention with peroxidase, the redox sensitivity could be further improved.

  With the fluorescent protein of the present invention, changes in redox state in cells can be conveniently monitored based on fluorescence intensity without measuring the excitation spectrum. In addition, by simultaneously using a plurality of the fluorescent proteins of the present invention having different fluorescence characteristics, it is possible to carry out analysis with better differentialness than before.

Claims (12)

Amino acid sequence selected from SEQ ID NO: 1,59, and 6 0 or Ranaru group, or 90% or more sequence identity to the amino acid sequence selected from SEQ ID NO: 1,59, and 6 0 or Ranaru group In the amino acid sequence, the 147th and 204th amino acid residues are substituted with cysteine (C), and between the 147th and 148th amino acid residues, glycine (G), aspartic acid (D), glutamic acid (E) an amino acid sequence in which any one amino acid selected from the group consisting of leucine (L) and arginine (R) is inserted , provided that the 66th amino acid residue is phenylalanine (F), tryptophan ( W) or a histidine (H), a fluorescent protein consisting of an amino acid sequence , wherein
When the fluorescence spectrum is measured with the excitation light wavelength fixed, the ratio of the other fluorescence intensity to the higher one of the fluorescence intensities at the oxidized and reduced fluorescence maximum wavelengths of the fluorescent protein is 80% or less.
Fluorescent protein .   In the amino acid sequence, the amino acid residue at position 146 is selected from the group consisting of leucine (L), tryptophan (W), asparagine (N), phenylalanine (F), serine (S) and glycine (G) The fluorescent protein according to claim 1 further substituted with one amino acid.   In the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, the 146th amino acid residue is substituted with asparagine (N) and the 147th and 204th amino acids The fluorescent protein according to claim 2, which consists of an amino acid sequence in which the residue is substituted with cysteine (C) and aspartic acid (D) is inserted between the 147th and 148th amino acid residues.   In the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, the 65th amino acid residue is substituted with serine (S) and the 66th amino acid residue is It is substituted with tryptophan (W), the 146th amino acid residue is substituted with glycine (G), the 147th and 204th amino acid residues are substituted with cysteine (C), and the 148th amino acid residue is aspartic acid. The fluorescent protein according to claim 2, consisting of an amino acid sequence substituted with (D) and in which glutamic acid (E) is inserted between the 147th and 148th amino acid residues.   In the amino acid sequence of SEQ ID NO: 59 or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59, the 146th amino acid residue is substituted with glycine (G), and the 147th and 204th amino acids The fluorescent protein according to claim 2, consisting of an amino acid sequence in which the residue is substituted by cysteine (C) and glutamic acid (E) is inserted between the 147th and 148th amino acid residues.   In the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 60, the 146th amino acid residue is substituted with leucine (L) and the 147th and 204th amino acids The fluorescent protein according to claim 2, which consists of an amino acid sequence in which the residue is substituted with cysteine (C) and aspartic acid (D) is inserted between the 147th and 148th amino acid residues.   In the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 59, the 146th amino acid residue is substituted with tryptophan (W), and the 147th and 204th amino acids The fluorescent protein according to claim 2, consisting of an amino acid sequence in which the residue is substituted by cysteine (C) and glycine (G) is inserted between the 147th and 148th amino acid residues. A fusion protein in which the fluorescent protein according to any one of claims 1 to 7 and a second protein are fused. A DNA encoding the fluorescent protein according to any one of claims 1 to 7 or the fusion protein according to claim 8 . A recombinant vector comprising the DNA according to claim 9 . A host cell transformed by the recombinant vector according to claim 10 . A method of monitoring the redox state in a cell, comprising
A fluorescent protein according to any one of claims 1 to 7 or a fusion protein according to claim 8 is present in a cell, the fluorescence of said fluorescent protein or fusion protein is detected, and the fluorescence of the detected fluorescence is detected. Monitoring the redox state of cells based on intensity.

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