WO2011096501A1 - Protéine fluorescente photosensibilisatrice - Google Patents

Protéine fluorescente photosensibilisatrice Download PDF

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Publication number
WO2011096501A1
WO2011096501A1 PCT/JP2011/052300 JP2011052300W WO2011096501A1 WO 2011096501 A1 WO2011096501 A1 WO 2011096501A1 JP 2011052300 W JP2011052300 W JP 2011052300W WO 2011096501 A1 WO2011096501 A1 WO 2011096501A1
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fluorescent protein
protein
photosensitized
seq
amino acid
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PCT/JP2011/052300
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Japanese (ja)
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健治 永井
知己 松田
里佳 高橋
研 竹本
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a photosensitized fluorescent protein.
  • the target protein can be efficiently destroyed in the chromophore-assisted photoinactivation method.
  • a so-called gene knockout method that destroys a gene encoding a target protein
  • an RNA interference method that binds to and degrades mRNA encoding the target protein
  • an antibody against the target protein A method of inactivating the function by acting is known. All of these methods are characterized in that they affect the target protein in all regions in the cell where the target protein is expressed. Therefore, it was impossible in principle to selectively inactivate only the target protein present at an arbitrary position in the cell.
  • chromophore-assisted photoinactivation is a method for functionally inactivating a functional site of a target protein by spatiotemporal inactivation by oxidation with active oxygen produced dependent on light irradiation.
  • assisted light inactivation hereinafter sometimes referred to as CALI
  • the chromophore-assisted photoinactivation method uses malachite green, rhodamine derivatives, or fluorescein derivatives as photosensitizers and binds to target molecules (for example, target proteins) to suppress the function of target molecules. This is a method for analyzing the function of the target molecule.
  • the chromophore-assisted photoinactivation method usually requires introduction of an antibody against a fluorescein-labeled target molecule or a fluorescein-labeled target molecule (for example, a target protein) into a cell by microinjection or the like, which is complicated. There was a problem.
  • KillerRed is a protein, it is possible to express a photosensitized fluorescent protein (CALI dye) in a cell by a method such as transformation or transfection by using a plasmid containing DNA encoding KillerRed. became.
  • KillerRed forms a dimer in the cell, many proteins can be fused with KillerRed, resulting in functional inactivation due to steric hindrance, etc., and light irradiation-dependent target protein functional failure. There was a problem that activation could not be performed. In addition, KillerRed has a problem that the amount of active oxygen produced is less than that of fluorescein because of its low photosensitizing activity. Furthermore, for example, when two or more target proteins in a biological sample are individually inactivated by light irradiation, photosensitized fluorescent proteins having different light absorption characteristics are necessary.
  • an object of the present invention is to provide a monomeric photosensitized fluorescent protein. Another object of the present invention is to provide a photosensitizing fluorescent protein having high photosensitizing activity. Furthermore, another object of the present invention is to provide a photosensitized fluorescent protein having an excitation wavelength and a fluorescence wavelength different from KillerRed.
  • the present inventor has developed a photosensitizing fluorescent protein having high photosensitizing activity capable of efficiently producing active oxygen by photostimulation in an individual organism, tissue, cell, intracellular organelle, or aqueous solution.
  • the fifth glycine (G) was changed to valine (V), the 147th asparagine (N) to serine (S), and the 162nd leucine.
  • (L) is threonine (T)
  • 164th phenylalanine (F) is threonine (T)
  • 174th leucine (L) is lysine (K)
  • M methionine
  • Supernova-Red which is a mutant substituted in (1)
  • Supernova-Orange which is a mutant in which the 68th tyrosine (Y) of Supernova-Red is replaced with tryptophan (W)
  • W tryptophan
  • V valine
  • A alanine
  • the present invention provides an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 9, and
  • the present invention relates to a photosensitized fluorescent protein comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 10, or a functional equivalent variant thereof.
  • the photosensitized fluorescent protein or functional equivalent variant thereof of the present invention constitutes a fusion protein bound to another protein.
  • the present invention also relates to DNA encoding the photosensitized fluorescent protein or a functionally equivalent variant thereof.
  • the base sequence represented by SEQ ID NO: 1 in the sequence listing, the base sequence represented by SEQ ID NO: 3, the base sequence represented by SEQ ID NO: 5, represented by SEQ ID NO: 19 A DNA comprising at least one base sequence selected from the group consisting of a base sequence and the base sequence represented by SEQ ID NO: 20.
  • the present invention relates to a plasmid containing the DNA.
  • the present invention provides a photosensitized active oxygen production method using the photosensitized fluorescent protein or a functional equivalent variant thereof, wherein the photosensitized fluorescent protein or a functional equivalent variant thereof is used. The method is characterized by irradiating excitation light.
  • the photosensitizing fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 2 or a functional equivalent variant thereof has a range of 490 to 620 nm.
  • the excitation light wavelength in the range of 400 to 550 nm is used.
  • the excitation light wavelength in the range of 390 to 440 nm is used, and the photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 9 or its In the functional equivalent variant, an excitation light wavelength in the range of 462 to 562 nm is used, and the amino acid sequence represented by SEQ ID NO: 10 is used.
  • an excitation light wavelength in the range of 459 ⁇ 559 nm is used, and the amino acid sequence represented by SEQ ID NO: 10 is used.
  • the present invention is a method for analyzing the function of a target molecule using a photosensitized fluorescent protein or a functional equivalent variant thereof, wherein excitation light is applied to the photosensitized fluorescent protein or a functional equivalent variant thereof. Irradiation relates to said method.
  • excitation light in the range of 490 to 620 nm.
  • the excitation light wavelength in the range of 400 to 550 nm is used, and the amino acid represented by SEQ ID NO: 6
  • an excitation light wavelength in the range of 390 to 440 nm is used, and the photosensitized fluorescent protein containing the amino acid sequence represented by SEQ ID NO: 9 or a functional thereof
  • the equivalent variant uses an excitation light wavelength in the range of 462 to 562 nm and includes the amino acid sequence represented by SEQ ID NO: 10
  • sensitizing fluorescent protein or a functional equivalent variant thereof using an excitation wavelength in the range of 459 ⁇ 559 nm.
  • the photosensitized fluorescent protein of the present invention can be present as a monomer in a cell and used as a photosensitized fluorescent protein in CALI for many proteins. It is possible.
  • the photosensitized fluorescent protein of the present invention and functionally equivalent variants thereof particularly Supernova-Orange, by replacing the 68th tyrosine of Supernova-Red with tryptophan (Y68W), the excitation maximum / fluorescence maximum is obtained. It shifts to 493 nm / 552 nm (visually orange), and the production of singlet oxygen is increased compared to KillerRed.
  • the photosensitized fluorescent protein of the present invention and functionally equivalent variants thereof, particularly Supernova-Green, by substituting valine No. 46 of Supernova-Orange with alanine, the excitation maximum / fluorescence maximum is 439 nm / 496 nm. It shifts to (green by visual observation), and the production amount of singlet oxygen is increased compared with KillerRed.
  • Non-Patent Document 2 Non-Patent Document 2 in which KillerRed's X-ray crystal structure analysis was performed, substitution of KillerRed's 46th valine with alanine (V46A) occurred due to the photosensitizing action of the chromophore. It describes the possibility of releasing active oxygen efficiently outside the KillerRed protein. However, as shown in Examples described later, when the V46A mutation was introduced into Supernova-Red, the amount of released active oxygen decreased.
  • FIG. 5 is a photomicrograph of localization of a fusion protein of Supernova-Red and tubulin, keratin, or connexin 43 (hereinafter sometimes referred to as Cx43) in HeLa cells.
  • a KillerRed fusion protein is shown as a control.
  • the respective photomicrographs are Supernova-Red-tubulin (a), Keratin-Supernova-Red (b), Cx43-Supernova-Red (c), KillerRed-tubulin (d), Keratin-KillerRed (e), (F). It is the microscope picture which showed cell degeneration by CALI with respect to actinin.
  • CALI was performed by irradiating a 561.5 nm laser beam into the blue circle of the protruding portion of HeLa cells expressing ⁇ -actinin-SNR.
  • Fluorescence image before irradiation (a), Fluorescence image immediately after irradiation (b), Differential interference image (c) before irradiation, Differential interference image (d) immediately after irradiation, Differential interference image (e) 3 minutes after irradiation, A differential interference image (f) 6 minutes after irradiation is shown.
  • the photosensitized fluorescent protein of the present invention comprises an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 9, And a protein comprising at least one amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 10.
  • the functional equivalent variant of the present invention is a functional equivalent variant of the photosensitized fluorescent protein.
  • “functionally equivalent variant” means that the amino acid sequence has one or more (particularly one or several) amino acids deleted, substituted, or added in the amino acid sequence of the original protein. It means a protein having an amino acid sequence and showing substantially the same activity as the original protein.
  • the number of amino acid deletions, substitutions or additions is, for example, 10, preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 2.
  • the first embodiment of the photosensitized fluorescent protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (hereinafter referred to as the first photosensitized fluorescent protein) or a functional equivalent variant thereof (hereinafter referred to as the first photosensitized fluorescent protein).
  • the first photosensitized fluorescent protein and the first functional equivalent modified protein may be collectively referred to as a first photosensitized fluorescent protein, etc.
  • a protein consisting of the amino acid sequence represented by No. 2 or a functionally equivalent variant thereof is preferred.
  • the fifth glycine (G) is replaced with valine (V) in the amino acid sequence of the photosensitizing fluorescent protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (ie, KillerRed)
  • SEQ ID NO: 8 ie, KillerRed
  • N147S serine
  • L 162nd leucine
  • T threonine
  • the 164th phenylalanine (F) may be replaced with threonine (T) (hereinafter may be referred to as F164T), and the 174th leucine (L) may be replaced with lysine (K).
  • L174K threonine
  • M206T threonine
  • a representative first photosensitized fluorescent protein is Supernova-Red, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
  • KillerRed is known to form a dimer, whereas Supernova-Red is in a sample (eg, in an individual organism, in a tissue, in a cell, in an intracellular organelle, or in an aqueous solution). ) In the monomer. Further, the excitation maximum wavelength and the fluorescence maximum wavelength of Supernova-Red are 579 nm and 610 nm, respectively.
  • the substitution of L162T and F164T (hereinafter, the KillerRed mutant having the substitution of L162T and F164T is referred to as KillerRed-L162T / F164T) as a dimer.
  • the existing KillerRed becomes a monomer. Therefore, when the KillerRed-L162T / F164T is expressed as a fusion protein with the target protein, it does not inhibit the function of the target protein.
  • substitution of L162T and F164T caused KillerRed-L162T / F164T to have reduced absorbance at 585 nm and fluorescence at 610 nm compared to KillerRed.
  • Supernova-Red having the same fluorescence intensity as KillerRed could be obtained by introducing further substitution of G5V, N147S, L174K, and M206T into the KillerRed-L162T / F164T.
  • the first functional equivalent variant of the present invention has substantially the same activity as Supernova-Red.
  • “Same activity as Supernova-Red” means that active oxygen is generated by light irradiation, fluorescence is generated by light irradiation, exists in a monomer in the sample, and the excitation maximum wavelength and fluorescence maximum wavelength are Meaning about 579 nm and 610 nm, respectively.
  • the excitation maximum wavelength and the fluorescence maximum wavelength are about 579 nm and 610 nm, respectively, preferably 569 to 589 nm and 600 to 620 nm, and more preferably 574 to 584 nm and 605 to 615 nm.
  • the first functional equivalent variant can use about 579 nm and 610 nm as the excitation wavelength and the fluorescence wavelength, respectively, which means that it has substantially the same function as Supernova-Red.
  • Examples of the first functional equivalent variant include a protein lacking the second glycine and the third serine in the amino acid sequence represented by SEQ ID NO: 2.
  • a second embodiment of the photosensitized fluorescent protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (hereinafter referred to as the second photosensitized fluorescent protein) or a functional equivalent variant thereof (hereinafter referred to as the second photosensitized fluorescent protein).
  • the second photosensitized fluorescent protein and the second functional equivalent modified protein may be collectively referred to as a second photosensitized fluorescent protein, etc.
  • It is preferably a protein consisting of the amino acid sequence represented by No. 4 or a functionally equivalent variant thereof.
  • the second photosensitizing fluorescent protein is a photosensitizing fluorescent protein comprising an amino acid sequence in which the 68th tyrosine (Y) is substituted with tryptophan (W) (hereinafter referred to as Y68W) in the Supernova-Red amino acid sequence. is there.
  • a representative second photosensitized fluorescent protein is Supernova-Orange, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 4.
  • the excitation maximum wavelength and the fluorescence maximum wavelength of Supernova-Orange are 493 nm and 552 m, respectively, and the production amount of singlet oxygen is increased as compared with KillerRed.
  • KillerRed which was a dimer, becomes a monomer by the substitution of L162T and F164T, and the excitation maximum wavelength and the fluorescence maximum wavelength are obtained by substitution of Y68W, respectively. These are approximately 493 nm and 552 nm.
  • the second functional equivalent variant of the present invention has substantially the same activity as Supernova-Orange. “The same activity as Supernova-Orange” means that active oxygen is generated by light irradiation, fluorescence is generated by light irradiation, exists in a monomer in the sample, and the excitation maximum wavelength and fluorescence maximum wavelength are Meaning about 493 nm and 552 nm, respectively.
  • the excitation maximum wavelength and the fluorescence maximum wavelength are about 493 nm and 552 nm, respectively, preferably 483 to 503 nm and 542 to 562 nm, and more preferably 488 to 498 nm and 547 to 557 nm. That is, the second functional equivalent variant can use about 493 nm and 552 nm as the excitation wavelength and the fluorescence wavelength, respectively, which means that it has substantially the same function as Supernova-Orange.
  • the second functional equivalent variant include a protein in which substitution of L162T, F164T, and Y68W is introduced in the amino acid sequence represented by SEQ ID NO: 8, and the second glycine and the second protein in the protein. Examples include a protein lacking the third serine and a protein lacking the second glycine and the third serine in the amino acid sequence represented by SEQ ID NO: 4.
  • a third embodiment of the photosensitized fluorescent protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 6 (hereinafter referred to as the third photosensitized fluorescent protein) or a functional equivalent variant thereof (hereinafter referred to as the third photosensitized fluorescent protein).
  • the third photosensitized fluorescent protein and the third functional equivalent modified protein may be collectively referred to as a third photosensitized fluorescent protein, etc.
  • a protein consisting of the amino acid sequence represented by No. 6 or a functionally equivalent variant thereof is preferable.
  • the third photosensitized fluorescent protein is a photosensitized fluorescent protein comprising an amino acid sequence in which the 46th valine (V) is replaced with alanine (A) (hereinafter referred to as V46A) in the Supernova-Orange amino acid sequence. is there.
  • a representative third photosensitizing fluorescent protein is Supernova-Green, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 6.
  • the excitation maximum wavelength and the fluorescence maximum wavelength of Supernova-Green are 439 nm and 496 nm, respectively, and the production amount of singlet oxygen is increased as compared with KillerRed.
  • KillerRed which was a dimer by substitution of L162T and F164T, becomes a monomer, and by exchanging Y68W and V46A, the excitation maximum wavelength and the fluorescence maximum wavelength are respectively 439 nm and 496 nm.
  • the third functional equivalent variant of the present invention has substantially the same activity as Supernova-Green. “Same activity as Supernova-Green” means that active oxygen is generated by light irradiation, fluorescence is generated by light irradiation, exists in a monomer in the sample, and the excitation maximum wavelength and fluorescence maximum wavelength are Meaning about 439 nm and 496 nm, respectively.
  • the excitation maximum wavelength and the fluorescence maximum wavelength are about 439 nm and 496 nm, respectively, preferably 429 to 449 nm and 486 to 506 nm, and more preferably 434 to 444 nm and 591 to 501 nm. That is, the third functional equivalent variant can use about 439 nm and 496 nm as the excitation wavelength and the fluorescence wavelength, respectively, which means that it has substantially the same function as Supernova-Green.
  • Examples of the third functional equivalent variant include, for example, a protein in which substitution of L162T, F164T, Y68W and V46A is introduced in the amino acid sequence represented by SEQ ID NO: 8, and the second glycine and Examples thereof include a protein lacking the third serine and a protein lacking the second glycine and the third serine in the amino acid sequence represented by SEQ ID NO: 6.
  • the fourth embodiment of the photosensitized fluorescent protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 9 (hereinafter referred to as the fourth photosensitized fluorescent protein) or a functional equivalent variant thereof (hereinafter referred to as the fourth photosensitized fluorescent protein).
  • the fourth photosensitized fluorescent protein and the fourth functional equivalent modified protein may be collectively referred to as a fourth photosensitized fluorescent protein, etc.
  • It is preferably a protein consisting of the amino acid sequence represented by No. 9 or a functionally equivalent variant thereof.
  • the fourth photosensitized fluorescent protein is a light containing an amino acid sequence having substitutions of L162T, F164T, and N147S in the amino acid sequence of the photosensitized fluorescent protein (that is, KillerRed) consisting of the amino acid sequence represented by SEQ ID NO: 8. It is a sensitizing fluorescent protein.
  • a representative fourth photosensitizing fluorescent protein is KillerRed_L162T_F164T_N147S, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 9.
  • the excitation maximum wavelength of KillerRed_L162T_F164T_N147S is 512 nm, and the fluorescence shows yellowish green (visually).
  • KillerRed which was a dimer by substitution of L162T and F164T, becomes a monomer, and by substitution of N147S, the excitation maximum wavelength is 512 nm and the fluorescence is yellow-green. Become.
  • the fourth functional equivalent variant of the present invention has substantially the same activity as KillerRed_L162T_F164T_N147S.
  • “The same activity as KillerRed_L162T_F164T_N147S” means that active oxygen is produced by light irradiation, fluorescence is generated by light irradiation, exists as a monomer in the sample, and the excitation maximum wavelength is about 512 nm and the fluorescence is yellow. Means green.
  • the excitation maximum wavelength of about 512 nm preferably means 502 to 522 nm, and more preferably 507 to 517 nm.
  • the fourth functional equivalent variant can use about 512 nm as the excitation wavelength, which means that it has substantially the same function as KillerRed_L162T_F164T_N147S.
  • Examples of the fourth functional equivalent variant include a protein lacking the second glycine and the third serine in the amino acid sequence represented by SEQ ID NO: 9.
  • the fifth embodiment of the photosensitized fluorescent protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 10 (hereinafter referred to as the fifth photosensitized fluorescent protein) or a functional equivalent variant thereof (hereinafter referred to as the fifth photosensitized fluorescent protein).
  • the fifth photosensitized fluorescent protein and the fifth functional equivalent modified protein may be collectively referred to as a fifth photosensitized fluorescent protein).
  • a protein consisting of the amino acid sequence represented by No. 10 or a functionally equivalent variant thereof is preferred.
  • the fifth photosensitizing fluorescent protein has a substitution of L162TF164T, G5V, N147S, L174K, M206T, and V46A in the amino acid sequence of the photosensitizing fluorescent protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (ie, KillerRed). It is a photosensitized fluorescent protein containing an amino acid sequence.
  • a representative fifth photosensitized fluorescent protein is Supernova-Red_V46A, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 10.
  • the excitation maximum wavelength and the fluorescence maximum wavelength of Supernova-Red_V46A are 509 nm and 594 nm, respectively.
  • KillerRed that was a dimer becomes a monomer by the substitution of L162T and F164T, and the excitation maximum wavelength is obtained by substitution of G5V, N147S, L174K, M206T, and V46A And the fluorescence maximum wavelength are 509 nm and 594 nm, respectively.
  • the fifth functional equivalent variant of the present invention has substantially the same activity as Supernova-Red_V46A.
  • “Same activity as Supernova-Red_V46A” means that active oxygen is generated by light irradiation, fluorescence is generated by light irradiation, exists in a monomer in the sample, and excitation maximum wavelength and fluorescence maximum wavelength are Meaning about 509 nm and 594 nm, respectively.
  • the excitation maximum wavelength and the fluorescence maximum wavelength are about 509 nm and 594 nm, respectively, preferably 499 to 519 nm and 584 to 604 nm, and more preferably 504 to 514 nm and 589 to 599 nm.
  • the fifth functional equivalent variant can use about 509 nm and 594 nm as the excitation wavelength and the fluorescence wavelength, respectively, which means that it has substantially the same function as Supernova-Red_V46A.
  • Examples of the fifth functional equivalent variant include a protein lacking the second glycine and the third serine in the amino acid sequence represented by SEQ ID NO: 10.
  • the photosensitized fluorescent protein and functionally equivalent variant of the present invention are activated oxygen (for example, singlet oxygen) upon irradiation with light. Can be produced. Therefore, by introducing the protein of the present invention into a cell and irradiating it with light having an appropriate excitation wavelength, singlet oxygen can be generated, the cell can be damaged, and the cell can be killed.
  • the protein of the present invention can also be used in a chromophore-assisted photoinactivation method.
  • the photosensitizing fluorescent protein of the present invention is bound to a target protein and active oxygen (for example, singlet oxygen) is produced by light irradiation, thereby suppressing the function of the target protein or the target By destroying the protein, the function of the target protein can be analyzed.
  • active oxygen for example, singlet oxygen
  • the photosensitized fluorescent protein and the functional equivalent variant according to the present invention can be obtained by various known methods.
  • the photosensitized fluorescent protein can be obtained by using a plasmid vector containing DNA encoding the photosensitized fluorescent protein KillerRed. It can be prepared by genetic engineering techniques. More specifically, it can be prepared by site-directed mutagenesis.
  • host cells such as E. coli can be transformed to obtain a transformant.
  • the obtained transformant (that is, a transformant having a plasmid containing DNA encoding the photosensitizing fluorescent protein or functionally equivalent variant according to the present invention) can be expressed as the photosensitizing fluorescent protein or the like.
  • It can be prepared by culturing under conditions and separating and purifying photosensitized fluorescent protein and the like from the culture by a method generally used for protein separation and purification.
  • the separation and purification method include a method of producing a photosensitized fluorescent protein with a His tag and using a nickel NTA column, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, and molecular sieve column using molecular sieve gel. Examples thereof include chromatography, affinity column chromatography using protein A-linked polysaccharide, dialysis, and lyophilization.
  • the photosensitized fluorescent protein or the like of the present invention can be a fusion protein bound to another protein.
  • Other proteins to be fused are not particularly limited as long as the activity of the photosensitized fluorescent protein or the like is not completely inhibited. For example, it binds to the target protein or target molecule in the chromophore-assisted photoinactivation method.
  • the binding protein to be made can be mentioned.
  • the target protein is a protein whose function is analyzed in the chromophore-assisted light inactivation method.
  • the target protein is a fusion protein with the photosensitized fluorescent protein or the like of the present invention
  • the active oxygen produced from the photosensitized fluorescent protein or the like by light irradiation can easily inactivate the target protein. This is because the target molecule present in a radius of about 10 to 50 cm is specifically inactivated by the produced active oxygen. Since the photosensitized fluorescent protein of the present invention is a monomer, it hardly interferes with the original localization or activity of the target protein in the cell.
  • the target protein and the photosensitized fluorescent protein of the present invention may be bound by a linker peptide.
  • a linker peptide By using a linker peptide, the function of the target protein may be prevented from being impaired, and inactivation of the target protein by active oxygen may be facilitated.
  • the amino acid sequence of the linker peptide is not particularly limited, and those commonly used can be used. For example, “G”, “GG”, “GGGS”, “GGS”, “GGGGS”, “ GGSGG "or” GGSGGGSGGS "can be mentioned.
  • the binding protein is a protein that binds the photosensitized fluorescent protein to the target molecule in the chromophore-assisted photoinactivation method.
  • the binding protein is not particularly limited as long as it is a protein that can bind to a target molecule.
  • an antibody and an antigen-binding fragment thereof eg, scFv, Fab, or gene product of the entire antibody variable region
  • Receptors eg.g, scFv, Fab, or gene product of the entire antibody variable region
  • the fusion protein can be prepared by a known genetic recombination technique. Specifically, DNA encoding a photosensitized fluorescent protein or the like and DNA encoding another protein are introduced into an expression plasmid according to a conventional method. The resulting plasmid can be expressed by introducing the resulting plasmid into a host cell by transformation or the like.
  • the DNA according to the present invention is not particularly limited as long as it encodes the photosensitizing fluorescent protein or the like of the present invention.
  • the base represented by SEQ ID NO: 1, 3, 5, 19, or 20 in the sequence listing A DNA comprising a sequence can be mentioned.
  • the DNA consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing encodes Supernova-Red consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
  • the DNA consisting of the base sequence represented by SEQ ID NO: 3 in the sequence listing encodes Supernova-Orange consisting of the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.
  • the DNA consisting of the base sequence represented by SEQ ID NO: 5 in the sequence listing encodes Supernova-Green consisting of the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing.
  • the DNA comprising the base sequence represented by SEQ ID NO: 19 in the sequence listing encodes KillerRed_L162T_F164T_N147S comprising the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing.
  • the DNA composed of the base sequence represented by SEQ ID NO: 20 in the sequence listing encodes Supernova-Red_V46A composed of the amino acid sequence represented by SEQ ID NO: 10 in the sequence listing.
  • a Kozak sequence (Kozak sequence) may be added before the start codon.
  • caccatgg can be used as the Kozak array. This is because the expression of eukaryotic proteins may be improved by adding a Kozak sequence.
  • the plasmid according to the present invention is not particularly limited as long as it contains the DNA according to the present invention.
  • the plasmid according to the present invention is not particularly limited as long as it contains the DNA according to the present invention.
  • a transformant can be obtained by transforming the obtained plasmid into a desired host cell.
  • the transformant is not particularly limited as long as it contains the plasmid according to the present invention.
  • Examples of the host cell include commonly used known microorganisms such as Escherichia coli or yeast (Saccharomyces cerevisiae), or known cultured cells such as animal cells (eg, CHO cells, HEK-293 cells, or COS). Cell) or insect cells (eg BmN4 cells).
  • Escherichia coli or yeast Sacharomyces cerevisiae
  • known cultured cells such as animal cells (eg, CHO cells, HEK-293 cells, or COS). Cell) or insect cells (eg BmN4 cells).
  • Examples of the known expression vector include pUC, pTV, pGEX, pKK, or pTrcHis for E. coli; pEMBLY or pYES2 for yeast; pcDNA3 or pMAMneo for CHO cells.
  • PcDNA3 for HEK-293 cells; pcDNA3 for COS cells; for BmN4 cells, vector with silkworm nuclear polyhedrosis virus (BmNPV) polyhedrin promoter (eg, pBK283) Can be mentioned.
  • the photosensitized fluorescent protein of the present invention is used.
  • active oxygen is produced from the photosensitized fluorescent protein or the like.
  • the photosensitizing active oxygen production method of the present invention for example, by introducing the photosensitizing fluorescent protein or the like into a cell and irradiating with excitation light, active oxygen is produced from the photosensitizing fluorescent protein or the like. And can kill the cells.
  • the wavelength of the excitation light is a wavelength capable of producing active oxygen from the photosensitized fluorescent protein of the present invention.
  • the excitation wavelength is preferably in the range of 490 to 620 nm, more preferably 490 to 600 nm, more preferably 490 to 580 nm, and still more preferably 540 to 580 nm. A range of 560 to 580 nm is most preferred.
  • the excitation wavelength is preferably in the range of 400 to 550 m, more preferably in the range of 420 to 550 nm, more preferably in the range of 440 to 540 nm, still more preferably 470 to 520 nm, A range of 485 to 505 nm is most preferred.
  • the excitation wavelength is preferably in the range of 390 to 480 nm, more preferably in the range of 390 to 460 nm, and most preferably in the range of 430 to 450 nm. Note that 390 to 440 nm can also be used.
  • the excitation wavelength is preferably in the range of 462 to 562 nm, more preferably in the range of 482 to 542 nm, and most preferably in the range of 502 to 522 nm.
  • the excitation wavelength is preferably in the range of 459 to 559 nm, more preferably in the range of 479 to 539 nm, and most preferably in the range of 499 to 519 nm.
  • the excitation wavelength to be used all wavelengths in the above range may be used, or a single wavelength in the above range may be used.
  • a 561.5 nm laser can be used as SuperNova-Red excitation light.
  • the excitation light may be light having a wavelength that is approximately an integral multiple of the excitation wavelength based on a multiphoton excitation phenomenon.
  • the photosensitizing active oxygen production method of the present invention it is possible to induce cell apoptosis.
  • the photosensitizing active oxygen production method of the present invention can be used for photodynamic therapy (PDT).
  • Photodynamic therapy is a method of treating a tumor in which a photosensitizing substance is taken into tumor cells and irradiated with light to kill the cells. More specifically, the photosensitizing fluorescent protein of the present invention is introduced into tumor cells or endothelial cells of new blood vessels in the tumor tissue, and active oxygen is generated by irradiating with appropriate light. This active oxygen damages tumor cells or tumor tissue, and the tumor disappears.
  • the function analysis method of the target molecule of the present invention uses the photosensitized fluorescent protein of the present invention.
  • active oxygen is produced from the photosensitized fluorescent protein or the like.
  • the produced active oxygen destroys the target molecule in the vicinity and can analyze the function of the target molecule destroyed thereby.
  • the wavelength of the excitation light is a wavelength capable of producing active oxygen from the photosensitized fluorescent protein of the present invention.
  • the excitation wavelength is preferably in the range of 490 to 620 nm, more preferably in the range of 490 to 600 nm, still more preferably in the range of 540 to 580 nm, and in the range of 560 to 580 nm. Most preferred.
  • the excitation wavelength is preferably in the range of 400 to 550 m, more preferably in the range of 420 to 550 nm, more preferably in the range of 440 to 540 nm, still more preferably 470 to 520 nm, A range of 485 to 505 nm is most preferred.
  • the excitation wavelength is preferably in the range of 390 to 480 nm, more preferably in the range of 390 to 460 nm, and most preferably in the range of 430 to 450 nm. Note that 390 to 440 nm can also be used.
  • the excitation wavelength is preferably in the range of 462 to 562 nm, more preferably in the range of 482 to 542 nm, and most preferably in the range of 502 to 522 nm.
  • the excitation wavelength is preferably in the range of 459 to 559 nm, more preferably in the range of 479 to 539 nm, and most preferably in the range of 499 to 519 nm.
  • the excitation wavelength to be used all wavelengths in the above range may be used, or a single wavelength in the above range may be used.
  • a 561.5 nm laser can be used as SuperNova-Red excitation light.
  • the excitation light may be light having a wavelength that is approximately an integral multiple of the excitation wavelength based on a multiphoton excitation phenomenon.
  • the target molecule functional analysis method of the present invention can be used as a chromophore-assisted photoinactivation method. That is, by introducing a fusion protein of the target protein and the photosensitized fluorescent protein of the present invention into the cell, the fusion protein moves to a position in the cell where the target protein is originally expressed. Then, singlet oxygen (active oxygen) is generated from the photosensitized fluorescent protein by irradiating the cells with light that excites the photosensitized fluorescent protein, and the function of the target protein is destroyed. When the function of the target protein is destroyed, a change appears in the cell, and the function of the target protein can be analyzed.
  • active oxygen active oxygen
  • the target molecule refers to a biomolecule that is a target for elucidating its physiological function, and is not particularly limited to, for example, protein, peptide, carbohydrate, lipid, DNA, RNA, sugar, signal Examples include transmitter substances.
  • a preferred target molecule in the method of the present invention is a protein, and examples thereof include an enzyme, a receptor protein, a ligand protein, a signal transduction protein, a transcription control protein, a skeletal protein, a cell adhesion protein, and a scaffold protein.
  • the active oxygen produced by the photosensitized fluorescent protein or the like is singlet oxygen.
  • the singlet oxygen is obtained by transferring energy to the ground state triplet oxygen existing in the vicinity of the chromophore by the transition of the photosensitized fluorescent protein chromophore in the excited state to the excited triplet state by intersystem crossing. appear.
  • the generated singlet oxygen can selectively inactivate a target molecule (for example, target protein) or a functional site thereof bound to a photosensitized fluorescent protein or the like with a radius of about 10 to 50 mm. In this way, the physiological function of the target substance can be analyzed by inactivating the target molecule itself or a specific site of the target molecule.
  • the function analysis method of the target molecule of the present invention can be used for in vitro and in vivo assays, and for target molecules inside and outside the cell.
  • a method for binding a photosensitized fluorescent protein or the like to a target molecule is not particularly limited, For example, a chemical bond is preferable.
  • a photosensitized fluorescent protein or the like can be covalently bound to a target molecule.
  • an antibody against a target molecule can be covalently bound to a photosensitized fluorescent protein or the like, and the photosensitized fluorescent protein or the like can be bound to the target molecule by the antibody.
  • a photosensitized fluorescent protein or the like and an antibody against the target molecule can be expressed as a fusion protein, and the photosensitized fluorescent protein or the like can be bound to the target molecule by the antibody.
  • the target molecule is a protein
  • a photosensitized fluorescent protein or the like and the target protein can be expressed as a fusion protein.
  • Example 1 an attempt was made to produce a photosensitized fluorescent protein that can exist as a monomer by introducing a mutation into the amino acid sequence of KillerRed.
  • the 5th glycine (G), the 147th asparagine (N), the 162nd leucine (L), the 164th phenylalanine (F), the 174th leucine (L) In order to mutate the 206th methionine (M) to valine (V), serine (S), threonine (T), threonine (T), lysine (K), threonine (T), respectively, the method of Sawano et al. According to (Sawano A and Miyawaki A. Nucleic Acid Research 28, E78, 2000), mutations were sequentially introduced by site-directed mutagenesis.
  • KillerRed / pRSETB obtained by cutting out with BamHI and EcoRI and inserting it into the plastic plasmid pRSET B (Inviologen) was used.
  • the mutant strand was synthesized as follows. 40 ⁇ L of a reaction solution having the following composition was prepared and synthesized in vitro by the PCR method. KillerRed / pRSET B 50 ng, Primer-L162T 10 pmol, dNTP 3.75 nmol, Pfu DNA polymerase 1.25U, Pfu DNA ligase 20U Pfu DNA polymerase is manufactured by STRATAGENE and dissolved in 10 ⁇ Pfu polymerase buffer. PfuDNA ligase is manufactured by STRATAGENE and dissolved in 10 ⁇ PfuDNA ligase buffer.
  • the thermal cycler was programmed as follows. That is, first, pre-incubation at 65 ° C.
  • the obtained PCR product was cleaved with DpnI according to the following reaction conditions, and a methylated or hemimethylated template plasmid DNA into which no mutation was introduced was selectively digested. Specifically, 1 ⁇ L (20 U) of DpnI (manufactured by New England BioLab) was added to 40 ⁇ L of the reaction solution after PCR and incubated at 37 ° C. for 2 hours. DpnI is an endonuclease that recognizes [5′-G m6 ATC-3 ′] and cleaves double-stranded DNA. Further, 20.4 ⁇ L of the reaction solution treated with DpnI was subjected to phenol chloroform extraction to purify the sample, and the supernatant was ethanol precipitated and dried.
  • E. coli competent cells JM109 (DE3) were transformed by the heat shock method using the single-stranded mutagenized circular DNA strand. Then, the transformed E. coli cells were cultured at 37 ° C. in an LB solid medium containing 100 ⁇ g / mL ampicillin, and the grown single colony was picked up and transferred to 2 mL of an LB culture solution containing 100 ⁇ g / mL ampicillin. The cells were grown overnight at 37 ° C and grown.
  • KillerRed_L162T_F164T / pRSET B protein was expressed using the T7 expression system (pRSET B / JM109 (DE3)). That is, E. coli competent cells (JM109 (DE3)) were transformed with KillerRed_L162T_F164T / pRSET B and cultured at 37 ° C. in an LB solid medium containing 100 ⁇ g / mL ampicillin. Thereafter, the grown single colony was picked up, inoculated into 300 mL of LB culture solution containing 100 ⁇ g / mL ampicillin, and cultured at 23 ° C. for 4 days.
  • JM109 E. coli competent cells
  • the obtained cells were disrupted with a French press, and the polyhistidine-labeled protein was purified from the resulting supernatant using a nickel chelate column (QIAGEN). Further, this eluate (100 mM imidazole, 50 mM Tris-Cl, pH 7.4, 300 mM NaCl) is used in a PD10 desalting / buffer exchange column (manufactured by GE healthcare Bio-Sciences) using 1 ⁇ PBS (pH 7.4). The protein KillerRed_L162T_F164T was obtained.
  • KillerRed_L162T_F164T Color development and confirmation of monomerization of KillerRed_L162T_F164T
  • the obtained KillerRed_L162T_F164T was analyzed by pseudo native PAGE.
  • the KillerRed_L162T_F164T band shifted to a lower molecular weight compared to the dimeric KillerRed band and was present as a monomer.
  • KillerRed_L162T_F164T attenuated fluorescence at 610 nm compared to KillerRed. The results are shown in Table 1.
  • Primer-G5V 5′-GGTTCAGAGGTCGGCCCCGCC-3 ′ (SEQ ID NO: 13)
  • Primer-N147S 5′-CAGACCGCCACCATCGGCTTC-3 ′
  • Primer-L174K 5′-GACGGCGGCAAGATGATGGGC-3 ′
  • Primer-M206T 5′-ACCAAGCAGACGAGGGACACT-3 ′ (SEQ ID NO: 16)
  • the fifth glycine (G) is replaced with valine (V) by the same procedure as in (1) except that KillerRed_L162T_F164T / pRSET B is used as a template plasmid DNA and Primer-G5V is used as a primer.
  • the plasmid KillerRed_L162T_F164T_G5V / pRSET B containing DNA encoding the protein substituted with the valine (V) of the fifth glycine (G) was obtained.
  • Comparative Examples 1 to 21 An attempt was made to introduce a mutation to make the dimer KillerRed a monomer. Specifically, in each comparative example, the following amino acid substitution was performed. Comparative Example 1: The 126th asparagine was replaced with arginine (N126R). Comparative Example 2: The 128th aspartic acid was replaced with threonine (D128T). Comparative Example 3: The 126th asparagine was substituted with arginine (N126R), and the 128th aspartic acid was substituted with threonine (N126R / D128T). Comparative Example 4 The 154th histidine was replaced with glutamic acid (H154E).
  • Comparative Example 5 The 165th isoleucine was replaced with arginine (I165R). Comparative Example 6: The 163rd alanine was replaced with lysine (A163K / I165R), and the 165th isoleucine was replaced with arginine (N126R / D128T). Comparative Example 7 The 176th methionine was replaced with aspartic acid (M176D). Comparative Example 8: The 196th proline was substituted with alanine (P196A). Comparative Example 9: The 198th phenylalanine was replaced with lysine (F198K). Comparative Example 10: The 140th glutamine was substituted with alanine (Q140A).
  • Comparative Example 11 The 26th lysine was replaced with glutamic acid (K26E).
  • Comparative Example 12 The 174th leucine was replaced with histidine (L174H).
  • Comparative Example 13 The 174th leucine was replaced with arginine (L174R).
  • Comparative Example 14 The 227th valine was replaced with lysine (V227K).
  • Comparative Example 15 The 230th isoleucine was replaced with lysine (I230K).
  • Comparative Example 16 The 234th isoleucine was replaced with lysine (I234K).
  • Comparative Example 17 The 152 th phenylalanine was replaced with threonine (F152T).
  • Comparative Example 18 The 162nd leucine was replaced with lysine (L162K). Comparative Example 19 The 164th phenylalanine was replaced with lysine (F164K). Comparative Example 20: The 167th phenylalanine was replaced with lysine (F167K). Comparative Example 21 The 167th phenylalanine was replaced with threonine (F167T).
  • primers corresponding to the respective substitutions were synthesized, and the respective mutations were obtained by repeating the operation of step (1) of Example 1 except that the respective primers were used. A plasmid containing the DNA encoding the protein was obtained.
  • Example 2 In this example, the 68th tyrosine (Y) of the protein Supernova-Red was substituted with tryptophan (W).
  • the following primers for mutagenesis were synthesized.
  • Primer-Y68W 5′-GCCACCTGATCCAGTGGGGCGAGCCC-3 ′ (SEQ ID NO: 17)
  • the procedure of Example 1 step 1) was repeated, and the plasmid Supernova-Orange / pRSET B was used.
  • the operation of the step (3) in Example 1 was repeated to obtain a protein Supernova-Orange (SEQ ID NO: 4).
  • Example 3 In this example, the 46th valine (V) of the protein Supernova-Orange was substituted with alanine (A).
  • the following primers for mutagenesis were synthesized.
  • Primer-V46A 5'-GGCGACTTCAACGCCCACGCCGTG-3 '(SEQ ID NO: 18) Except for using Supernova-Orange / pRSET B as the template plasmid DNA and using Primer-V46A as the primer, the procedure of Step (1) of Example 1 was repeated, and the plasmid Supernova-Green / pRSET B Got. Further, except that the plasmid Supernova-Green / pRSET B was used, the operation of the step (3) of Example 1 was repeated to obtain a protein Supernova-Green (SEQ ID NO: 6).
  • Example 4 substitution of N147S was further introduced into the protein KillerRed_L162T_F164T, and an attempt was made to produce KillerRed_L162T_F164T_N147S. Except for using KillerRed_L162T_F164T / pRSET B as a template plasmid DNA and using Primer-V46A as a primer, the procedure of Step (1) of Example 1 was repeated to obtain a plasmid KillerRed_L162T_F164T_V46A / pRSET B.
  • Example 5 In this example, the 46th valine (V) of the protein Supernova-Red was substituted with alanine (A). Except for using Supernova-Red / pRSET B as the template plasmid DNA and using Primer-V46A as the primer, the procedure of Step (1) of Example 1 was repeated, and the plasmid Supernova-Red_V46A / pRSET B Got. Further, except that the plasmid Supernova-Red_V46A / pRSET B was used, the operation of the step (3) of Example 1 was repeated to obtain a protein Supernova-Red_V46A (SEQ ID NO: 10).
  • Comparative Examples 22 to 27 An attempt was made to introduce a mutation for changing the color development of the Supernova-Red chromophore. Specifically, in each comparative example, the following amino acids were further substituted for L162T / F164T. Comparative Example 22: The 150th histidine was replaced with threonine (H150T). Comparative Example 23: The 148th glutamic acid was replaced with threonine (E148T). Comparative Example 24: The 148th glutamic acid was replaced with lysine (E148K). Comparative Example 25: The 148th glutamic acid was replaced with valine (E148V).
  • Comparative Example 26 The 226th serine was replaced with isoleucine (S226I).
  • Comparative Example 27 The 148th glutamic acid was replaced with valine (E148V), and the 226th serine was replaced with isoleucine (S226I).
  • a primer corresponding to each substitution was synthesized, and each of the mutations was obtained by repeating the operation of step (5) of Example 1 except that each primer was used.
  • a plasmid containing the DNA encoding the protein was obtained. Except for using the obtained plasmid, coloration and monomerization of each protein were confirmed by repeating the operations of steps (3) and (4) of the above-mentioned Examples.
  • the proteins obtained in Comparative Examples 22 to 27 showed no shift in color development from red, and no enhancement of color development. The results are summarized in Table 2.
  • Example 6 It was confirmed by measuring the excitation wavelength and fluorescence wavelength of the protein and comparing it with the excitation wavelength and fluorescence wavelength of an existing protein (KillerRed etc.) that the obtained protein was introduced with the desired mutation. .
  • the excitation wavelength of the obtained protein was measured and normalized, and the curve is shown in FIG.
  • the fluorescence wavelength of the obtained protein was measured and normalized, and the curve in FIG. FIGS. 1A and 1B show the excitation wavelength and fluorescence wavelength for KillerRed and Supernova-Red for comparison. As shown in FIGS.
  • the protein Supernova-Orange that has been mutated by the method described above has an excitation wavelength peak of 493 nm and a fluorescence wavelength peak of 552 nm, and Supernova-Green represents the excitation wavelength. It can be seen that the peak is 439 nm and the fluorescence wavelength peak is 469 nm. In contrast, the peak of the excitation wavelength of KillerRed is 585 nm, the peak of the fluorescence wavelength is 610 nm, the peak of the excitation wavelength of Supernova-Red is 579 nm, and the peak of the fluorescence wavelength is 610 nm.
  • ⁇ abs represents the peak of the absorbance spectrum in nanometer units. ( ⁇ ) is the molar extinction coefficient in units of 10 3 M ⁇ 1 cm ⁇ 1 .
  • ⁇ em represents the peak of the fluorescence spectrum in nanometer units.
  • KillerRed_L162T_F164T_N147S obtained in Example 4
  • Supernova-Red_V46A obtained in Example 5.
  • KillerRed_L162T_F164T_N147S had an excitation wavelength peak of 512 nm and fluorescence of yellow-green
  • Supernova-Red_V46A had an excitation wavelength peak of 509 nm and a fluorescence wavelength peak of 594 nm.
  • Example 7 the amount of active oxygen was measured for KillerRed, Supernova-Red, Supernova-Orange, and Supernova-Green.
  • 100 ⁇ L each of 50 ⁇ Supernova-Red, Supernova-Red, Supernova-Orange, and Supernova-Green protein solutions each containing 10 ⁇ M anthracene-9,10-dipropionic acid (hereinafter referred to as ADPA) was prepared.
  • ADPA is a singlet oxygen detection reagent having an excitation maximum of 380 nm and a fluorescence maximum of 430 nm.
  • Singlet oxygen is a kind of active oxygen and has a very strong oxidizing ability.
  • ADPA When ADPA reacts with singlet oxygen, ADPA changes to peroxide and the fluorescence peak at 430 nm is lost. .
  • the obtained sample solution was subjected to active oxygen measurement according to the following measurement conditions. That is, 15 ⁇ L is added to the Terrasaki dish, and the third harmonic (355 nm) of the YAG laser (LOTIS, LS-2137U-YAG, frequency 10 Hz, pulse width 15 ns) is optical parametric oscillator (LOTIS, LT-2214-OPO).
  • FIG. 2 shows a comparison of the amount of active oxygen of four fluorescent proteins including the fluorescent protein into which the mutation described above is introduced.
  • FIG. 2 shows the ADPA fading rate, that is, the amount of active oxygen released by KR various photosensitizing fluorescent proteins.
  • FITC fluorescein isothiocyanate
  • Example 8 In this example, Supernova-Red obtained in Example 1 was expressed as a fusion protein with tubulin, keratin, or Cx43. DNA encoding tubulin, keratin, or Cx43 obtained by PCR was inserted into the Supernova-Red / pRSET B. The resulting plasmids are referred to as tublin-Supernova-Red / pRSET B , keratin-Supernova-Red / pRSET B , and Cx43-Supernova-Red / pRSET B , respectively.
  • tublin-Supernova-Red / pRSET B keratin-Supernova-Red / pRSET B
  • Cx43-Supernova-Red / pRSET B were transfected into HeLa cells, and the intracellular localization was examined with a fluorescence microscope.
  • Tubulin, keratin, or Cx43 is localized in the microtubules, intermediate filaments, and gap junctions of cells, and is expressed at the position where each protein is present. There was no effect.
  • Example 9 apoptosis was induced by ligating the mitochondrial matrix translocation signal to Supernova-Red to introduce it into intracellular mitochondria and inactivating mitochondrial function by light irradiation.
  • 1 ⁇ g of plasmid_Supernova-Red / pcDNA3 was introduced into Hela cells cultured on 35 mm glass bottom dishes using transfection reagent (Superfect QIAGEN). After culturing for 2 days, light at 580 nm was irradiated at a power density of 8 W / cm 2 for 90 seconds, and then cells at 0, 3 and 6 hours were observed using a differential interference microscope. Cells in which Supernova-Red was expressed in mitochondria regressed and died after 6 hours.
  • Example 10 Supernova-Red was expressed as a fusion protein with tubulin, keratin, or connexin 43 using a linker peptide, and cell localization of each fusion protein was examined.
  • a vector for expression of a fusion protein with tubulin was prepared as follows. The EGFP gene between the NheI and XhoI sites of the vector pEGFP-tub (Clontech) for expressing a protein in which cytoskeletal protein ⁇ -tubulin is fused to the C-terminal side of EGFP in mammalian cells is transferred to SuperNova-Red.
  • Preparation of a vector for expression of a fusion protein with keratin was performed as follows. EcoRI of the vector pmTFP1-Keratin (Ai HW et al., Biochem J. 400, 531-540, 2006) for expressing a protein in which cytoskeletal protein keratin is fused to the N-terminal side of the fluorescent protein mTFP1 in mammalian cells.
  • the mTFP1 gene between NotI sites was replaced with the SuperNova-Red gene to prepare an expression vector for a keratin and SuperNova-Red fusion protein (keratin-SuperNova-Red).
  • a kozak sequence (ccaccatgg) was added before the start codon of the gene sequence of SuperNova-Red.
  • a fusion protein expression vector with connexin 43 was performed as follows.
  • Vector pmRFP1-Cx43 Campbell RE et al., Proc. Natl. Acad. Sci. USA 99, 7877-7882, for expressing a protein in which the gap junction protein connexin 43 is fused to the N-terminal side of the fluorescent protein mRFP1 in mammalian cells.
  • the mRFP1 gene between the AgeI and NotI sites was replaced with the SuperNova-Red gene to prepare a vector for expressing the connexin 43 and SuperNova-Red fusion protein (Cx43-SuperNova-Red).
  • a kozak sequence (ccaccatgg) was added before the start codon of the gene sequence of SuperNova-Red.
  • Each of the prepared expression vectors was transduced into HeLa cells cultured on a 35 mm glass bottom dish using Superfect (QIAGEN) and observed 24 to 48 hours later. Imaging was performed with a confocal inverted microscope A1 (Nikon) equipped with a PlanApo60x (NA1.4) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. A 561.5 nm laser was used as excitation light. Cells expressing the SuperNova-Red fusion protein were observed under a microscope.
  • SuperNova-Red-tubulin is expressed in microtubules of cells, keratin-SuperNova-Red is expressed in intermediate filaments, and Cx43-SuperNova-Red is expressed in gap junctions. Existed. (FIGS. 4a, 4b, and 4c).
  • Example 28 The procedure of Example 10 was repeated except that a gene encoding KillerRed was used in place of the gene encoding SuperNova-Red, and a KillerRed-tubulin fusion protein (KillerRed-tubulin), keratin And KillerRed fusion proteins (keratin-KillerRed) and connexin43 and KillerRed fusion proteins (Cx43-KillerRed) were obtained, and the localization of each fusion protein in the cells was confirmed. As shown in FIGS. 4d, 4e, and 4f, KillerRed-tubulin, keratin-KillerRed, and Cx43-KillerRed did not express tubulin, keratin, and connexin 43 at their original locations.
  • KillerRed-tubulin, keratin-KillerRed, and Cx43-KillerRed did not express tubulin, keratin, and connexin 43 at their original locations.
  • Example 11 CALI for actinin was performed on intracellular adhesion plaques to confirm degeneration of the cytoskeleton.
  • Vector ⁇ -actinin-EGFP / pcDNA3 for expressing a protein in which myofibrillar protein ⁇ -actinin is fused to the N-terminal side of EGFP in mammalian cells (Rajfur Z et al., Nat Cell Biol. 4, 286-293, 2002)
  • the EGFP gene was replaced with the SuperNova-Red gene, and a SuperNova-Red and ⁇ -actinin fusion protein ( ⁇ -actinin-SuperNova-Red) expression vector was prepared.
  • the produced expression vector was transduced into HeLa cells cultured on a 35 mm glass bottom dish using Superfect (QIAGEN) and observed 24 to 48 hours later. Imaging was performed with a confocal inverted microscope A1 (Nikon) equipped with a PlanApo 60x (NA1.4) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. A 561.5 nm laser (power 6.0%) was used as excitation light for SuperNova-Red fluorescence observation, and a 561.5 nm laser (power 100%) was used for CALI. Differential interference images were used for observation of cell morphology.
  • the projection region of cells expressing ⁇ -actinin-SuperNova-Red was irradiated with a 561.5 nm laser beam for 10 seconds to destroy ⁇ -actinin localized in the region.
  • the SuperNova-Red fluorescence in the region disappeared, and after 3 minutes of irradiation, cell degeneration accompanied by the collapse of the actinin bridge was observed (FIG. 5).
  • the photosensitized fluorescent protein of the present invention or a functional equivalent variant thereof can be used for the chromophore-assisted photoinactivation method and can be used for functional analysis of the target molecule.
  • this invention was demonstrated along the specific aspect, the deformation

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Abstract

L'invention porte sur une protéine fluorescente photosensibilisatrice, sur une protéine fluorescente photosensibilisatrice monomère ayant une activité élevée de photosensibilisation ; et sur une protéine fluorescente photosensibilisatrice qui est différente du Killer Red, et présente une longueur d'onde d'excitation et une longueur d'onde de fluorescence. Elle porte plus précisément sur une protéine fluorescente photosensibilisatrice qui contient au moins une séquence d'acides aminés choisie dans le groupe consistant en la séquence d'acides aminés représentée par SEQ ID NO : 2, la séquence d'acides aminés représentée par SEQ ID NO : 4, la séquence d'acides aminés représentée par SEQ ID NO : 9, la séquence d'acides aminés représentée par SEQ ID NO : 6 et la séquence d'acides aminés représentée par SEQ ID NO : 10, chacune étant présentée dans la liste des séquences, ou sur une modification, fonctionnellement équivalente, de la protéine fluorescente photosensibilisatrice. La protéine fluorescente photosensibilisatrice peut être utilisée pour une inactivation à la lumière assistée par un chromophore.
PCT/JP2011/052300 2010-02-03 2011-02-03 Protéine fluorescente photosensibilisatrice WO2011096501A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016537008A (ja) * 2013-09-06 2016-12-01 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｃａｓ９バリアントおよびその使用
WO2017155101A1 (fr) * 2016-03-10 2017-09-14 国立大学法人大阪大学 Protéine fluorescente

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2599443C2 (ru) * 2013-07-29 2016-10-10 Федеральное государственное бюджетное образовательное учреждение высшего образования "Нижегородская государственная медицинская академия" Министерства здравоохранения Российской Федерации (ФГБОУ ВО НижГМА Минздрава России) Модифицированный генетически кодируемый фотосенсибилизатор

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006117694A2 (fr) * 2005-02-08 2006-11-09 Evrogen Sensibilisateurs genetiquement codes et procedes d'utilisation associes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006117694A2 (fr) * 2005-02-08 2006-11-09 Evrogen Sensibilisateurs genetiquement codes et procedes d'utilisation associes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BULINA ME. ET AL.: "A genetically encoded photosensitizer", NAT. BIOTECHNOL., vol. 24, pages 95 - 99 *
CARPENTIER P. ET AL.: "Structural basis for the phototoxicity of the fluorescent protein KillerRed", FEBS LETT., vol. 583, no. 17, 2009, pages 2839 - 2842 *
PLETNEV S. ET AL.: "Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed", J. BIOL. CHEM., vol. 284, no. 46, 2009, pages 32028 - 32039 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016537008A (ja) * 2013-09-06 2016-12-01 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｃａｓ９バリアントおよびその使用
WO2017155101A1 (fr) * 2016-03-10 2017-09-14 国立大学法人大阪大学 Protéine fluorescente
CN108834418A (zh) * 2016-03-10 2018-11-16 国立大学法人大阪大学 荧光蛋白质
US10899804B2 (en) 2016-03-10 2021-01-26 Osaka University Fluorescent protein

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