WO2011096501A1 - Photosensitizing fluorescent protein - Google Patents

Abstract

Disclosed are a monomeric photosensitizing fluorescent protein; a photosensitizing fluorescent protein having high photosensitizing activity; and a photosensitizing fluorescent protein which is different from Killer Red and has an excitation wavelength and a fluorescence wavelength. Specifically disclosed is a photosensitizing fluorescent protein which contains at least one amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 9, and the amino acid sequence represented by SEQ ID NO: 10, each shown in the Sequence Listing, or a functionally equivalent modification of the photosensitizing fluorescent protein. The photosensitizing fluorescent protein can be used for chromophore-assisted light inactivation.

Description

Photosensitized fluorescent protein

The present invention relates to a photosensitized fluorescent protein. According to the present invention, the target protein can be efficiently destroyed in the chromophore-assisted photoinactivation method.

As a method for examining the function of an arbitrary protein in a biological sample, 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, or 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.

On the other hand, chromophore-assisted photoinactivation (chromophore-inactivation) 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) is known. Specifically, 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. For example, when a fluorescein derivative is used, it is known that singlet oxygen (active oxygen) generated from the fluorescein derivative destroys the function of the target protein.
However, 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.

Recently, Lukyanov et al. Reported “KillerRed”, which is a photosensitizing fluorescent protein (CALI dye) that can be used in the chromophore-assisted photoinactivation method (Patent Document 1 and Non-Patent Document 1). Since 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.

International Publication No. 2006/117694

However, because 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. However, no photosensitized fluorescent protein having an absorption spectrum other than KillerRed has been reported, and development of a photosensitized fluorescent protein having different excitation wavelength and fluorescence wavelength has been desired.
Accordingly, 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. Surprisingly, as a result of intensive research on the amino acid sequence of killer red, 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), and 206th methionine (M) is threonine (T). It was found that Supernova-Red, which is a mutant substituted in (1), is present in the cells as a monomer. Furthermore, Supernova-Orange, which is a mutant in which the 68th tyrosine (Y) of Supernova-Red is replaced with tryptophan (W), increases the production amount of singlet oxygen, and has an excitation maximum wavelength and a fluorescence maximum wavelength. It was found to shift to 493 nm and 552 nm, respectively.
Furthermore, Supernova-Green, which is a mutant in which valine (V) of Supernova-Orange No. 46 is substituted with alanine (A), has an increased singlet oxygen production compared to KillerRed, and has an excitation maximum wavelength and fluorescence. It has been found that the maximum wavelength shifts to 439 nm and 496 nm, respectively.
The present invention is based on these findings.

Accordingly, 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.
In a preferred embodiment of the photosensitized fluorescent protein or functional equivalent variant thereof of the present invention, the photosensitized fluorescent protein or functional equivalent variant thereof 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.
In a preferred embodiment of the DNA of the present invention, 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.
Furthermore, the present invention relates to a plasmid containing the DNA.
Furthermore, 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.
In a preferred embodiment of the photosensitizing active oxygen production method of the present invention, 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. In the photosensitized fluorescent protein containing the amino acid sequence represented by SEQ ID NO: 4 or the functional equivalent variant thereof using the excitation light wavelength, the excitation light wavelength in the range of 400 to 550 nm is used. In the photosensitized fluorescent protein comprising the amino acid sequence or a functionally equivalent variant thereof, 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. In Muhikarizo sensitive fluorescent protein or a functional equivalent variant thereof, using an excitation wavelength in the range of 459 ~ 559 nm.
Furthermore, 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.
In a preferred embodiment of the target molecule functional analysis method of the present invention, in the photosensitized fluorescent protein containing the amino acid sequence represented by SEQ ID NO: 2 or a functional equivalent variant thereof, excitation light in the range of 490 to 620 nm. In the photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 4 or a functional equivalent variant thereof using the wavelength, the excitation light wavelength in the range of 400 to 550 nm is used, and the amino acid represented by SEQ ID NO: 6 In a photosensitized fluorescent protein containing a sequence or a functional equivalent variant thereof, 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 In sensitizing fluorescent protein or a functional equivalent variant thereof, using an excitation wavelength in the range of 459 ~ 559 nm.

According to the photosensitized fluorescent protein of the present invention or a functionally equivalent variant thereof, it can be present as a monomer in a cell and used as a photosensitized fluorescent protein in CALI for many proteins. It is possible.
According to 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. Therefore, it can be used as an enhanced photosensitized fluorescent protein in CALI, and by using it simultaneously with KillerRed, Supernova-Red, Supernova-Green, etc., the functional correlation of two or more target proteins is analyzed. It is possible.
According to 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. Therefore, it can be used as an enhanced photosensitized fluorescent protein in CALI, and by using it together with KillerRed, Supernova-Red, and Supernova-Orange, etc., the functional correlation of two or more target proteins can be analyzed. Is possible.
According to a paper (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. On the other hand, in Supernova-Green in which the V46A mutation was introduced into Supernova-Orange into which the Y68W mutation was introduced, the amount of active oxygen released increased more than twice, and the fluorescence wavelength changed to green. Therefore, even in the case of those skilled in the art, it is anticipated that the release of active oxygen will increase or decrease due to the introduction of mutations in the photosensitized fluorescent protein, and that the fluorescence wavelength will change. It is not easy and requires trial and error.

It is the graph which showed the excitation spectrum (A) and fluorescence spectrum (B) of KillerRed, Supernova-Red, Supernova-Orange, and Supernova-Green. It is the graph which showed the production amount of active oxygen of KillerRed, Supernova-Red, Supernova-Orange, Supernova-Green, and Supernova-Red-V46A. FITC (fluorescein isothiocyanate) represents a positive control and Buffer (1 × PBS) represents a negative control. It is the photograph which showed the differential interference image of the cell after introduce | transducing Supernova-Red into a Hela cell, performing light irradiation, and after 0 hour, 3 hours, and 6 hours. Cells in which Supernova-Red is expressed in mitochondria have died due to apoptosis after 6 hours. 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 α-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. In the present specification, “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). Hereinafter, 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.
In the first photosensitizing fluorescent protein, 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) ( The 147th asparagine (N) is replaced with serine (S) (hereinafter sometimes referred to as N147S), and the 162nd leucine (L) is replaced with threonine (T) (hereinafter sometimes referred to as G5V). Hereinafter, 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). Hereinafter, it may be referred to as L174K), and the 206th methionine (M) is replaced with threonine (T) (hereinafter referred to as M206T). It is photosensitizing fluorescent protein comprising lies) amino acid sequence referred to. 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.

In the first photosensitized fluorescent protein of the present invention, 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. On the other hand, substitution of L162T and F164T caused KillerRed-L162T / F164T to have reduced absorbance at 585 nm and fluorescence at 610 nm compared to KillerRed.
However, 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. That is, 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). Hereinafter, 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.

In the second photosensitized fluorescent protein or the like of the present invention, 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. Examples of 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). Hereinafter, 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.

In the third photosensitized fluorescent protein or the like of the present invention, 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). Hereinafter, 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).

In the fourth photosensitized fluorescent protein of the present invention, 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. That is, 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). Hereinafter, 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.

In the fifth photosensitized fluorescent protein and the like of the present invention, 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. That is, 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 (hereinafter sometimes collectively referred to as the photosensitized fluorescent protein 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. Specifically, 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.

The photosensitized fluorescent protein and the functional equivalent variant according to the present invention can be obtained by various known methods. For example, 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.
Furthermore, using the obtained plasmid, 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. Examples of 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. When 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. As shown in Examples below, when Supernova-Red and a target protein (for example, tubulin, keratin, or Cx43) were expressed as a fusion protein, the localization of the target protein was not affected. .
Moreover, when preparing the fusion protein used for the chromophore assisted photoinactivation method, the target protein and the photosensitized fluorescent protein of the present invention may be bound by 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. For example, an antibody and an antigen-binding fragment thereof (eg, scFv, Fab, or gene product of the entire antibody variable region) ), Receptors, or ligands.

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. For example, 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. Further, 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. Furthermore, 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.
In addition, when the DNA of the present invention is expressed using, for example, the above-described expression plasmid, a Kozak sequence (Kozak sequence) may be added before the start codon. For example, 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. For example, by inserting the DNA according to the present invention into a known expression vector appropriately selected according to the host cell to be used. Mention may be made of the resulting plasmid.
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).

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.

In the photosensitized active oxygen production method of the present invention, the photosensitized fluorescent protein of the present invention is used. By irradiating the photosensitized fluorescent protein or the like with excitation light, active oxygen is produced from the photosensitized fluorescent protein or the like.
According to 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. In the first photosensitizing 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. In the second photosensitized fluorescent protein and the like of the present invention, 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. In the third photosensitizing fluorescent protein of the present invention, 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. In the fourth photosensitized fluorescent protein of the present invention, 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. In the fifth photosensitized fluorescent protein of the present invention, 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.
In the active oxygen production method of the present invention, as 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. For example, as shown in an example described later, a 561.5 nm laser can be used as SuperNova-Red excitation light.
In addition to the light having the excitation wavelength, 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.

According to the photosensitizing active oxygen production method of the present invention, it is possible to induce cell apoptosis. By inducing 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. By irradiating the photosensitized fluorescent protein or the like with excitation light, 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. In the first 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. In the second photosensitized fluorescent protein and the like of the present invention, 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. In the third photosensitizing fluorescent protein of the present invention, 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. In the fourth photosensitized fluorescent protein of the present invention, 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. In the fifth photosensitized fluorescent protein of the present invention, 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.
In the active oxygen production method of the present invention, as 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. For example, as shown in an example described later, a 561.5 nm laser can be used as SuperNova-Red excitation light.
In addition to the light having the excitation wavelength, 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.
More specifically, 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.

In the method of the present invention, 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. In particular, 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. For example, by such inactivation, identification of the functional site of the protein, confirmation of the function of the functional site, confirmation of the function of the ligand, confirmation of the effect of the functional site on the life of the protein, protein of the functional site Confirmation of effects on kinetics, confirmation of effects of functional sites on protein folding, and the like.
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.

In the method for analyzing the function of a target molecule of the present invention, a method for binding a photosensitized fluorescent protein or the like to a target molecule (eg, protein, carbohydrate, lipid, DNA, RNA, sugar, signal transmitter) 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. Further, 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. Furthermore, 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. Furthermore, when the target molecule is a protein, a photosensitized fluorescent protein or the like and the target protein can be expressed as a fusion protein.

Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
In this example, 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.
Specifically, in 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.

(1) Replacement of 162nd leucine (L) with threonine (T) In order to replace the 162nd leucine (L) with threonine (T), the following primers for mutagenesis were synthesized.
Primer-L162T: 5′-GTGCGCCAGACCGCCACCATC-3 ′ (SEQ ID NO: 11)
The end of Primer-L162T was phosphorylated by T4 polynucleotide kinase. As a template plasmid DNA for introducing the mutation, BamHI and EcoRI are added upstream and downstream of the KillerRed gene, respectively, by polymerase chain reaction using a plasmid vector containing the KillerRed gene as a template, and then the restriction enzyme is amplified. 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 μL 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. for 5 minutes was performed to repair the nick of the template DNA by Pfu DNA ligase, and then the first denaturation at 94 ° C. for 1 minute was performed. Subsequently, the DNA denaturation at 94 ° C. for 10 seconds, the annealing reaction at 55 ° C. for 30 seconds, and the extension / ligation reaction at 65 ° C. for 10 minutes were performed as 35 cycles for 35 cycles. Finally, post-incubation at 75 ° C. for 10 minutes was performed.

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 μL (20 U) of DpnI (manufactured by New England BioLab) was added to 40 μL 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 μL 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.

Next, transformation and expression were performed using the obtained DNA. 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 μg / mL ampicillin, and the grown single colony was picked up and transferred to 2 mL of an LB culture solution containing 100 μg / mL ampicillin. The cells were grown overnight at 37 ° C and grown.
Substitution of the 162nd leucine (L) to threonine (T) was performed by sequencing the nucleotide sequence according to a conventional method, and the mutation-introduced plasmid was named KillerRed_L162T / pRSET _B.

(2) Replacement of 164th phenylalanine (F) with threonine (T) To replace 164th phenylalanine (F) of KillerRed_L162T / pRSET _B with threonine (T), the following primers for mutagenesis were synthesized. .
Primer-F164T: 5′-CAGACCGCCACCATCGGCTTC-3 ′ (SEQ ID NO: 12)
The substitution of 164th phenylalanine (F) with threonine (T) is carried out as described in (1) above, except that KillerRed_L162T / pRSET _B is used as the template plasmid DNA and Primer-F164T is used as the primer. The plasmid KillerRed_L162T_F164T / pRSET _B containing DNA encoding a protein in which the 164th phenylalanine (F) was replaced with threonine (T) was obtained.

(3) Expression of KillerRed_L162T_F164T The resulting plasmid 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 μg / mL ampicillin. Thereafter, the grown single colony was picked up, inoculated into 300 mL of LB culture solution containing 100 μg / mL ampicillin, and cultured at 23 ° C. for 4 days. 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.

(4) 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.
However, KillerRed_L162T_F164T attenuated fluorescence at 610 nm compared to KillerRed. The results are shown in Table 1.

(5) Introduction of substitution of G5V, N147S, L174K, and M206T In order to introduce substitution of G5V, N147S, L174K, and M206T into KillerRed_L162T / F164T, the following primers were synthesized.
Primer-G5V: 5′-GGTTCAGAGGTCGGCCCCGCC-3 ′ (SEQ ID NO: 13)
Primer-N147S: 5′-CAGACCGCCACCATCGGCTTC-3 ′ (SEQ ID NO: 14)
Primer-L174K: 5′-GACGGCGGCAAGATGATGGGC-3 ′ (SEQ ID NO: 15)
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.
Thereafter, mutations were sequentially introduced in the same manner, and in the amino acid sequence of KillerRed, the 147th asparagine (N) was changed to serine (S), the 174th leucine (L) was changed to lysine (K), and the 206th position. Plasmid Supernova-Red / pRSET _B containing a DNA encoding a protein obtained by substituting methionine (M) with threonine (T) was obtained.

(6) Expression of Supernova-Red For Supernova-Red protein, the purified protein Supernova-Red (SEQ ID NO: 2) was repeated except that Supernova-Red / pRSET _B was used as a plasmid, except that Supernova-Red / pRSET _B was used. Got.

<< Comparative Examples 1 to 21 >>
In 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).
In each of the comparative examples, 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.
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. In Comparative Examples 1 to 21, the obtained protein remained a dimer and was not monomerized. The results are summarized in Table 1.

Example 2
In this example, the 68th tyrosine (Y) of the protein Supernova-Red was substituted with tryptophan (W).
In order to replace the 68th tyrosine (Y) with tryptophan (W), the following primers for mutagenesis were synthesized.
Primer-Y68W: 5′-GCCACCTGATCCAGTGGGGCGAGCCC-3 ′ (SEQ ID NO: 17)
Except for using Supernova-Red / pRSET _B as the template plasmid DNA and using Primer-Y68W as the primer, the procedure of Example 1 (step 1) was repeated, and the plasmid Supernova-Orange / pRSET _{B was used.} Got.
Further, except that 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).
In order to replace the 46th valine (V) 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
In this example, 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.
Further, except for using the plasmid KillerRed_L162T_F164T_V46A / pRSET _B , the operation of the step (3) of Example 1 was repeated to obtain a protein KillerRed_L162T_F164T_N147S (SEQ ID NO: 9).

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 >>
In 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).
In each of the comparative examples, 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. Moreover, 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. 1 (A) and 1 (B), 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.
In Table 3, λabs represents the peak of the absorbance spectrum in nanometer units. (Ε) is the molar extinction coefficient in units of 10 ³ M ⁻¹ cm ⁻¹ . λem represents the peak of the fluorescence spectrum in nanometer units.

Furthermore, the excitation wavelength and the fluorescence wavelength were measured for KillerRed_L162T_F164T_N147S obtained in Example 4 and Supernova-Red_V46A obtained in Example 5. KillerRed_L162T_F164T_N147S had an excitation wavelength peak of 512 nm and fluorescence of yellow-green, and Supernova-Red_V46A had an excitation wavelength peak of 509 nm and a fluorescence wavelength peak of 594 nm.

Example 7
In addition, the amount of active oxygen was measured for KillerRed, Supernova-Red, Supernova-Orange, and Supernova-Green. Here, 100 μL each of 50 μ Supernova-Red, Supernova-Red, Supernova-Orange, and Supernova-Green protein solutions each containing 10 μM 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. 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 μL 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). ), And after irradiation for 5 minutes at an energy density of 20 mJ / cm ² / pulse, this was diluted with 1 mL of 1 × PBS, and ADPA was used with a spectrofluorometer F-2500 (Hitachi). The fading rate was measured. Laser wavelengths are 580 nm for KillerRed and Supernova-Red, 450 nm for Supernova-Orange, and 440 nm for Supernova-Green, respectively. 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. In FIG. 2, for the purpose of comparison, the amount of active oxygen of fluorescein isothiocyanate (FITC) was normalized.

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.
The resulting plasmids tublin-Supernova-Red / pRSET _B , keratin-Supernova-Red / pRSET _B , and 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
In this example, 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 μg 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
In this example, 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 α-tubulin is fused to the C-terminal side of EGFP in mammalian cells is transferred to SuperNova-Red. Substitution was performed, and an expression vector of a SuperNova-Red-tubulin fusion protein (SuperNova-Red-tubulin) was prepared. In addition, a nucleotide sequence (GGCGGSCGGSGGGGCCTCGGCGCTCT; sequence encoding a GGSGGGSGGS linker sequence (SEQ ID NO: 21) immediately before the XhoI site on the 3 ′ end side is added before the start codon of the SuperNova-Red gene sequence. Number 22) was added.

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). In addition, a kozak sequence (ccaccatgg) was added before the start codon of the gene sequence of SuperNova-Red.

Preparation of 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. In 2002), 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). In addition, 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).

<< Comparative 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.

Example 11
In this example, CALI for actinin was performed on intracellular adhesion plaques to confirm degeneration of the cytoskeleton.
Vector α-actinin-EGFP / pcDNA3 for expressing a protein in which myofibrillar protein α-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 α-actinin fusion protein (α-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 α-actinin-SuperNova-Red was irradiated with a 561.5 nm laser beam for 10 seconds to destroy α-actinin localized in the region. In the acquired image immediately after irradiation, 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.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

Claims (10)

1. The amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 9, and the amino acid sequence represented by SEQ ID NO: 10 A photosensitized fluorescent protein comprising at least one amino acid sequence selected from the group consisting of:
2. The protein according to claim 1 or a functional equivalent variant thereof, wherein the photosensitized fluorescent protein or a functional equivalent variant thereof constitutes a fusion protein bound to another protein.
3. DNA encoding the photosensitized fluorescent protein according to claim 1 or 2 or a functional equivalent variant thereof.
4. The nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence represented by SEQ ID NO: 3, the nucleotide sequence represented by SEQ ID NO: 5, the nucleotide sequence represented by SEQ ID NO: 19, and the nucleotide sequence represented by SEQ ID NO: 20. The DNA according to claim 3, comprising at least one base sequence selected from the group consisting of:
5. A plasmid containing the DNA according to claim 3 or 4.
6. A photosensitized active oxygen production method using the photosensitized fluorescent protein according to claim 1 or 2 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.
7. The photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 2 or a functional equivalent variant thereof includes an amino acid sequence represented by SEQ ID NO: 4 using an excitation light wavelength in the range of 490 to 620 nm. In the photosensitized fluorescent protein or a functional equivalent variant thereof, the photosensitizing fluorescent protein containing the amino acid sequence represented by SEQ ID NO: 6 using an excitation light wavelength in the range of 400 to 550 nm or a functional equivalent variant thereof In the present invention, an excitation light wavelength in the range of 390 to 440 nm is used, and in the photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 9 or a functional equivalent variant thereof, the excitation light wavelength in the range of 462 to 562 nm And a photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 10 or a functional equivalent variant thereof , Active oxygen production method of photosensitizing according to claim 6 using an excitation wavelength in the range of 459 ~ 559 nm.
8. A method for analyzing the function of a target molecule using the photosensitized fluorescent protein or functional equivalent variant thereof according to claim 1 or 2, wherein the photosensitized fluorescent protein or functional equivalent variant thereof is excited. Irradiating light, said method.
9. The photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 2 or a functional equivalent variant thereof includes an amino acid sequence represented by SEQ ID NO: 4 using an excitation light wavelength in the range of 490 to 620 nm. In the photosensitized fluorescent protein or a functional equivalent variant thereof, the photosensitizing fluorescent protein containing the amino acid sequence represented by SEQ ID NO: 6 using an excitation light wavelength in the range of 400 to 550 nm or a functional equivalent variant thereof In the present invention, an excitation light wavelength in the range of 390 to 440 nm is used, and in the photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 9 or a functional equivalent variant thereof, the excitation light wavelength in the range of 462 to 562 nm And a photosensitized fluorescent protein comprising the amino acid sequence represented by SEQ ID NO: 10 or a functional equivalent variant thereof , Functional analysis method of a target molecule according to claim 8 using an excitation wavelength in the range of 459 ~ 559 nm.
10. The method for analyzing a function of a target molecule according to claim 9, wherein the target molecule is at least one target molecule selected from the group consisting of protein, DNA, RNA, carbohydrate, and lipid.

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