WO2017155101A1 - 蛍光タンパク質 - Google Patents
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- WO2017155101A1 WO2017155101A1 PCT/JP2017/009759 JP2017009759W WO2017155101A1 WO 2017155101 A1 WO2017155101 A1 WO 2017155101A1 JP 2017009759 W JP2017009759 W JP 2017009759W WO 2017155101 A1 WO2017155101 A1 WO 2017155101A1
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- 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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Definitions
- the present disclosure relates to a green-yellow fluorescent protein having high pH stability, a photoswitching mutant thereof, a fusion protein thereof, a molecular sensor thereof, DNA, an expression cassette, a vector, and a transformant.
- a fluorescent protein is used as a marker capable of tracking the behavior of molecules in a living body in real time, or as a fluorescence resonance energy transfer (FRET) type biosensor preparation tool for detecting an ion concentration or a physiologically active substance (for example, Patent Document 1). ) Is universal.
- the present disclosure provides a green-yellow fluorescent protein having high pH stability (having reduced pH sensitivity).
- the present disclosure is a fluorescent protein having an amino acid sequence in which at least the following mutation is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1 It is also called body fluorescent protein.
- the mutation is selected from the group consisting of F149 TorSor A, L158 TorSor A, H160 TorSor A, Y174 TorSorA, Y192 TorSorA, and combinations of 2 to 5 thereof.
- the fluorescent protein having the second to 232th sequence of the amino acid sequence of SEQ ID NO: 1, or the second to 232th sequence of the amino acid sequence of SEQ ID NO: 1 Fluorescence having an amino acid sequence in which 1 to several amino acids have been deleted, substituted, and / or added, pH sensitivity (pKa) of 4.0 or less, a dimer type, and a fluorescent color of green-yellow
- the present invention relates to a protein (hereinafter, a dimeric fluorescent protein according to the present disclosure).
- the light in which “a mutation that becomes a light switching type” is further introduced into the monomeric fluorescent protein according to the present disclosure and the dimeric fluorescent protein according to the present disclosure.
- the present invention relates to a fluorescent protein of a switching mutant type.
- the present disclosure relates to a fusion protein or a molecular sensor including the fluorescent protein according to the present disclosure in one or a plurality of other embodiments.
- the present disclosure includes a nucleic acid including a base sequence encoding a fluorescent protein, a fusion protein, or a molecular sensor according to the present disclosure, an expression cassette including the base sequence, a vector or trait including them. Concerning the converter.
- a green-yellow fluorescent protein having high pH stability having reduced pH sensitivity
- imaging can be performed in an acidic environment
- cell imaging can be performed in an acidic cell environment such as lysosomes, secretory granules, and autophagosomes.
- by combining with a cyan or red fluorescent protein having high pH stability for example, multiple types of fluorescence observation can be performed in an acidic environment.
- biosensors for detecting ion concentrations and physiologically active substances in an acidic environment for example, FRET biosensors, biosensors using circular permutation technology, molecular sensors using split GFP technology, etc. are possible It becomes.
- a green-yellow fluorescent protein having high pH stability can be provided as a monomer type.
- the influence of dimer formation in the case of using a fusion protein with another protein can be eliminated, and effective imaging and detection can be performed.
- FIG. 1 shows the results of size-removed gel filtration chromatography of purified fluorescent protein.
- the dotted spectrum shows the absorption of 10 ⁇ M DsRed (tetramer), tdTomato (dimer), and mCherry (monomer) in order from the left.
- the solid line spectrum shows the absorption of mfGFP.
- FIG. 2 shows the results of measuring the fluorescence characteristics of purified mfGFP.
- FIG. 3 shows the results of measuring fluorescence intensity by dissolving mfGFP or EGFP in a buffer having a pH of 3 to 9.
- FIG. 4 shows the results of measuring the photostability of mfGFP or EGFP.
- FIG. 1 shows the results of size-removed gel filtration chromatography of purified fluorescent protein.
- the dotted spectrum shows the absorption of 10 ⁇ M DsRed (tetramer), tdTomato (dimer), and mCherry (monomer
- FIG. 5 shows the results of measuring the maturation rate ratio of mfGFP or EGFP fluorophores.
- FIG. 6 is an image showing the results of fusing the localization signals to various organelle proteins with mfGFP and expressing them in HeLa cells.
- FIG. 7 is an image after mfGFP or EGFP is expressed in the cytoplasm of HeLa cells and cultured at 37 ° C./5% for 3 days.
- FIG. 8 shows the results of measuring the fluorescence intensity of the mfGFP photoswitching mutant in pH 5, 6, 7, and 8 buffers.
- FIG. 9 shows an example of continuous light switching of mfGFP light switching mutants in pH 5, 6, 7, and 8 buffers.
- the present disclosure is based on the finding that a green-yellow fluorescent protein derived from the cherry tree jellyfish (scientific name: Olindias formosa) exhibits good pH stability (ie, reduced pH sensitivity).
- the wild-type green-yellow fluorescent protein (hereinafter, also referred to as dfGFP, SEQ ID NO: 1) cloned from the cherry tree jellyfish is a dimer.
- SEQ ID NO: 2 also referred to as mfGFP, was obtained.
- the “green-yellow fluorescent protein” refers to a fluorescent protein having a fluorescent color of green or yellow-green, and in one or a plurality of embodiments, the wavelength at which the fluorescence intensity of the emission spectrum is maximum is included in 495 to 570 nm. Say things.
- the “monomer-type green-yellow fluorescent protein” refers to all mutants of the monomer-type green-yellow fluorescent protein originating from the amino acid sequence (SEQ ID NO: 1) of the green-yellow fluorescent protein (dfGFP, dimer type). Can be included.
- the phrase that the fluorescent protein is monomeric means that the same type of fluorescent protein does not polymerize and exists as a single substance. Further, in the present disclosure, that the fluorescent protein is in a dimer form means that two fluorescent proteins of the same species interact with each other to form a stable complex.
- fluorescent protein having amino acid sequence A means, in one or more embodiments, other amino acids such as N-terminal methionine at the N-terminal or C-terminal position of amino acid sequence A, a signal peptide sequence, It means that amino acid sequences that allow protein purification and combinations thereof can be included. A person skilled in the art can select an amino acid sequence that enables protein purification.
- the mutation represented by “X 1 NX 2 ” is a general notation method for mutation, and the N-th amino acid residue X 1 (single-letter amino acid residue) of the amino acid sequence is an amino acid. Represents a mutation to be substituted with residue X 2 (single letter amino acid residue).
- the mutation represented by “X 1 NX 2 or X 3 or X 4 ” indicates that the Nth amino acid residue X 1 (single letter amino acid residue) of the amino acid sequence is amino acid residues X 2 , X 3. Or X 4 (both are amino acid residues represented by one letter).
- X 1 NX 2 orX 3 orX 4" mutation and is represented by the "X 1 N / X 2 ( N + 1) / X 3 (N + 2) ⁇ Y 1 Y 2 Y 3 Y 4"
- Each mutation may be counted as one mutation.
- X 1 and N represented by “X 1 NX 2 ” and the like are the N-th amino acid residue X 1 of the amino acid sequence of SEQ ID NO: 1 in the sequence listing, or the sequence listing, unless otherwise specified.
- the pH sensitivity of the fluorescent protein refers to a decrease in the fluorescence intensity of the fluorescent protein when the pH of the medium in which the fluorescent protein exists changes from a basic pH to an acidic pH.
- the pH stability of the fluorescent protein refers to a decrease in the fluorescence intensity of the fluorescent protein when the pH of the medium in which the fluorescent protein is present changes from a basic pH to an acidic pH.
- the “fluorescence intensity” of a fluorescent protein can be measured using a spectrofluorometer.
- the pH sensitivity represents a pH at which the fluorescence intensity of the fluorescent protein becomes half of the maximum value when the pH of the medium in which the fluorescent protein exists changes from a basic pH to an acidic pH. Specifically, it can be measured by the method described in the examples.
- the present disclosure relates to a monomeric green-yellow fluorescent protein having an amino acid sequence in which a monomer-like mutation is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1.
- the “monomer-type mutation” is a mutation selected from the group consisting of F149 TorSorA, L158 TorSorA, H160 TorSorA, Y174 TorSorA, Y192 TorSorA, and combinations of these 2 to 5 mutations. It is.
- F149TorSorA means “F149T, F149S, or F149A”
- L158TorSorA means “L158T, L158S, or L158A”
- H160TorSorA means “H160T, H160S, or H160A”.
- Y174TorSorA means “Y174T, Y174S, or Y174A”
- Y192TorSorA means “Y192T, Y192S, or Y192A", respectively.
- the present disclosure relates to a fluorescent protein having an amino acid sequence in which at least the following mutation is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1 in one or a plurality of embodiments.
- a mutation selected from the group consisting of F149 TorSorA, L158 TorSorA, H160 TorSorA, Y174 TorSorA, and Y192 TorSorA, and combinations of these 2-5.
- the “mutation that becomes a monomer type” or the “mutation introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1” is any one of F149 TorSorA, L158 TorSorA, H160 TorSorA, Y174TorSorA, or Y192TorSorA. Or a combination of 2, 3, 4 or 5 mutations selected from F149 TorSorA, L158 TorSorA, H160 TorSorA, Y174 TorSorA, and Y192 TorSorA.
- the combination of two mutations includes, in a non-limiting embodiment, a combination of Y174 TorSorA and Y192 TorSorA.
- Other embodiments include a combination of two mutations, Y174T and Y192A.
- the three mutation combinations include, in a non-limiting embodiment, a combination of F149 TorSorA, L158 TorSorA, and H160 TorSorA.
- Other embodiments include combinations of three mutations, F149T, L158T and H160T.
- the five mutation combinations include, but are not limited to, F149 TorSor A, L158 TorSorA, H160 TorSorA, Y174 TorSorA, and Y192 TorSorA.
- Other embodiments include combinations of 5 mutations of F149T, L158T, H160T, Y174T and Y192A.
- dfGFP SEQ ID NO: 1
- SEQ ID NO: 1 which is a dimer form
- the monomer-type green-yellow fluorescent protein according to the present disclosure may further include K112EorDorQ, E147KorRorQ, Q145EorD in addition to the “monomer-type mutation” from the viewpoint of improving luminance and / or solubility.
- K166EorDorQ, P162LorVorIorA, P223LorVorIorA, N37SorAorG, E140KorRorQ, K185RorAorG, A150 / E151 / G152 ⁇ PHGPorPHA, and K180TorS, and a combination of 2 to 11 of these may be selected.
- the positions of these mutations are based on SEQ ID NO: 1.
- the monomer-type green-yellow fluorescent protein according to the present disclosure may further include K112E, E147K, Q145E in addition to the “monomer-type mutation” from the viewpoint of improving luminance and / or solubility.
- K112E, E147K, Q145E in addition to the “monomer-type mutation” from the viewpoint of improving luminance and / or solubility.
- a mutation selected from the group consisting of K166E, P162L, P223L, N37S, E140K, K185R, A150 / E151 / G152 ⁇ PHGP, and K180T, and combinations of these 2-11 may be introduced.
- the monomer-type green-yellow fluorescent protein according to the present disclosure has an N-terminal and a C-terminal of the second to 225th sequences of the amino acid sequence of SEQ ID NO: 1 from the viewpoint of protein folding stability.
- the amino acid sequences of the N-terminal and C-terminal of other fluorescent proteins may be added to each of the above.
- the length of the added amino acid sequence is, for example, 6 to 8 amino acids or 7 amino acids.
- a fluorescent protein having the 8th to 232nd sequence of the amino acid sequence of SEQ ID NO: 2 A fluorescent protein having the 2nd to 232nd sequences of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the 8th to 239th sequence of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the second to 239th sequences of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the amino acid sequence of SEQ ID NO: 2, Is mentioned.
- the pH sensitivity (pKa) of the monomer type green-yellow fluorescent protein according to the present disclosure is 4.0 or less, preferably less than 4.0, and more preferably 3.9 or less.
- the monomer-type green-yellow fluorescent protein according to the present disclosure has a pH sensitivity (pKa) of 4.0 or less so long as the function of the monomer-type green-yellow fluorescent protein can be maintained. It may have a mutation. Such mutations include deletions, substitutions, and / or additions of one to several amino acids, and “one to several” refers to 1 to 4, 1 to 3, in one or more embodiments, Includes 1-2, or one.
- the monomer type green-yellow fluorescent protein according to the present disclosure is at least 90%, 91%, 92%, 93%, 94% and the 8th to 232th sequence of the amino acid sequence of SEQ ID NO: 2. , 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence having a pH sensitivity (pKa) of 4.0 or less and a monomeric green-yellow fluorescent protein It is.
- the monomer type green-yellow fluorescent protein according to the present disclosure may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique.
- a method for producing a recombinant protein by a gene recombination technique a method for producing a recombinant protein using a host transformed with an expression vector containing a gene encoding a monomeric green-yellow fluorescent protein according to the present disclosure, Or the method of producing by a cell-free system is mentioned.
- the monomer-type green-yellow fluorescent protein according to the present disclosure may further have “a mutation that becomes a light switching type”.
- Examples of the “mutation that becomes an optical switching type” include T197AorG, that is, “T197A or T197G”. The position of this mutation is based on the amino acid sequence of SEQ ID NO: 1.
- the “mutation that becomes an optical switching type” is T204AorG.
- a fluorescent protein having the 8th to 232nd sequence of the amino acid sequence of SEQ ID NO: 2 A fluorescent protein having the 2nd to 232nd sequences of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the 8th to 239th sequence of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the second to 239th sequences of the amino acid sequence of SEQ ID NO: 2, A fluorescent protein having the amino acid sequence of SEQ ID NO: 2, Having a mutation of T204AorG in the amino acid sequence of SEQ ID NO: 2, Is mentioned.
- the light-switching mutant monomer type green-yellow fluorescent protein according to the present disclosure has at least 90% and 91% of the 8th to 232th sequence of the amino acid sequence of SEQ ID NO: 2 having the mutation of T204AorG. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity and pH sensitivity (pKa) of 4.0
- pKa pH sensitivity
- the light-switching fluorescent protein refers to a fluorescent protein (Reversibly Photo-Switchable Fluorescent Protein, RSFP) that can reversibly switch fluorescence.
- fluorescent protein Reversibly Photo-Switchable Fluorescent Protein, RSFP
- “light-switching type fluorescent protein capable of reversibly switching light” means that in one or a plurality of embodiments, on and off of fluorescence can be controlled by two light irradiations having different wavelengths, and on A fluorescent protein that can be repeatedly switched between and off.
- the light-switching mutant monomer type green-yellow fluorescent protein according to the present disclosure having the above-mentioned “mutation that becomes a light-switching type” changes from non-fluorescence to fluorescence upon irradiation with light of a specific wavelength that does not excite fluorescence, Therefore, it is a negative light switching type RSFP that changes from fluorescent to non-fluorescent by light irradiation.
- the term “monomer-type green-yellow fluorescent protein according to the present disclosure” may include a light-switching mutant monomer-type green-yellow fluorescent protein.
- the present disclosure is a fusion protein in which the monomeric green-yellow fluorescent protein according to the present disclosure is fused with another protein or peptide, and the fluorescent protein portion has a pH sensitivity (pKa) of 4.0. It is a fusion protein that can function as the following monomeric green-yellow fluorescent protein.
- the protein to be bound (fused) to the monomer type green-yellow fluorescent protein according to the present disclosure may be a signal sequence, an expression tag, or a protein (necessary) in one or a plurality of non-limiting embodiments. Corresponding linker sequences).
- the signal sequence and the protein include a cell membrane, an intracellular cytoskeleton (microfilament, intermediate filament, microtubule) and an organelle (nucleus, endoplasmic reticulum) from the viewpoint of imaging. , Golgi, mitochondria, endosome, lysosome, etc.) and signal sequences and proteins that can be localized.
- the monomeric green-yellow fluorescent protein and the fusion protein according to the present disclosure are monomeric and have reduced pH sensitivity
- acidic substances such as lysosomes, secretory granules, and autophagosomes are used. It can be used as a fluorescent function indicator or fluorescent marker in a cellular environment, or can be used for cell imaging (for example, in vivo imaging).
- the monomeric green-yellow fluorescent protein and the fusion protein according to the present disclosure are green-yellow fluorescent proteins with high pH stability, in one or a plurality of non-limiting embodiments, a cyan or red fluorescent protein with high pH stability and By combining them, for example, multiple types of fluorescence observation are possible in an acidic environment, and a biosensor for detecting ion concentration, physiologically active substance, and the like in an acidic environment becomes possible. Therefore, in another aspect, the present disclosure relates to a molecular sensor that has a part or all of the amino acid sequence of the monomer type green-yellow fluorescent protein or fusion protein according to the present disclosure and uses the fluorescence characteristics of the protein.
- the molecular sensor according to the present disclosure may include all of the molecular sensors that utilize the fluorescence characteristics of the monomer-type green-yellow fluorescent protein according to the present disclosure or the fusion protein according to the present disclosure.
- Examples of the molecular sensor according to the present disclosure include, but are not limited to, a FRET biosensor, a biosensor using a circular permutation technique, a molecular sensor using a split GFP technique, and the like.
- Examples of the fluorescence resonance energy transfer (FRET) biosensor include a FRET biosensor in which at least one fluorescent protein component used in FRET is a monomeric green-yellow fluorescent protein according to the present disclosure.
- the molecular sensor according to the present disclosure may be in a form composed of a protein or a peptide, and is a form in which the monomer-type green-yellow fluorescent protein or fusion protein according to the present disclosure is bound to a substance other than a protein. Also good.
- Biosensors using circular permutation technology include biosensors that include circular permutants of monomeric green-yellow fluorescent protein according to the present disclosure.
- circular permutation refers to the production of a new N-terminus and C-terminus within the monomeric green-yellow fluorescent protein according to the present disclosure (ie, the protein is divided into two internally), and the original The mutation which connects the C-terminal and N-terminal of the above by an appropriate linker sequence.
- Circular permutation in fluorescent proteins has been performed conventionally, see, for example, Baird et al. (Proc. Natl. Acad. Sci. USA, vol96, pp11241-11246 1999).
- the biosensor using the circular permutation technology in the present disclosure includes a peptide or protein that binds to the N-terminal and C-terminal of the circular permutation of the monomer-type green-yellow fluorescent protein according to the present disclosure.
- the peptide or protein can be appropriately selected depending on the object to be sensed.
- the molecular sensor using the split GFP technology examples include a biosensor using the monomer type green-yellow fluorescent protein according to the present disclosure divided into two.
- a conventional division GFP technique can be referred to.
- the biosensor using the split GFP technology in the present disclosure includes a configuration in which a peptide or protein is bound to each of the monomer-type green-yellow fluorescent protein according to the present disclosure that is split into two.
- the peptide or protein can be appropriately selected depending on the object to be sensed.
- the present disclosure relates to a nucleic acid encoding a monomeric green-yellow fluorescent protein according to the present disclosure, a fusion protein according to the present disclosure, or a molecular sensor according to the present disclosure.
- the nucleic acid includes single-stranded or double-stranded DNA selected from synthetic DNA, cDNA, genomic DNA, and plasmid DNA, and transcription products of these DNAs.
- An example of the base sequence of DNA encoding the monomeric green-yellow fluorescent protein according to the present disclosure is the base sequence represented by SEQ ID NO: 3.
- the present disclosure relates to an expression cassette comprising a nucleic acid encoding a monomeric green-yellow fluorescent protein, fusion protein, or molecular sensor according to the present disclosure.
- the nucleic acid is operably linked to an expression regulatory sequence according to the host cell to be introduced.
- expression regulatory sequences include promoters, enhancers, transcription terminators, and the like, and other examples include start codons, intron splicing signals, and stop codons.
- the present disclosure relates to a vector capable of expressing a monomer type green-yellow fluorescent protein, a fusion protein, or a molecular sensor according to the present disclosure.
- the present disclosure relates to a vector capable of expressing the monomer type green-yellow fluorescent protein according to the present disclosure, the fusion protein according to the present disclosure, or the molecular sensor according to the present disclosure.
- the vector according to the present disclosure is an expression vector having the nucleic acid or the expression cassette according to the present disclosure.
- the vector according to the present disclosure can be used by appropriately selecting an expression vector system according to the cell (host) to be expressed.
- One or more non-limiting embodiments include plasmids, cosmids, YACS, viral (eg, adenovirus, retrovirus, episomal EBV, etc.) vectors and phage vectors.
- the present disclosure relates to a transformant that expresses a monomeric green-yellow fluorescent protein, a fusion protein, or a molecular sensor according to the present disclosure.
- the transformant of the present disclosure is a cell that expresses the disclosed monomer-type green-yellow fluorescent protein, fusion protein, or molecular sensor, or a tissue, organ, or living body that includes the cell. .
- this indication is related with the transformant which has the nucleic acid or vector which concerns on this indication in one or some embodiment.
- the transformant of the present disclosure can be produced by introducing the nucleic acid, expression cassette or vector of the present disclosure into a host.
- the host include animal cells, animal cells that do not contain human organisms, plant cells, insect cells, microorganisms, and the like.
- the present disclosure relates to an imaging method using a monomer type green-yellow fluorescent protein, a fusion protein, or a molecular sensor according to the present disclosure.
- the imaging method of the present disclosure includes introducing a monomer type green-yellow fluorescent protein, a fusion protein, or a molecular sensor according to the present disclosure into a cell or the like, and the monomer type green-yellow according to the present disclosure. Detecting the fluorescent signal of a fluorescent protein, fusion protein, or molecular sensor.
- an imaging method for a human living body is not included.
- the present disclosure relates to an imaging method using the photoswitching mutant monomer type green-yellow fluorescent protein according to the present disclosure, a fusion protein thereof, or a molecular sensor using them.
- an imaging method for a human living body is not included.
- the imaging method of the present disclosure introduces the light-switching mutant monomer type green-yellow fluorescent protein of the present disclosure into a cell or the like, and performs light switching of the light-switching mutant-type fluorescent protein to perform fluorescence. On / off sex and / or detecting the fluorescence signal of the light-switching mutant fluorescent protein.
- the imaging method of the present disclosure is, in one or more embodiments, super-resolution imaging or super-resolution live imaging, and in one or more embodiments, PALM (photoactivated localization microscopy), STORM (stochastic optical reconstruction microscopy). ), RESOLFT (reversible saturable optical fluorescence transition), NL-SIM (Nonlinear structured illumination microscopy), or SOFI (stochastic optical fluctuation imaging).
- the present disclosure provides, in other embodiments, a wild-type dimeric green-yellow fluorescent protein (dfGFP, the second to 232th sequences of SEQ ID NO: 1) cloned from the cherry tree jellyfish (scientific name: Olindias formosa), and the dfGFP It is related with the mutant of the dimer type
- pKa pH sensitivity
- the dimeric green-yellow fluorescent protein according to the present disclosure has a pH sensitivity (pKa) of 4.0 or less and can maintain the function of the dimeric green-yellow fluorescent protein. It may have a mutation. Such mutations include deletions, substitutions, and / or additions of one to several amino acids, and “one to several” refers to 1 to 4, 1 to 3, in one or more embodiments, Includes 1-2, or one.
- the dimer-type green-yellow fluorescent protein according to the present disclosure is at least 90%, 91%, 92%, 93%, 94% and the second to 232th sequences of the amino acid sequence of SEQ ID NO: 1. 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence having a pH sensitivity (pKa) of 4.0 or less and a dimeric green-yellow fluorescent protein It is.
- a mutation that becomes a light switching type may be further introduced.
- the “mutation that becomes an optical switching type” include T197AorG, that is, “T197A or T197G”. The position of this mutation is based on the amino acid sequence of SEQ ID NO: 1.
- the “dimeric green-yellow fluorescent protein according to the present disclosure” may include a light-switching mutant dimer-type green-yellow fluorescent protein.
- the present disclosure also relates to a fusion protein, a molecular sensor, a nucleic acid, an expression cassette, a vector, and a transformant related to the dimeric green-yellow fluorescent protein according to the present disclosure, or an imaging method using the molecular sensor using them. These can be the same as those of the monomer type green-yellow fluorescent protein according to the present disclosure.
- a fluorescent protein having an amino acid sequence in which at least the following mutation is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1.
- the mutation is selected from the group consisting of F149 TorSor A, L158 TorSor A, H160 TorSor A, Y174 TorSorA, Y192 TorSorA, and combinations of 2 to 5 thereof.
- the fluorescent protein according to [1] which has an amino acid sequence in which the following mutation is further introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1.
- the mutation consists of K112EorDorQ, E147KorRorQ, Q145EorD, K166EorDorQ, P162LorVorIorA, P223LorVorIorA, N37SorAorG, E140KorRorQ, K185RorAorG, A150 / P151A, GPH, A150 / P151A Selected.
- the fluorescent protein according to [1] or [2] which has an 8th to 232nd sequence of the amino acid sequence of SEQ ID NO: 2.
- It has an amino acid sequence in which one to several amino acids are deleted, substituted, and / or added in the amino acid sequence of the fluorescent protein according to any one of [1] to [4], and is pH sensitive (pKa ) Is 4.0 or less, is a monomer type, and has a fluorescent color of green-yellow.
- [6] The amino acid sequence of the fluorescent protein according to any one of [1] to [4] and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A fluorescent protein having an amino acid sequence showing 99% or 99.5% identity, having a pH sensitivity (pKa) of 4.0 or less, a monomeric type, and a fluorescent color of green-yellow.
- the fluorescent protein according to any one of [1] to [6], which has an amino acid sequence in which a mutation of T197AorG is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1.
- the function according to [8] which can function as a fluorescent protein that changes from non-fluorescence to fluorescence by irradiation with light of a specific wavelength that does not excite fluorescence, and changes from fluorescence to non-fluorescence by irradiation with light for fluorescence excitation. Fluorescent protein.
- a molecular sensor having a part or all of the amino acid sequence of the protein according to any one of [1] to [10] and utilizing the fluorescence characteristics of the protein.
- [12] A nucleic acid having a base sequence encoding the protein according to any one of [1] to [10] or the molecular sensor according to [11].
- An expression cassette comprising a base sequence encoding the protein according to any one of [1] to [10] or the molecular sensor according to [11].
- the protein according to any one of [1] to [10] or the molecular sensor according to [11] can be expressed, or the nucleic acid according to [12] or the expression cassette according to [13] Having a vector.
- [15] A transformant expressing the protein according to any one of [1] to [10] or the molecular sensor according to [11].
- the fluorescent protein according to [16] having a pH sensitivity (pKa) of 4.0 or less, a dimer type, and a fluorescent color of green-yellow.
- It has an amino acid sequence in which 1 to several amino acids are deleted, substituted, and / or added in the 2nd to 232nd sequences of the amino acid sequence of SEQ ID NO: 1, and pH sensitivity (pKa) is 4.
- a fluorescent protein that is 0 or less, is dimeric, and has a fluorescent color of green-yellow.
- the fluorescent protein according to any one of [16] to [20] which has an amino acid sequence in which a mutation of T197AorG is introduced into the 2nd to 225th sequences of the amino acid sequence of SEQ ID NO: 1.
- a molecular sensor having a part or all of the amino acid sequence of the protein according to any one of [16] to [24] and utilizing the fluorescence characteristics of the protein.
- [26] A nucleic acid having a base sequence encoding the protein according to any one of [16] to [24] or the molecular sensor according to [25].
- An expression cassette comprising a base sequence encoding the protein according to any one of [16] to [24] or the molecular sensor according to [25].
- the protein according to any one of [16] to [24] or the molecular sensor according to [25] can be expressed, or the nucleic acid according to [26] or the expression cassette according to [27] Having a vector.
- dfGFP is a fluorescent protein derived from the cherry tree jellyfish (scientific name: Olindias formosa).
- RNA was extracted from the cherry tree jellyfish, and mRNA was reverse transcribed into DNA using an oligo T primer to prepare a cDNA library.
- This cDNA library was amplified by PCR and then inserted into a bacterial expression vector. These plasmids were transformed into Escherichia coli, and colonies emitting fluorescence were selected to identify a gene encoding the fluorescent protein dfGFP.
- the amino acid sequence of dfGFP encoded by the gene is SEQ ID NO: 1 in the sequence listing.
- MfGFP having a polyhistidine tag at the N-terminus was introduced into the bacterial expression vector pRSET B and expressed in E. coli. After culturing in LB medium at 23 ° C. for 65 hours, the cells were disrupted with a French press, and the supernatant was gel-filtrated with a Ni-NTA agarose affinity column (Qiagen) and a PD-10 column (GE Healthcare). And re-purified on an AKTA Superdex200 10/300 GL (GE Healthcare) column.
- FIG. 1 shows the result of size-removal gel filtration chromatography using a Superdex 200 10/30 column.
- the solvent is a 20 mM HEPES solution of pH 7.4 containing 150 mM NaCl.
- the spectrum of the dotted line in FIG. 1 shows the absorption of 10 ⁇ M DsRed (tetramer), tdTomato (dimer), and mCherry (monomer) in order from the left.
- the solid line spectrum shows the absorption of 10 ⁇ M mfGFP. Since the peak position of mfGFP is almost the same as that of mCherry, it can be said that mfGFP is a monomer.
- Table 1 summarizes the optical properties of mfGFP and EGFP.
- MfGFP Physical property 5 of mfGFP
- MfGFP was fused with localization signals to various organelle proteins and expressed in HeLa cells (actin, Golgi apparatus, phosphorus, paxillin, peroxisome, dixin, mitochondria, fibrillarin, vimentin, tubulin, connexin 43, histone H2B, Life Act, VAMP2, LAMP3. Catthepsin B, LC3).
- FIG. 6 the mfGFP fusion protein showed the correct localization to the target organelle protein.
- the successful labeling of tubulin which is difficult to localize, suggests the property of mfGFP as a high monomer.
- High fluorescence intensity was also exhibited in secretory vesicles and lysosomes (pH 4.5-5, 5) whose inside was kept acidic.
- [MfGFP photoswitching mutant] By introducing a T197A (or T197G) amino acid mutation into mfGFP, a mutant (light switching mutant) capable of controlling the fluorescence / non-fluorescence state in a light wavelength-dependent manner while maintaining the acid resistance of mfGFP was obtained.
- a mutant light switching mutant capable of controlling the fluorescence / non-fluorescence state in a light wavelength-dependent manner while maintaining the acid resistance of mfGFP was obtained.
- the T197A mutant was expressed in Escherichia coli and the extract was mixed with buffers of pH 5, 6, 7, and 8, it was found that the fluorescence intensity was not affected by the pH in the above range (FIG. 8).
- SEQ ID NO: 1 amino acid sequence of dfGFP
- SEQ ID NO: 2 amino acid sequence of mfGFP
- SEQ ID NO: 3 base sequence of mfGFP
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Abstract
Description
ここで、前記変異は、F149TorSorA、L158TorSorA、H160TorSorA、Y174TorSorA、及び、Y192TorSorA、並びにこれらの2~5個の組合せからなる群から選択される。
また、該蛍光タンパク質によれば、限定されない一又は複数の実施形態において、pH安定性の高いシアン色又は赤色の蛍光タンパク質と組み合わせることで、例えば、酸性環境下で複数種類の蛍光観察が可能となり、また、酸性環境におけるイオン濃度や生理活性物質等を検出するためのバイオセンサ(例えば、FRET型バイオセンサ、円順列変異技術を利用したバイオセンサ、分割GFP技術を利用した分子センサなど)が可能となる。
本開示において、“X1NX2orX3orX4”で表される変異は、アミノ酸配列のN番目のアミノ酸残基X1(一文字表記のアミノ酸残基)が、アミノ酸残基X2、X3又はX4(いずれも、一文字表記のアミノ酸残基)に置換される変異を表す。
本開示において、“X1N/X2(N+1)/X3(N+2)⇒Y1Y2Y3Y4”で表される変異は、アミノ酸配列のNから(N+2)番目の3アミノ酸残基X1X2X3がY1Y2Y3Y4の4アミノ酸残基に置換される変異を表す。また、“X1N/X2(N+1)/X3(N+2)⇒Y1Y2Y3”で表される変異は、アミノ酸配列のNから(N+2)番目の3アミノ酸残基X1X2X3がY1Y2Y3の3アミノ酸残基に置換される変異を表す。
本開示において、“X1NX2orX3orX4”で表される変異及び“X1N/X2(N+1)/X3(N+2)⇒Y1Y2Y3Y4”で表される変異はそれぞれ、1個の変異としてカウントすることがある。
本開示において、“X1NX2”等で表されるX1及びNは、特に言及のない場合、配列表の配列番号1のアミノ酸配列のN番目のアミノ酸残基X1、又は、配列表の配列番号1のアミノ酸配列のN番目に相当する位置のアミノ酸残基X1をいう。
本開示は、一態様において、配列番号1のアミノ酸配列の2番目から225番目の配列に単量体型となる変異が導入されたアミノ酸配列を有する単量体型緑黄色蛍光タンパク質に関する。
F149TorSorA、L158TorSorA、H160TorSorA、Y174TorSorA、及び、Y192TorSorA、並びにこれらの2~5個の組合せからなる群から選択される変異。
前記「変異の組合せ」のうち、2変異の組合せとして、限定されない一実施形態において、Y174TorSorA及びY192TorSorAの組合せが挙げられる。その他の実施形態として、Y174T及びY192Aの2変異の組合せが挙げられる。
前記「変異の組合せ」のうち、3変異の組合せとして、限定されない一実施形態において、F149TorSorA、L158TorSorA、及びH160TorSorAの組合せが挙げられる。その他の実施形態として、F149T、L158T及びH160Tの3変異の組合せが挙げられる。
前記「変異の組合せ」のうち、5変異の組合せとして、限定されない一実施形態において、F149TorSorA、L158TorSorA、H160TorSorA、Y174TorSorA及びY192TorSorAが挙げられる。その他の実施形態として、F149T、L158T、H160T、Y174T及びY192Aの5変異の組合せが挙げられる。
本開示に係る単量体型緑黄色蛍光タンパク質は、一又は複数の実施形態において、輝度向上及び/又は溶解度向上の観点から、前記「単量体型となる変異」に加えてさらに、K112E、E147K、Q145E、K166E、P162L、P223L、N37S、E140K、K185R、A150/E151/G152→PHGP、及びK180T、並びにこれらの2~11個の組合せからなる群から選択される変異が導入されてもよい。
配列番号2のアミノ酸配列の8番目から232番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の2番目から232番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の8番目から239番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の2番目から239番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列を有する蛍光タンパク質、
が挙げられる。
本開示に係る単量体型緑黄色蛍光タンパク質は、一又は複数の実施形態において、配列番号2のアミノ酸配列の8番目から232番目の配列と少なくとも90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、又は99.5%の同一性を示すアミノ酸配列を有し、pH感受性(pKa)が4.0以下である単量体型緑黄色蛍光タンパク質である。
本開示に係る単量体型緑黄色蛍光タンパク質は、一又は複数の実施形態において、さらに、「光スイッチング型となる変異」が導入されていてもよい。前記「光スイッチング型となる変異」としては、T197AorG、すなわち、「T197A又はT197G」が挙げられる。なおこの変異の位置、配列番号1のアミノ酸配列を基準としている。配列番号2のアミノ酸配列を基準とすると、前記「光スイッチング型となる変異」は、T204AorGとなる。
したがって、本開示に係る光スイッチング変異型単量体型緑黄色蛍光タンパク質の限定されない一又は複数の実施形態として、
配列番号2のアミノ酸配列の8番目から232番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の2番目から232番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の8番目から239番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列の2番目から239番目の配列を有する蛍光タンパク質、
配列番号2のアミノ酸配列を有する蛍光タンパク質、
であって、配列番号2のアミノ酸配列においてT204AorGの変異を有するもの、
が挙げられる。
本開示に係る光スイッチング変異型単量体型緑黄色蛍光タンパク質は、一又は複数の実施形態において、T204AorGの変異を有する配列番号2のアミノ酸配列の8番目から232番目の配列と少なくとも90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、又は99.5%の同一性を示すアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、光スイッチング型の単量体型緑黄色蛍光タンパク質である。
上述した「光スイッチング型となる変異」を有する本開示に係る光スイッチング変異型単量体型緑黄色蛍光タンパク質は、蛍光励起しない特定の波長の光照射によって無蛍光性から蛍光性になり、蛍光励起のための光照射によって蛍光性から無蛍光性になるネガティブ光切替型のRSFPである。
本開示は、その他の態様において、本開示に係る単量体型緑黄色蛍光タンパク質が他のタンパク質又はペプチドと融合した融合タンパク質であって、該蛍光タンパク質の部分が、pH感受性(pKa)が4.0以下の単量体型緑黄色蛍光タンパク質として機能可能な融合タンパク質である。
本開示に係る単量体型緑黄色蛍光タンパク質及び融合タンパク質は、pH安定性の高い緑黄色蛍光タンパク質であるため、限定されない一又は複数の実施形態において、pH安定性の高いシアン色又は赤色の蛍光タンパク質と組み合わせることで、例えば、酸性環境下で複数種類の蛍光観察が可能となり、また、酸性環境におけるイオン濃度や生理活性物質等を検出するためのバイオセンサが可能となる。
したがって、本開示は、その他の態様において、本開示に係る単量体型緑黄色蛍光タンパク質又は融合タンパク質のアミノ酸配列の一部又は全部を有し、該タンパク質の蛍光特性を利用する分子センサに関する。
本開示に係る分子センサは、本開示に係る単量体型緑黄色蛍光タンパク質又は本開示に係る融合タンパク質の蛍光特性を利用する分子センサの全てを含みうる。
本開示に係る分子センサ-としては、限定されない一又は複数の実施形態として、FRET型バイオセンサ、円順列変異技術を利用したバイオセンサ、分割GFP技術を利用した分子センサなどが挙げられる。
本開示は、一態様において、本開示に係る単量体型緑黄色蛍光タンパク質、本開示に係る融合タンパク質、又は本開示に係る分子センサをコードする核酸に関する。本開示において、核酸は、合成DNA,cDNA、ゲノムDNA及びプラスミドDNAから選択される一本鎖又は二本鎖DNA、並びにこれらのDNAの転写生成物が挙げられる。
本開示に係る単量体型緑黄色蛍光タンパク質をコードするDNAの塩基配列の一例が、配列番号3で示される塩基配列である。
本開示は、一態様において、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサをコードする核酸を含む発現カセットに関する。該発現カセットにおいて、前記核酸は、導入する宿主細胞に応じた発現調節配列が作動的に連結されている。発現調節配列としては、プロモーター、エンハンサー、転写ターミネーター等が挙げられ、その他には、開始コドン、イントロンのスプライシングシグナル、及び停止コドンなどが挙げられる。
本開示は、一態様において、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサを発現可能なベクターに関する。本開示は、その他の態様において、本開示に係る単量体型緑黄色蛍光タンパク質、本開示に係る融合タンパク質、又は本開示に係る分子センサを発現可能なベクターに関する。本開示に係るベクターは、一又は複数の実施形態において、本開示に係る核酸又は発現カセットを有する発現ベクターである。
本開示は、一態様において、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサを発現する形質転換体に関する。本開示の形質転換体は、一又は複数の実施形態において、開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサを発現する細胞、又は、該細胞を含む組織、器官、生体である。また、本開示は、一又は複数の実施形態において、本開示に係る核酸又はベクターを有する形質転換体に関する。本開示の形質転換体は、一又は複数の実施形態において、本開示の核酸、発現カセット又はベクターを宿主に導入することによって作成することができる。宿主としては、動物細胞、ヒト生体を含まない動物細胞、植物細胞、昆虫細胞、微生物等が挙げられる。
本開示は、一態様において、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサを用いるイメージング方法に関する。本開示のイメージング方法は、一又は複数の実施形態において、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサを細胞等に導入すること、及び、本開示に係る単量体型緑黄色蛍光タンパク質、融合タンパク質、又は分子センサの蛍光シグナルを検出することを含む。なお、本態様の一又は複数の実施形態において、ヒト生体に対するイメージング方法は含まれない。
本開示は、一態様において、本開示に係る光スイッチング変異型単量体型緑黄色蛍光タンパク質、その融合タンパク質、又はそれらを用いた分子センサを用いるイメージング方法に関する。なお、本態様の一又は複数の実施形態において、ヒト生体に対するイメージング方法は含まれない。本開示のイメージング方法は、一又は複数の実施形態において、本開示の光スイッチング変異型単量体型緑黄色蛍光タンパク質を細胞等に導入すること、前記光スイッチング変異型蛍光タンパク質の光切替を行って蛍光性をon/offすること、及び/又は、前記光スイッチング変異型蛍光タンパク質の蛍光シグナルを検出することを含む。本開示のイメージング方法は、一又は複数の実施形態において、超解像イメージング若しくは超解像ライブイメージングであって、一又は複数の実施形態において、PALM(photoactivated localization microscopy)、STORM(stochastic optical reconstruction microscopy)、RESOLFT(reversible saturable optical fluorescence transition)、NL-SIM (Nonlinear structured illumination microscopy) 又は、SOFI(stochastic optical fluctuation imaging)が挙げられる。
本開示は、その他の態様において、ハナガサクラゲ(学名:Olindias formosa)からクローニングされた野生型の二量体型緑黄色蛍光タンパク質(dfGFP、配列番号1の2番目から232番目の配列)、及び、このdfGFPのアミノ酸配列(配列番号1)を起源とするpH感受性(pKa)が4.0以下である二量体型緑黄色蛍光タンパク質の変異体に関する。
本開示に係る二量体型緑黄色蛍光タンパク質は、一又は複数の実施形態において、配列番号1のアミノ酸配列の2番目から232番目の配列と少なくとも90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、又は99.5%の同一性を示すアミノ酸配列を有し、pH感受性(pKa)が4.0以下である二量体型緑黄色蛍光タンパク質である。
〔1〕 配列番号1のアミノ酸配列の2番目から225番目の配列に少なくとも下記変異が導入されたアミノ酸配列を有する蛍光タンパク質。
ここで、前記変異は、F149TorSorA、L158TorSorA、H160TorSorA、Y174TorSorA、及び、Y192TorSorA、並びにこれらの2~5個の組合せからなる群から選択される。
〔2〕 配列番号1のアミノ酸配列の2番目から225番目の配列に、さらに下記の変異が導入されたアミノ酸配列を有する、〔1〕記載の蛍光タンパク質。
ここで、前記変異は、K112EorDorQ、E147KorRorQ、Q145EorD、K166EorDorQ、P162LorVorIorA、P223LorVorIorA、N37SorAorG、E140KorRorQ、K185RorAorG、A150/E151/G152→PHGPorPHA、及び、K180TorS、並びにこれらの2~11個の組合せからなる群から選択される。
〔3〕 配列番号2のアミノ酸配列の8番目から232番目の配列を有する、〔1〕又は〔2〕に記載の蛍光タンパク質。
〔4〕 pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色である、〔1〕から〔3〕のいずれかに記載の蛍光タンパク質。
〔5〕 〔1〕から〔4〕のいずれかに記載の蛍光タンパク質のアミノ酸配列において1から数個のアミノ酸が欠失、置換、及び/又は付加されたアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
〔6〕 〔1〕から〔4〕のいずれかに記載の蛍光タンパク質のアミノ酸配列と少なくとも90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、又は99.5%の同一性を示すアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
〔7〕 〔1〕から〔6〕のいずれかに記載の蛍光タンパク質が融合された融合タンパク質であって、該蛍光タンパク質の部分は、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色の蛍光タンパク質である、融合タンパク質。
〔8〕 配列番号1のアミノ酸配列の2番目から225番目の配列に、T197AorGの変異が導入されたアミノ酸配列を有する、〔1〕から〔6〕のいずれかに記載の蛍光タンパク質。
〔9〕 蛍光励起しない特定の波長の光照射によって無蛍光性から蛍光性になり、蛍光励起のための光照射によって蛍光性から無蛍光性になる蛍光タンパク質として機能可能な、〔8〕記載の蛍光タンパク質。
〔10〕 〔8〕又は〔9〕に記載の蛍光タンパク質が融合された融合タンパク質であって、該蛍光タンパク質の部分は、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色であり、蛍光励起しない特定の波長の光照射によって無蛍光性から蛍光性になり、蛍光励起のための光照射によって蛍光性から無蛍光性になる蛍光タンパク質として機能可能である、融合タンパク質。
〔11〕 〔1〕から〔10〕のいずれかに記載のタンパク質のアミノ酸配列の一部又は全部を有し、該タンパク質の蛍光特性を利用する、分子センサ。
〔12〕 〔1〕から〔10〕のいずれかに記載のタンパク質又は〔11〕に記載の分子センサをコードする塩基配列を有する核酸。
〔13〕 〔1〕から〔10〕のいずれかに記載のタンパク質又は〔11〕に記載の分子センサをコードする塩基配列含む発現カセット。
〔14〕 〔1〕から〔10〕のいずれかに記載のタンパク質又は〔11〕に記載の分子センサを発現可能な、或いは、〔12〕に記載の核酸又は〔13〕に記載の発現カセットを有する、ベクター。
〔15〕 〔1〕から〔10〕のいずれかに記載のタンパク質又は〔11〕に記載の分子センサを発現する形質転換体。
〔16〕 配列番号1のアミノ酸配列の2番目から232番目の配列を有する蛍光タンパク質。
〔17〕 pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色である、〔16〕に記載の蛍光タンパク質。
〔18〕 配列番号1のアミノ酸配列の2番目から232番目の配列において1から数個のアミノ酸が欠失、置換、及び/又は付加されたアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
〔19〕 〔16〕から〔18〕のいずれかに記載の蛍光タンパク質のアミノ酸配列において1から数個のアミノ酸が欠失、置換、及び/又は付加されたアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
〔20〕 〔16〕から〔18〕のいずれかに記載の蛍光タンパク質のアミノ酸配列と少なくとも90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、又は99.5%の同一性を示すアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
〔21〕 〔16〕から〔20〕のいずれかに記載の蛍光タンパク質が融合された融合タンパク質であって、該蛍光タンパク質の部分は、pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色の蛍光タンパク質である、融合タンパク質。
〔22〕 配列番号1のアミノ酸配列の2番目から225番目の配列に、T197AorGの変異が導入されたアミノ酸配列を有する、〔16〕から〔20〕のいずれかに記載の蛍光タンパク質。
〔23〕 蛍光励起しない特定の波長の光照射によって無蛍光性から蛍光性になり、蛍光励起のための光照射によって蛍光性から無蛍光性になる蛍光タンパク質として機能可能な、〔22〕記載の蛍光タンパク質。
〔24〕 〔22〕又は〔23〕に記載の蛍光タンパク質が融合された融合タンパク質であって、該蛍光タンパク質の部分は、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色であり、蛍光励起しない特定の波長の光照射によって無蛍光性から蛍光性になり、蛍光励起のための光照射によって蛍光性から無蛍光性になる蛍光タンパク質として機能可能である、融合タンパク質。
〔25〕 〔16〕から〔24〕のいずれかに記載のタンパク質のアミノ酸配列の一部又は全部を有し、該タンパク質の蛍光特性を利用する、分子センサ。
〔26〕 〔16〕から〔24〕のいずれかに記載のタンパク質又は〔25〕に記載の分子センサをコードする塩基配列を有する核酸。
〔27〕 〔16〕から〔24〕のいずれかに記載のタンパク質又は〔25〕に記載の分子センサをコードする塩基配列含む発現カセット。
〔28〕 〔16〕から〔24〕のいずれかに記載のタンパク質又は〔25〕に記載の分子センサを発現可能な、或いは、〔26〕に記載の核酸又は〔27〕に記載の発現カセットを有する、ベクター。
〔29〕 〔16〕から〔24〕のいずれかに記載のタンパク質又は〔25〕に記載の分子センサを発現する形質転換体。
〔30〕 〔1〕から〔10〕及び〔16〕から〔24〕のいずれかに記載のタンパク質、〔11〕若しくは〔25〕に記載の分子センサ、〔12〕若しくは〔26〕に記載の核酸、〔13〕若しくは〔27〕に記載の発現カセット、又は〔14〕若しくは〔28〕に記載のベクターが導入された細胞から、前記タンパク質又は前記分子センサの蛍光シグナルを検出することを含む、イメージング方法。
〔31〕 〔8〕から〔10〕及び〔22〕から〔24〕のいずれかに記載の光スイッチング変異型緑黄色蛍光タンパク質が導入された細胞において、前記光スイッチング変異型蛍光タンパク質の光切替を行って蛍光性をon/offすること、及び/又は、前記光スイッチング変異型蛍光タンパク質の蛍光シグナルを検出することを含む、超解像イメージング又は超解像ライブイメージング方法。
dfGFPはハナガサクラゲ(学名:Olindias formosa)由来の蛍光タンパク質である。まずハナガサクラゲより全RNA抽出し、mRNAをオリゴTプライマーを用いてDNAへと逆転写し、cDNAライブラリーを作成した。このcDNAライブラリーをPCRで増幅したのち、バクテリア発現ベクターへと挿入した。これらのプラスミドを大腸菌に形質転換し、蛍光を発するコロニーを選別することで、ハナガサクラゲの蛍光タンパク質dfGFPをコードする遺伝子を同定した。該遺伝子にコードされるdfGFPのアミノ酸配列が、配列表の配列番号1である。
配列表の配列番号1のアミノ酸配列にF149T、L158T、H160T、Y174T、Y192A、K112E、E147K、Q145E、K166E、P162L、P223L、N37S、E140K、K185R、A150/E151/G152→PHGP、及びK180Tの変異を部位特異的に導入し、さらに、配列番号1のN末のMをEGFPのN末端7アミノ酸残基(MVSKGEE)に置換し、かつ、配列番号1のC末7アミノ酸残基(EPSASAV)をEGFPのC末端7アミノ酸残基(GMDELYK)に置換し、配列表の配列番号2のアミノ酸配列をコードする遺伝子を作製した(配列番号3)。該遺伝子にコードされるmfGFPのアミノ酸配列が、配列表の配列番号2である。
N末にポリヒスチジンタグを持つmfGFPをバクテリア発現ベクターpRSETBに導入し、大腸菌で発現させた。23℃65時間LB培地で培養した後、菌体をフレンチプレスで破砕し、上清をNi-NTAアガロースアフィニティカラム(Qiagen社製)、及びPD-10カラム(GE Healthcare社製)によるゲルフィルトレーションで精製し、さらに、AKTA Superdex200 10/300 GL(GE Healthcare)カラムで再精製した。
図1に、Superdex200 10/30カラムを用いた、サイズ除去ゲルろ過クロマトグラフフィーの結果を示す。溶媒は150mM NaClを含むpH7.4の20mM HEPES溶液。分子量・分子体積の大きいものほどカラム内を速く流れるため、図中では左側にピークが現れる。
図1の点線のスペクトルは左から順に10μMのDsRed(四量体)、tdTomato(二量体)、mCherry(単量体)の吸収を示す。実線のスペクトルは10μMの mfGFPの吸収を示す。mfGFPのピーク位置はmCherryのものとほぼ同じなので、mfGFPは単量体であるといえる。
精製した蛍光タンパク質の性質を測定した。その結果を図2に示す。蛍光スペクトルメーターを用いた測定により、mfGFPは504nmの励起ピーク、519nmの蛍光ピークを持つことがわかった。吸収スペクトル測定によりmfGFPの吸収ピークにおけるモル吸光係数は83,000M-1・cm-1、絶対蛍光量子収率の測定によりmfGFPの量子収率は0.90であることがわかった。
30mMクエン酸ナトリウムと30mMホウ酸の混合溶液を用いて、pH3から9のpHバッファーを0.5きざみに作製した。mfGFP又はEGFPを上記のバッファーに溶解させ、蛍光スペクトルメーターを用いて、蛍光強度を測定した(図3)。得られたデータを下に、ヒルの式上にフィッティングしたところ、mfGFPのpKa(蛍光タンパク質が存在する媒体のpHが塩基性pHから酸性pHになるときの、該蛍光タンパク質の蛍光強度が最大値の半分となるpH)としてpKa=3.5という値が得られた。
mfGFP又はEGFPをHeLa細胞の細胞質に発現させ、水銀ランプ光と440-480nmバンドパスフィルターを用いて光照射した際の光安定性を調べた(図4)。mfGFPとEGFPの蛍光強度半減時間はそれぞれ1.2分、0.6分であった。この結果は、mfGFPの光安定性はEGFPの約2倍高いことを示唆している。
mfGFP、EGFPそれぞれがもつ蛍光団の成熟速度比を調べた。それぞれの蛍光タンパク質を大腸菌に形質転換、無酸素環境下・37℃で1日―1.5日培養させることで、蛍光団が非成熟なタンパク質を作成した。タンパク質を空気に暴露することに伴う蛍光強度の時間変化を測定した(図5)。mfGFPとEGFPの蛍光団成熟にかかる半飽和時間は、それぞれ8.0分、14.9分であったことから、mfGFPの優れた蛍光団生成能が示唆される。
mfGFPに様々なオルガネラ・タンパク質への局在シグナルを融合させHeLa細胞に発現させた(アクチン、ゴルジ体、リン、パキシリン、ペルオキシソーム、ジキシン、ミトコンドリア、フィブリラリン、ビメンチン、チューブリン、コネキシン43、ヒストンH2B、ライフアクト、VAMP2、LAMP3.CathepsinB,LC3)。その結果を図6に示す。
図6に示すように、mfGFPの融合タンパク質は、目的のオルガネラ・タンパク質に正しい局在を示した。中でも局在の難しいチューブリンのラベル化に成功したことは、mfGFPの高い単量体としての性質を示唆している。また。内部が酸性に保たれている分泌小胞・リソソーム(pH4.5-5,5)内でも高い蛍光強度を示した。
mfGFP又はEGFPをHeLa細胞の細胞質に発現させ、3日間37℃/5%で培養したところ、mfGFPのみリソソーム様の蛍光ドット画像が得られた(図7)。これは、細胞質に存在していた蛍光タンパク質が、非選択的なマクロオートファジーによってリソソームへと運搬された結果だと考えられる。同様に、mfGFPが細胞質からリソソームへ蓄積させる過程をタイムラプスイメージングすることにも成功した(図7)。HeLa細胞のリソソームのpHは約4.7と低く、pH感受性の高いEGFPではこのようなドットは観察されなかった。
mfGFPに、T197A(またはT197G)アミノ酸変異を導入することで、mfGFPの耐酸性能を保ちながら、その蛍光・非蛍光状態を光波長依存的にコントロールできる変異体(光スイッチング変異体)が得られた。
T197A変異体を大腸菌に発現させ、その抽出物をpH5、6、7、8のバッファーと混合させたところ、蛍光強度が上記範囲のpHに影響を受けないことが判明した(図8)。また、同サンプルを顕微鏡下で観察したところ、500/24nm光照射により無蛍光状態へと移行、370/36nm光照射により蛍光状態に移行することが判明した(図9)。
配列番号2:mfGFPのアミノ酸配列
配列番号3:mfGFPの塩基配列
Claims (13)
- 配列番号1のアミノ酸配列の2番目から225番目の配列に少なくとも下記変異が導入されたアミノ酸配列を有する蛍光タンパク質。
ここで、前記変異は、F149TorSorA、L158TorSorA、H160TorSorA、Y174TorSorA、及び、Y192TorSorA、並びにこれらの2~5個の組合せからなる群から選択される。 - 配列番号1のアミノ酸配列の2番目から225番目の配列に、さらに下記の変異が導入されたアミノ酸配列を有する、請求項1記載の蛍光タンパク質。
ここで、前記変異は、K112EorDorQ、E147KorRorQ、Q145EorD、K166EorDorQ、P162LorVorIorA、P223LorVorIorA、N37SorAorG、E140KorRorQ、K185RorAorG、A150/E151/G152→PHGPorPHA、及び、K180TorS、並びにこれらの2~11個の組合せからなる群から選択される。 - 配列番号1のアミノ酸配列の2番目から225番目の配列に、さらに下記の変異が導入されたアミノ酸配列を有する、請求項1又は2に記載の蛍光タンパク質。
ここで、前記変異は、T197AorGである。 - 配列番号2のアミノ酸配列の8番目から232番目の配列を有する、請求項1から3のいずれかに記載の蛍光タンパク質。
- pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色である、請求項1から4のいずれかに記載の蛍光タンパク質。
- 請求項1から5のいずれかに記載の蛍光タンパク質のアミノ酸配列において1から数個のアミノ酸が欠失、置換、及び/又は付加されたアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色である、蛍光タンパク質。
- 請求項1から6のいずれかに記載の蛍光タンパク質が融合された融合タンパク質であって、該蛍光タンパク質の部分は、pH感受性(pKa)が4.0以下であり、単量体型であり、蛍光色が緑黄色の蛍光タンパク質である、融合タンパク質。
- 配列番号1のアミノ酸配列の2番目から232番目の配列を有する蛍光タンパク質、
或いは、
配列番号1のアミノ酸配列の2番目から232番目の配列において1から数個のアミノ酸が欠失、置換、及び/又は付加されたアミノ酸配列を有し、pH感受性(pKa)が4.0以下であり、二量体型であり、蛍光色が緑黄色である、蛍光タンパク質。 - 請求項1から8のいずれかに記載のタンパク質のアミノ酸配列の一部又は全部を有し、該タンパク質の蛍光特性を利用する、分子センサ。
- 請求項1から8のいずれかに記載のタンパク質又は請求項9に記載の分子センサをコードする塩基配列を有する核酸。
- 請求項1から8のいずれかに記載のタンパク質又は請求項9に記載の分子センサをコードする塩基配列含む発現カセット。
- 請求項1から8のいずれかに記載のタンパク質又は請求項9に記載の分子センサを発現可能な、或いは、請求項10に記載の核酸又は請求項11に記載の発現カセットを有する、ベクター。
- 請求項1から8のいずれかに記載のタンパク質又は請求項9に記載の分子センサを発現する形質転換体。
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SHINODA, H. ET AL.: "Novel green fluorescent protein from Olindias formosa with exceptional pH stability", BIOPHYSICS, vol. 55, no. Suppl. 1-2, 2015, pages S298 * |
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