JP2011097930A - Photoactivatable physiological function sensor protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein observable for concentration change of a biological material such as Ca<SP>2+</SP>as fluorescent intensity change by FRET (Fluorescence Resonance Energy Transfer) and a method for measuring concentration of the biological material. <P>SOLUTION: There is provided a chimera protein having a donor molecule of FRET and an acceptor molecule of FRET bonded to both terminals of a biological material-binding linker, wherein the donor molecule is a photoactivatable fluorescent protein, the acceptor molecule is a pigment protein or a weakly fluorescent protein, and the biological material binding linker can induce FRET by binding with the biological material. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a photoactivated physiological function sensor protein. This protein is useful as a reagent for basic research such as biological research, pharmaceutical research, medical research, and agricultural research.

For measuring the Ca ²⁺ ion concentration in biological samples, a Ca ²⁺ sensitive small electrode, a small molecule synthetic probe, and a protein probe have been used.

The Ca ²⁺ sensitive small electrode is a method for measuring the Ca ²⁺ concentration based on the Nernst potential generated between thin films having Ca ²⁺ selectivity. Although it is easy to measure the Ca ²⁺ concentration in the extracellular fluid, in order to measure the ion concentration inside the cell, it is necessary to insulate the small electrode and the plasma membrane of the cell with high resistance. There was a problem that it was not suitable for measuring the internal Ca ²⁺ concentration change.

Small molecule synthetic probes and protein probes are methods that measure Ca ²⁺ concentration based on changes in fluorescence or luminescence properties induced by changes in the microenvironment and conformational changes of the probe due to Ca ²⁺ binding. is there. Since it can be introduced into the cell, it is possible to measure Ca ²⁺ ions inside the cell. However, it has been difficult for small molecule synthetic probes to appear at specific locations within an organism or tissue. When a gene encoding a proteinaceous probe is introduced and expressed, it can be easily realized by placing the gene under the control of a promoter capable of causing gene expression at a specific location. is there.

As a proteinaceous probe, a fluorescent probe cameleon encoded by a fusion gene of a green fluorescent protein (GFP) mutant and a Ca ²⁺ binding protein has already been reported (Non-Patent Documents 1 to 4).

Proteinaceous probes are easy to localize in cells and organelles and can be measured under low excitation conditions, making them very suitable for noninvasive measurement of Ca ²⁺ concentration in living cells ing. cameleon is a chimeric protein consisting of a short-wave mutant of GFP, calmodulin (CaM), a glycylglycine linker (Gly-Gly), a CaM-binding peptide of myosin light chain kinase (M13), and a long-wave mutant of GFP. It is. Binding of Ca ²⁺ to CaM initiates an intermolecular interaction between CaM and M13, which changes the chimeric protein from an elongated conformation to a smaller conformation, resulting in a short wavelength mutation The efficiency of FRET <fluorescence resonance transfer> from the body GFP to the long wavelength mutant GFP increases.

  In addition, proteinaceous fluorescent probes that are chimeric proteins composed of a short-wave mutant of GFP, a F0F1-ATP synthase ε subunit, and a long-wave mutant of GFP have already been used as proteinaceous probes for measuring ATP concentration. It has been reported (Patent Document 1).

  However, even when these proteinaceous probes are used, there are cases where a specific promoter suitable for appearance in the cell at the place to be observed cannot be found, and the degree of freedom is limited.

  Regarding other biological substances, no technique has been reported that can measure the concentration of a biological sample, in particular, a specific place in an individual or a living organism in a noninvasive manner with a high degree of freedom.

WO2008 / 117792 International publication pamphlet

Miyawaki A et al., Nature 388,882-887,1997 Miyawaki A and others PNAS 96, 2135-2140,1999 Nagai T et al. Nature Biotechnol. 20, 87-90, 2002 Nagai T et al. PNAS 101, 10554-10559, 2004

The present invention relates to changes in the concentration of biological substances such as Ca ²⁺ concentration and ATP concentration by FRET at an arbitrary position by light stimulation in an individual organism, tissue, cell, intracellular organelle, and aqueous solution as a change in fluorescence intensity. Provided are a protein that can be observed and a method for measuring a concentration of a biological substance.

  The present invention uses a photoactivated fluorescent protein as a donor molecule for FRET, and a non-fluorescent chromoprotein or weak fluorescent protein (a fluorescent protein having a fluorescence quantum yield of 0.05 or less) as an acceptor molecule. It is characterized in that it was made into a model.

So far, DsRed (Bevis BJ et al., Nature Biotechnol.
20, 83-87, 2002), mCherry, mOrange, tdTomato (Shaner NC et al., Nature Biotechnol. 22, 1567-72, 2004), mKO (Karasawa
S. et al., Biochem. J. 381, 307-312, 2004), mKO2 (Sakurai-Sawano A et al., Cell 132, 487-498, 2008) However, when fluorescence measurement is performed after photoactivation in the cells, changes in fluorescence intensity due to changes in Ca ²⁺ concentration are not observed, or small changes that are difficult to distinguish from changes in fluorescence intensity due to noise. I could only get up. On the other hand, it is possible to produce a probe that causes a large change in fluorescence intensity by using a non-fluorescent chromoprotein or a fluorescent protein (weak fluorescent protein) having a fluorescence quantum yield of 0.05 or less.
Item 1. A chimeric protein in which a FRET donor molecule and a FRET acceptor molecule are bonded to both ends of a biological substance-binding linker, wherein the donor molecule is a photoactivated fluorescent protein, and the acceptor molecule is a chromoprotein or a weak fluorescent protein And the biological substance-binding linker is a chimeric protein capable of inducing FRET by binding to a biological substance.
Item 2. Item 2. The chimeric protein according to Item 1, wherein the donor molecule is photoactivated green fluorescent protein (PA-GFP), photoactivated red fluorescent protein (PA-mCherry), or a functional equivalent variant thereof.
Item 3. Item 3. The chimeric protein according to Item 1 or 2, wherein the acceptor molecule is asCP, REACh, Venus-Y145W, Venus-Y145W / H148V, or a functional equivalent variant thereof.
Item 4. Item 4. The chimeric protein according to any one of Items 1 to 3, wherein the biological substance is calcium ion or ATP.
Item 5. Item 5. The chimeric protein according to any one of Items 1 to 4, wherein the biological substance-binding linker has a structure in which calmodulin and a CaM-binding peptide (M13) of myosin light chain kinase are linked via a peptide as necessary. .
Item 6. Item 5. The chimeric protein according to any one of Items 1 to 4, wherein the biological substance-binding linker is F0F1-ATP synthase ε subunit protein or a functionally equivalent variant thereof.
Item 7. Item 7. A DNA encoding the chimeric protein according to any one of Items 1 to 6.
Item 8. Item 8. A recombinant vector having the DNA of Item 7.
Item 9. Item 9. A transformant having the DNA according to Item 7 or the recombinant vector according to Item 8.
Item 10. Item 7. A method for measuring the concentration of a biological material, wherein the chimeric protein according to any one of Items 1 to 6 and a biological material are allowed to coexist and luminescence from a donor molecule is detected.

According to the present invention, without the need for expression control by a gene promoter for site-specific expression, fluorescence is generated by light stimulation in any cell such as a biological solid or tissue, and Ca ²⁺ ions inside the cell It is possible to obtain a proteinaceous photoactivated probe that enables noninvasive measurement of the concentration of a biological substance such as the concentration and ATP concentration. By using them, highlighting only remarkable cells in a complex network of neurons and observing changes in the concentration of biological substances such as Ca ²⁺ ion concentration and ATP concentration inside Can do.

It is also possible to change the range of Ca ²⁺ ion concentration that can be measured by improving the CaM part, and to create a Ca ²⁺ probe using a Ca ²⁺ ion binding protein other than CaM such as troponin. It is. Furthermore, the CaM moiety can be applied to the production of a light-activated probe by changing it to a protein that causes structural changes by binding to another metal or substrate (Miyawaki A et al., Develop. Cell 4, 295- 305, 2003).

1 is a conceptual diagram of a chimeric protein of the present invention when CaM-M13 is used as a biological material binding linker. FIG. Intracellular Ca ²⁺ imaging using photo-activated Ca ²⁺ probe PA-GFP-asCP using non-fluorescent chromoprotein asCP572 derived from Anemonia sulcata (a) Activity of 405 nm light-stimulated probe expressed in HeLa cells Conversion. (Left) Before activation (center) After selecting and activating 3 cells (Right) After selecting and activating 3 cells. (b) Fluorescence intensity change accompanying intracellular Ca ²⁺ concentration change after histamine stimulation of the probe after activation. Intracellular Ca ²⁺ imaging with the light-activated Ca ²⁺ probe PA-GFP-cp173REACh using the weakly fluorescent protein REACh modified with EYFP (a) Activation of the probe expressed in HeLa cells by 405 nm light stimulation. (Left) Before activation (Right) After activation. (b) Fluorescence intensity change accompanying intracellular Ca ²⁺ concentration change after histamine stimulation of the probe after activation. Intracellular Ca ²⁺ imaging with photoactivated Ca ²⁺ probe cp173PA-GFP- (Venus-Y145W / H148V) using Venus-modified chromoprotein Venus-Y145W / H148V (a) Probe expressed in HeLa cells Activation by light stimulation of 405nm. (Left) Before activation (Right) After activation. (b) Fluorescence intensity change accompanying intracellular Ca ²⁺ concentration change after histamine stimulation of the probe after activation. Intracellular ATP imaging with photo-activated ATP probe PA-ATeam using non-fluorescent chromoprotein asCP572 (a) Activation of probe expressed in HeLa cells by light stimulation at 405 nm. (Left) Before activation (Right) After activation. (b) Fluorescence intensity change accompanying intracellular ATP concentration change after deoxyglucose stimulation of the probe after activation.

  Hereinafter, embodiments of the present invention will be described in detail.

(1)
Biological material-binding linker The biological material-binding linker used in the chimeric protein of the present invention is a linker that can promote or induce FRET by changing the three-dimensional structure when the biological material is bonded. This linker may contain calcium-binding proteins such as calmodulin and troponin, and may contain ATP-binding proteins such as the F0F1-ATP synthase ε subunit, metals other than calcium, and substrates other than ATP. It may be a protein that binds to and causes a structural change. The relationship between the biological substance to be bound and the binding protein is exemplified below.

A preferred biological material-binding linker is a calcium ion-binding linker that undergoes a structural change by binding to calcium ions. Examples of such a calcium ion-binding linker include a proteinaceous linker in which calmodulin (CaM) and a myosin light chain kinase CaM-binding peptide (M13) are connected by a peptide. The peptide is composed of 2 to 12, preferably 3 to 8, more preferably 3 to 5 amino acids. This biological material-binding linker changes the structure of the biological material-binding linker when Ca ²⁺ binds to CaM, so that both ends (N-terminal and C-terminal) of the biological material-binding linker are close together. When a donor molecule and an acceptor molecule are connected to both ends of the functional linker, transfer from the donor molecule to the acceptor molecule (FRET) occurs.

The calmodulin (CaM), can be combined with Ca ^2+, and, Ca ²⁺ bound CaM can be widely used natural calmodulin and variants thereof as long as it can bind to M13.

  M13 is a peptide having 26 amino acids: KRRWKKNFIAVSAANRFKKISSSGAL.

  CaM and M13 are linked by a peptide. Conventionally, GlyGly has been used as this linker peptide. The amino acid constituting the linker peptide is arbitrary, but preferred amino acids are Gly, Ala, Ser, Thr, Leu, Ile, Val, Pro, Cys, Met, Phe, Tyr, Trp, Asn, Gln, Asp, Glu , Lys, Arg, His, etc., preferably Gly, Ala, Ser, Thr, more preferably Gly, Ser. The linker peptide preferably contains at least one of Gly and Ser, for example GlyGlySer, GlyGlyGlySer, GlyGlyGlyGlySer and the like.

Another preferred biological substance-binding linker is an ATP-binding linker that undergoes a structural change by binding to ATP. Examples of such ATP-binding linkers include F0F1-ATP synthase ε subunit protein. The ε subunit protein used in the present invention may be derived from any species, but for example, derived from Bacillus sp. PS3 (GenBank accession No. AB044942) or derived from Bacillus subtilis (GenBank accession
The ε subunit protein shown in No. Z28592) can be preferably used.

  The biological material-binding linker used in the present invention may also be a functional equivalent variant of the biological material-binding linker. “Functional equivalent variant” is an amino acid sequence in which one or more (particularly one or several) amino acids are deleted, substituted, or added in the amino acid sequence of the original protein. In addition, it means a protein that exhibits substantially the same activity as the original fusion 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.

(2) Chimeric protein The chimeric protein of the present invention has a structure in which a donor molecule and an acceptor molecule are linked directly or via a linker peptide to the N-terminus and C-terminus of a biological substance-binding linker.

  Donor molecules include fluorescent proteins, particularly photoactivated fluorescent proteins. Acceptor molecules include chromoproteins or weakly fluorescent proteins.

  Examples of fluorescent proteins include CFP, YEP, GFP, RFP, BFP, DsRed, and the like, and mutants thereof may be used. Examples of the mutant include ECFP, EYFP, EGFP, ERFP, and EBFP. Preferred fluorescent proteins are photoactivated fluorescent proteins, such as photoactivated green fluorescent protein (PA-GFP), photoactivated red fluorescent protein (PA-mCherry: Subach FV et al., Nat. Methods, 6, 153-159 , 2009), and these mutants, especially circular permutations.

  As the chromoprotein, a non-fluorescent chromoprotein is preferable. For example, asCP (Lukyanov KA et al., J. Biol. Chem. 275, 25879-25882, 2000), tyrosine at position 145 of the yellow fluorescent protein Venus is substituted with tryptophan. A chromoprotein Venus-Y145W which is a mutant, a chromoprotein Venus-Y145W / H148V which is a mutant obtained by substituting tyrosine at position 145 with tryptophan and histidine at position 148 with valine in yellow fluorescent protein Venus, and variants thereof, In particular, circular permutants and the like can be mentioned. asCP is a chromoprotein derived from the sea anemone Anemonia sulcata, and amino acid sequence information is described as wild-type asFP595 in the literature of Lukyanov et al. (Lukyanov KA et al., J. Biol. Chem. 275, 25879-25882, 2000).

As weak fluorescent protein, REACh (Ganasan
S, et al., Proc. Natl. Acad. Sci. USA 103, 4089-4094, 2006), and mutants thereof, particularly circular permutants. REACh is a weak fluorescent protein produced by further introducing a mutation in EYFP, a modified GFP derived from Aequorea jellyfish. The amino acid sequence information is Ganasan et al. (Ganasan S et al., Proc. Natl. Acad. Sci. USA 103, 4089). -4094, 2006).

  The donor molecule and the acceptor molecule may also be a functional equivalent variant of the fluorescent protein, chromoprotein, or weak fluorescent protein.

  Either the donor molecule or the acceptor molecule may be linked to the N-terminus of the biological material-binding linker, but it is preferable to bind the donor molecule to the N-terminus.

  The biological substance-binding linker and the donor molecule or acceptor molecule can be linked directly or via a linker peptide. The linker peptide is not particularly limited, as is the case with the linker peptide connecting CaM and M13 described above, and a peptide linker having a length of about 2 to 20 amino acids can be used. In addition, the amino acid used for the peptide linker is not limited, but for example, glycine (G), serine (S), threonine (T) and the like can be used. As specific linker peptides, GlyGlySer, Preferred examples include GlyGlyGlySer and GlyGlyGlyGlySer.

  In the chimeric protein of the present invention, when the donor molecule (fluorescent protein) is excited by light irradiation, the energy transfer efficiency from the donor molecule to the acceptor molecule changes depending on the concentration of the biological substance, and the concentration of the biological substance is detected by detecting it. It functions as a sensor for measuring.

  The method for obtaining the chimeric protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique.

  When producing a recombinant protein, it is necessary to first obtain DNA encoding the chimeric protein. The amino acid sequences and base sequences of various proteins used as donor molecules and acceptor molecules are known to those skilled in the art, and DNAs encoding them can be obtained from commercial products, or can be obtained by ordinary genetic recombination such as PCR. Can be cloned by techniques. The DNA encoding the chimeric protein of the present invention can be constructed by linking the DNA encoding the donor molecule and the acceptor molecule thus obtained with the DNA encoding the biological material binding linker by genetic recombination technology. . By introducing this DNA into an appropriate expression system, the chimeric protein of the present invention can be produced.

(3) DNA
According to the present invention, DNA encoding the chimeric protein of the present invention is provided.

The GFP gene has been isolated and sequenced (Prasher, DC. Et al. (1992), “Primary
structure of the Aequorea victoria
green fluorescent protein ", Gene 111: 229-233). Many other amino acid sequences of fluorescent proteins or variants thereof have also been reported. For example, Roger Y. Tsien, Annu. Rev. Biochem. 1998, 67: 509- 44, as well as the references cited therein, GFP, YFP or a variant thereof can be derived from, for example, Aequorea victoria (eg, Aequorea victoria).

  The base sequence of the gene encoding the fluorescent protein used in the present invention is known. A commercially available gene can be used as the gene encoding the fluorescent protein. For example, an EGFP vector, an EYFP vector, an ECFP vector, an EBFP vector, etc., commercially available from Clontech can be used.

  The DNA of the present invention can be synthesized by, for example, the phosphoramidite method or can be produced by polymerase chain reaction (PCR) using specific primers.

  A method for introducing a desired mutation into a predetermined nucleic acid sequence is known to those skilled in the art. For example, constructing DNA having mutations by appropriately using known techniques such as site-directed mutagenesis, PCR using degenerate oligonucleotides, exposure of cells containing nucleic acids to mutagens or radiation, etc. Can do. Such known techniques include, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplements 1-38, John. Wiley &amp; Sons (1987-1997).

(4) Recombinant vector The DNA of the present invention can be used by inserting it into an appropriate vector. The type of the vector used in the present invention is not particularly limited, and may be, for example, an autonomously replicating vector (for example, a plasmid), or it is integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes.

  Preferably, the vector used in the present invention is an expression vector. In the expression vector, the DNA of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

Promoters that can operate in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene (Bacillus stearothermophilus maltogenic amylase gene), the Bacillus licheniformis alpha-amylase gene (Bacillus licheniformis alpha-amylase gene).
gene), Bacillus amyloliquefaciens BAN amylase gene (Bacillus amyloliquefaciens)
BAN amylase gene), Bacillus Subtilis alkaline protease gene or Bacillus pumilus xylosldase gene promoter, phage lambda PR or PL promoter, E. coli lac, trp Or a tac promoter etc. are mentioned.

  Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa calihornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are promoters. Examples of a promoter operable in a yeast host cell include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, an ADH2-4c promoter, and the like.

Moreover, the DNA of the present invention may be operably linked to an appropriate terminator such as human growth hormone terminator or TPI1 terminator or ADH3 terminator for fungal hosts, if necessary. The recombinant vectors of the present invention further comprise a polyadenylation signal (eg, from SV40 or the adenovirus 5E1b region), a transcription enhancer sequence (eg, SV40 enhancer) and a translation enhancer sequence (eg, adenovirus VA).
Such as those encoding RNA).

  The recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). .

  The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin.

  Methods for ligating the DNA of the present invention, promoter, and optionally terminator and / or secretory signal sequence, respectively, and inserting them into a suitable vector are well known to those skilled in the art.

(5) Transformant A transformant can be prepared by introducing the DNA or recombinant vector of the present invention into an appropriate host.

  The host cell into which the DNA or recombinant vector of the present invention is introduced may be any cell as long as it can express the DNA construct of the present invention, and examples thereof include bacteria, yeast, fungi, and higher eukaryotic cells.

  Examples of bacterial cells include Gram positive bacteria such as Bacillus or Streptomyces or Gram negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method.

  Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, for example, Saccharomyces cerevisiae or Saccharomyces kluybergi (Saccharomyces
kluyveri) and the like. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  Examples of other fungal cells are cells belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  The transformant is cultured in a suitable nutrient medium under conditions that allow expression of the introduced DNA construct. In order to isolate and purify the chimeric protein of the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used.

  For example, when the protein of the present invention is expressed in a dissolved state in the cell, after the culture is completed, the cell is collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Use a hydrophobic chromatography method, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination to obtain a purified preparation be able to.

(5) Method for Measuring Concentration of Biological Substance The present invention further provides a method for measuring the concentration of biological substance, characterized in that the chimeric protein and the biological substance coexist to detect luminescence (fluorescence) from a donor molecule. .

  The measurement target is not particularly limited, and examples thereof include solid surfaces such as metals or plastics, liquids such as aqueous solutions or organic solvents, individual organisms, tissues, cells, intracellular organelles, and microorganisms. However, it can be suitably used for measuring the concentration of biological substances in individual organisms, tissues, cells, intracellular organelles, and microorganisms, which are difficult to measure temperature, particularly with ordinary measurement methods. .

  In order for the chimeric protein to coexist with a biological substance, the chimeric protein itself may be introduced into the measurement target, and the DNA encoding the chimeric protein, the recombinant vector having the DNA, or the transformant having the DNA or vector may be used. It may be introduced into the measurement target and the chimeric protein may be expressed inside the target.

  Examples of methods for introducing the chimeric protein and DNA into an individual organism include, for example, oral administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intravaginal administration, intracapsular administration, intradermal administration, intrapulmonary administration Administration, inhalation, subcutaneous administration, ophthalmic administration, intravitreal administration, subconjunctival administration, intraconjunctival sac administration, or transdermal administration may be mentioned. Examples of the method for introduction into a tissue include transfection, gene gun, viral vector, electroporation, photoporation, injection by injection, and the like. Examples of methods for introducing into cells, intracellular organelles, and microorganisms include transformation of competent cells, electroporation, or transfection.

  The biological material concentration is measured by detecting light emitted from the donor molecule after the chimeric protein and the biological material are allowed to coexist. First, the chimeric protein is irradiated with light of a specific wavelength, and photoactivation is performed so that the photoactivated fluorescent protein that is a donor molecule emits fluorescence. The wavelength of light used for photoactivation is determined by the photoactivated fluorescent protein used. For example, PA-GFP is 380 to 420 nm, preferably 405 nm, and PA-mCherry is 380 to 420 nm, preferably 405 nm. .

  When the chimeric protein is photoactivated and irradiated with excitation light having a predetermined wavelength, the photoactivated fluorescent protein of the donor molecule emits fluorescence. When the concentration of the biological substance to be measured is low, the fluorescence from the donor molecule is not affected by the chromoprotein or weak fluorescent protein that are acceptor molecules. On the other hand, when the concentration of the biological substance to be measured increases, the biological substance binds to the biological substance-binding linker moiety of the chimeric protein and the three-dimensional structure of the chimeric protein changes, and fluorescence from the donor molecule is absorbed by the acceptor molecule by FRET. As a result, the fluorescence intensity decreases. FIG. 1 shows a conceptual diagram of the present invention when PA-GFP is used as a donor molecule, asCP is used as an acceptor molecule, and CaM-M13 is used as a biological material binding linker.

  Unlike the normal FRET pair, the acceptor molecule is a chromoprotein or weakly fluorescent protein, so that the acceptor molecule does not emit fluorescence at all or does not emit much. Therefore, the fluorescence emitted from the donor molecule of the chimeric protein can be measured, and the concentration of the biological substance can be calculated and determined from the intensity change. Specifically, by preparing a calibration curve using a standard substance whose concentration is known in advance, the fluorescence intensity emitted from the actual measurement object can be calculated from the calibration curve.

  The excitation light is determined by the photoactivated fluorescent protein used, for example, PA-GFP is 460 to 490 nm, preferably 488 nm, and PA-mCherry is 540 to 580 nm, preferably 564 nm.

  The wavelength of the fluorescence emitted from the chimeric protein is determined by the photoactivated fluorescent protein used. However, in the measurement method of the present invention, the fluorescence intensity of a specific wavelength may be measured, and the fluorescence spectrum (wavelength of a certain range of wavelengths) May be measured. For example, for example, PA-GFP is 517 nm or 505 to 540 nm, and PA-mCherry is 595 nm or 590 to 650 nm.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Intracellular Ca ²⁺ imaging with a photoactivated Ca ²⁺ probe PA-GFP-asCP using a non-fluorescent chromoprotein asCP572 derived from Anemonia sulcata Calcium ion sensor cameleonYC3.60 (GenBank, AB178712) Mammalian cell expression vector cameleonYC3.6 / pcDNA3 (Nagai) in which the encoding gene is inserted between the BamHI and EcoRI sites of plasmid pcDNA3 (Invitrogen)
T et al., Proc. Natl. Acad. Sci.
USA 101, 10554-10559, 2004), a photoactivated Ca ²⁺ sensor protein expression vector was prepared. Photoactivated green fluorescent protein PA-GFP was used as the FRET donor molecule, and non-fluorescent chromoprotein asCP was used as the acceptor molecule.
A gene encoding ECFP with deletion of the C-terminal 11 residues between BamHI and SphI sites, which are FRET donor molecules of CameleonYC3.6 / pcDNA3, and a deletion of the C-terminal 11 residues of PA-GFP. It was replaced with the encoding gene. Furthermore, the gene sequence encoding the Venus circular permutation cp173Venus between the SacI and EcoRI sites, which are FRET acceptor molecules, was categorized into a non-fluorescent chromoprotein asCP (Lukyanov KA et al., J. Biol. Chem. 275 , 25879-25882, 2000).
A stable expression strain of HeLa cells transformed with this expression vector was cultured on a 35 mm glass bottom dish containing 1.5 mL medium and observed. Imaging was performed with a confocal inverted microscope FV1000 (Olympus) equipped with a UPLSAPO60x (NA1.35) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. Laser light of 488 nm was used as the excitation light wavelength and 405 nm was used as the stimulation light for photoactivation, and images were acquired at intervals of 3 seconds in the time lapse measurement.

Under microscopic observation, 6 cells that were not light-stimulated on the dish were selected (Fig. 2-a left). When 3 cells were irradiated with stimulating light of 405nm, FRET donors were selectively used with the irradiated 3 cells. The fluorescence of the molecule PA-GFP was able to appear (Fig. 2-a center). Furthermore, the remaining 3 cells were irradiated with stimulating light, and fluorescence was able to appear in all 6 cells (FIG. 2-a right).
In these 6 cells emitting fluorescence, intracellular Ca ²⁺ by histamine stimulation was further time-lapse observed. After obtaining 20 frames from the start of measurement, 0.5 mM histamine solution was added dropwise in the vicinity of the cells to be observed so that the final concentration was 5 μM. When the relative fluorescence intensity obtained by dividing the average value of the fluorescence intensity of 20 frames before dropping histamine by the fluorescence intensity at each time is plotted against time, the increase in relative fluorescence intensity with increasing Ca ²⁺ concentration immediately after histamine stimulation Was observed (FIG. 2-b). Further, from about 5 minutes after the addition, an increase in spike-like relative fluorescence intensity (about 10%) accompanying the Ca ²⁺ concentration oscillation was observed. These behaviors were similar to those seen in existing HeLa cells expressing cameleon YC3.60. The measurable range of Ca ²⁺ concentration that can be predicted is about several hundreds nM as in cameleon YC3.60, and the dynamic range is about 10%.

[Example 2] Intracellular Ca2 + imaging using the light-activated Ca2 + probe PA-GFP-cp173REACh using the weakly fluorescent protein REACh modified with EYFP The gene encoding the calcium ion sensor cameleonYC3.60 (GenBank, AB178712) Mammalian cell expression vector cameleonYC3.6 / pcDNA3 (Nagai) inserted between BamHI and EcoRI sites of Invitrogen
T et al., Proc. Natl. Acad. Sci.
USA 101, 10554-10559, 2004), a light-activated Ca ²⁺ sensor protein expression vector was prepared. Weakly fluorescent protein REACh2 (Ganesan S et al., Proc. Natl.) Using PA-GFP as a FRET donor molecule and a Y145W / H148V mutation introduced into EYFP as a yellow fluorescent protein
Acad. Sci. USA 103, 4089-4094, 2006), a circular permutation mutant cp173REACh2.
The gene encoding ECFP in which the C-terminal 11 residues between the BamHI and SphI sites of Cameleon YC3.6 / pcDNA3 were deleted was replaced with the gene encoding PA-GFP from which the C-terminal 11 residues were deleted. . Furthermore, the Venus circular permutation cp173Venus between SacI and EcoRI sites, which are FRET acceptor molecules, was replaced with a gene encoding cp173REACh2. The prepared expression vector was transduced into HeLa cells cultured on a 35 mm glass bottom dish using Superfect (QIAGEN), and observed 24-48 hours later.
Imaging was performed with a confocal inverted microscope FV1000 (Olympus) equipped with a UPLSAPO60x (NA1.35) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. Laser light of 488 nm was used as the excitation light wavelength and 405 nm was used as the stimulation light for photoactivation, and images were acquired at intervals of 3 seconds in the time lapse measurement.

Under microscopic observation, two cells that were not light-stimulated on the dish were selected (Figure 3-a, left). When irradiated with 405 nm stimulation light, the FRET donor molecule PA- GFP fluorescence could appear (Fig. 3-a right).
With respect to these two cells emitting fluorescence, a time-lapse observation of changes in intracellular Ca ²⁺ concentration by histamine stimulation was further performed. After obtaining 20 frames from the start of measurement, 0.5 mM histamine solution was added dropwise in the vicinity of the cells to be observed so that the final concentration was 5 μM. Plotting the relative fluorescence intensity divided by the fluorescence intensity at each time with the average value of the fluorescence intensity of 20 frames before dropping histamine, the increase in fluorescence intensity with increasing Ca ²⁺ concentration immediately after histamine stimulation Observed (FIG. 3-b). In addition, a spike-like increase in relative fluorescence intensity (about 15% of the average fluorescence intensity before stimulation) accompanying the Ca ²⁺ concentration oscillation was observed after addition. These behaviors were similar to those seen in existing HeLa cells expressing cameleon YC3.60. The measurable range of Ca ²⁺ concentration that can be predicted is about several hundreds nM as in cameleon YC3.60, and the dynamic range is about 10%.

[Example 3] Intracellular Ca ²⁺ imaging with photoactivated Ca ²⁺ probe cp173PA-GFP- (Venus-Y145W / H148V) using chromoprotein Venus-Y145W / H148V modified with Venus Calcium ion sensor cameleonYC3.60 Mammalian cell expression vector cameleonYC3.6 / pcDNA3 (Nagai) in which the gene encoding (GenBank, AB178712) is inserted between the BamHI and EcoRI sites of plasmid pcDNA3 (Invitrogen)
T et al., Proc. Natl. Acad. Sci.
USA 101, 10554-10559, 2004), a photoactivated Ca ²⁺ sensor protein expression vector was prepared. The circular permutation cp173PA-GFP of PA-GFP was used as the FRET donor molecule, and the mutant Venus-Y145W / H148V in which the Y145W / H148V mutation was introduced into the yellow fluorescent protein Venus was used as the acceptor molecule.
Replacing the gene encoding ECFP with the C-terminal 11 residues deleted between the BamHI and SphI sites of CameleonYC3.6 / pcDNA3 with the gene encoding Venus-Y145W / H148V with the C-terminal 11 residues deleted did. Furthermore, the Venus circular permutation cp173Venus between the SacI and EcoRI sites, which are FRET acceptor molecules, was replaced with a gene encoding the PA-GFP circular permutation cp173PA-GFP. The prepared expression vector was transduced into HeLa cells cultured on a 35 mm glass bottom dish using Superfect (QIAGEN), and observed 24-48 hours later.
Imaging was performed with a confocal inverted microscope FV1000 (Olympus) equipped with a UPLSAPO60x (NA1.35) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. Laser light of 488 nm was used as the excitation light wavelength and 405 nm was used as the stimulation light for photoactivation, and images were acquired at intervals of 3 seconds in the time lapse measurement.

Under microscopic observation, 2 cells that were not light-stimulated on the dish were selected (Fig. 4-a left). When irradiated with 405 nm stimulating light, FRET donor molecule PA- GFP fluorescence could appear (Fig. 4-a right).
With respect to these two cells emitting fluorescence, a time-lapse observation of changes in intracellular Ca ²⁺ concentration by histamine stimulation was further performed. After obtaining 20 frames from the start of measurement, 0.5 mM histamine solution was added dropwise in the vicinity of the cells to be observed so that the final concentration was 5 μM. Plotting the relative fluorescence intensity divided by the fluorescence intensity at each time with the average value of the fluorescence intensity of 20 frames before dropping histamine, the increase in fluorescence intensity with increasing Ca ²⁺ concentration immediately after histamine stimulation Observed (FIG. 4-b). In addition, a spike-like increase in relative fluorescence intensity (about 15% of the average fluorescence intensity before stimulation) accompanying the Ca ²⁺ concentration oscillation was observed after addition. These behaviors were similar to those seen in existing HeLa cells expressing cameleon YC3.60. The measurable range of Ca ²⁺ concentration that can be predicted is about several hundreds nM as in cameleon YC3.60, and the dynamic range is about 10%.

[Example 4] Intracellular ATP imaging with a photoactivated ATP probe using non-fluorescent chromoprotein asCP572
Mammalian cell expression vector pCDNA-AT1.03 (Imamura H et al., Proc. Natl) in which the gene encoding ATP sensor ATeam (Patent Document 1) is inserted between XhoI and HindIII sites of plasmid pcDNA3.1 (-) (Invitrogen) Acad. Sci. USA 106, 15651-15656, 2009), a photoactivated ATP sensor protein expression vector was prepared. Cp173Venus-Y145W which used PA-GFP as a FRET donor molecule, and introduced a circular permutation into a chromoprotein mutant Venus-Y145W that was introduced into the yellow fluorescent protein Venus as a acceptor molecule.
Was used.
The gene encoding ECFP from which the C-terminal 11 residues between the XhoI and ClaI sites of pCDNA-AT1.03 were deleted was replaced with a gene encoding PA-GFP from which the C-terminal 11 residues were deleted. Furthermore, the Venus circular permutation cp173Venus between the SacI and EcoRI sites, which are FRET acceptor molecules, was replaced with the gene encoding the circular permutation cp173Venus-Y145W of Venus-Y145W. The prepared expression vector was transduced into HeLa cells cultured on a 35 mm glass bottom dish using Superfect (QIAGEN), and observed 24-48 hours later.
Imaging was performed with a confocal inverted microscope FV1000 (Olympus) equipped with a UPLSAPO60x (NA1.35) oil objective lens, a multi-argon laser light source for observation, and a semiconductor laser for light stimulation. Laser light of 488 nm was used as the excitation light wavelength and 405 nm was used as the stimulation light for photoactivation, and images were acquired at 1-minute intervals in the time lapse measurement.

Under microscopic observation, two cells that were not light-stimulated on the dish were selected (Fig. 5-a left). When irradiated with 405 nm stimulating light, the FRET donor molecule PA- GFP fluorescence could appear (Fig. 5-a right).
These two cells emitting fluorescence were further observed with a time lapse of changes in intracellular ATP concentration by 2-deoxyglucose stimulation. 1M 2-deoxyglucose after 10 frames from the start of measurement
The solution was dropped in the vicinity of the cells to be observed so that the final concentration was 10 mM. When the relative fluorescence intensity divided by the fluorescence intensity at each time was plotted against the average value of the fluorescence intensity of 10 frames before 2-deoxyglucose was added, a decrease in the relative fluorescence intensity with a decrease in ATP concentration was observed. (Fig. 5-b). The dynamic range is about 30%.

Claims (10)

A chimeric protein in which a FRET donor molecule and a FRET acceptor molecule are bonded to both ends of a biological substance-binding linker, wherein the donor molecule is a photoactivated fluorescent protein, and the acceptor molecule is a chromoprotein or a weak fluorescent protein And the biological substance-binding linker is a chimeric protein capable of inducing FRET by binding to a biological substance. The chimeric protein according to claim 1, wherein the donor molecule is photoactivated green fluorescent protein (PA-GFP), photoactivated red fluorescent protein (PA-mCherry), or a functional equivalent variant thereof. The chimeric protein according to claim 1 or 2, wherein the acceptor molecule is asCP, REACh, Venus-Y145W, Venus-Y145W / H148V, or a functional equivalent variant thereof. The chimeric protein according to claim 1, wherein the biological substance is calcium ion or ATP. The chimera according to any one of claims 1 to 4, wherein the biological substance-binding linker has a structure in which calmodulin and a CaM-binding peptide (M13) of myosin light chain kinase are linked via a peptide as necessary. protein. The chimeric protein according to any one of claims 1 to 4, wherein the biological substance-binding linker is F0F1-ATP synthase ε subunit protein or a functionally equivalent variant thereof. DNA encoding the chimeric protein according to any one of claims 1 to 6. A recombinant vector comprising the DNA according to claim 7. A transformant comprising the DNA according to claim 7 or the recombinant vector according to claim 8. A method for measuring a concentration of a biological material, wherein the chimeric protein according to any one of claims 1 to 6 and a biological material are allowed to coexist and light emission from a donor molecule is detected.