WO2013094359A1 - セルロース/キチン系高分子発光材料 - Google Patents
セルロース/キチン系高分子発光材料 Download PDFInfo
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- WO2013094359A1 WO2013094359A1 PCT/JP2012/080241 JP2012080241W WO2013094359A1 WO 2013094359 A1 WO2013094359 A1 WO 2013094359A1 JP 2012080241 W JP2012080241 W JP 2012080241W WO 2013094359 A1 WO2013094359 A1 WO 2013094359A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Definitions
- the present invention relates to a chimeric protein capable of binding to cellulose and / or chitin, DNA encoding the chimeric protein, a complementary strand thereof, and a luminescent material.
- Polymeric hydrolase often has a binding domain for a substrate, for example, cellulase is known to have a cellulose binding domain, and chitinase is known to have a chitin binding domain.
- Patent Document 1 Patent No. 4604185 discloses a domain that is heat resistant and binds to both chitin and cellulose.
- Patent Document 2 discloses a fusion protein of luciferase and fluorescent protein with high efficiency of bioluminescence resonance energy transfer (BRET) based on BAF (BRET-based Auto-illuminated Fluorescent-protein) technology. ing.
- BRET bioluminescence resonance energy transfer
- An object of the present invention is to provide a new technique using a light emitting domain.
- the present invention provides the following chimeric protein capable of binding to cellulose and / or chitin, DNA encoding the chimeric protein, its complementary strand, and a luminescent material.
- Item 1 A chimeric protein comprising a luminescent domain and a cellulose and / or chitin binding domain, wherein the luminescent domain comprises at least one photoprotein selected from the group consisting of luciferase and fluorescent protein.
- Item 2 The chimeric protein according to Item 1, wherein the luminescent domain and the cellulose and / or chitin-binding domain are bound directly or via a first linker.
- Item 3 The chimeric protein according to Item 1 or 2, wherein the luminescent domain includes luciferase and a fluorescent luminescent protein, and energy transfer (BRET) from the luciferase to the fluorescent luminescent protein can occur.
- Item 4 The chimeric protein according to Item 3, wherein the luciferase and the fluorescent protein are bound via a second linker.
- Item 5 The chimeric protein according to any one of Items 1 to 4, wherein the fluorescent protein is GFP, YFP, BFP, CFP, OFP, DsRED, or RFP.
- Item 6 The chimeric protein according to Item 5, wherein the fluorescent protein is YFP or RFP.
- Item 7 The chimeric protein according to any one of Items 1 to 6, wherein the first linker and / or the second linker includes a protease cleavage sequence.
- Item 8 DNA encoding the chimeric protein according to any one of Items 1 to 7, or a complementary strand thereof.
- Item 9 A luminescent material obtained by binding the chimeric protein according to any one of Items 1 to 7 to particles, beads, sheets, or films containing cellulose or chitin.
- the chimeric protein of the present invention can be bound to materials such as particles of biopolymer materials such as cellulose and chitin, beads, sheets, films, etc., and can retain activity for a long time in a dry state. Since the photoprotein generally deactivates when dried, the chimeric protein of the present invention is excellent as a luminescent material.
- the procedure for evaluating the drying resistance (storage at room temperature) after air-drying of the chimeric protein-coated / bonded paper pieces is shown. Each process is as follows. (A) An aqueous solution of the chimeric protein is dropped onto a circular filter paper piece cut out by a punch to bind the chimeric protein to the filter paper. (A-1) Dry the filter paper piece. (A-2) Store the filter paper pieces at room temperature. (B) Add buffer. (B) This wets the filter paper pieces. (C) Add the aqueous luciferin solution to the buffer containing the filter paper pieces. (C) The luciferase activity is measured on the obtained wet filter paper piece. The room temperature dry storage performance of CBD-BAF bonded paper pieces is shown.
- A An example in which a CBD-BAF-Ym3 binding paper piece (filter paper) stored dry at room temperature is reacted with a buffer and a luciferin aqueous solution is shown.
- A A bright-field image, (b) a merged image, and (c) a chemiluminescent image are shown.
- a a circular area at the center of the filter paper piece indicates a coated area of CBD-BAF-Ym3.
- chemiluminescence image (c) green light emission was observed in the coated area of CBD-BAF.
- the horizontal axis represents the storage period (weeks) of the filter paper pieces in a room temperature dry state.
- the vertical axis represents the measured emission intensity (x10 8 RLU).
- the dried filter paper was stored at a room temperature of 27 ° C.
- the long-term room temperature dry storage performance of hCBD-eBAF-Ym3 bonded paper pieces is shown.
- A A schematic diagram of the structure of hCBD-eBAF-Ym3 is shown. In order from the N-terminal side, hCBD which is a cellulose and / or chitin-binding domain and eBAF-Ym3 which is a luminescent domain are included.
- the horizontal axis represents the storage period (weeks) of the filter paper pieces in a room temperature dry state.
- the vertical axis represents the measured light emission intensity (RLU).
- the dried filter paper was stored at room temperature of 22-27 ° C.
- the storage period of 52 weeks indicates a storage period of one year.
- the long-term room-temperature dry storage performance of hCBD-eBAF-R 3 binding paper pieces having a protease recognition sequence is shown.
- a schematic diagram of the structure of hCBD-eBAF-R 3 is shown.
- HRV-3C cleavage sequence is a protease recognition sequence is a cellulose and / or chitin-binding domain.
- the horizontal axis represents the storage period (weeks) of the filter paper pieces in a room temperature dry state.
- the vertical axis represents the measured light emission intensity (RLU).
- the dried filter paper was stored at room temperature of 22-26 ° C.
- the storage period of 52 weeks indicates a storage period of one year.
- FIG. 1 A schematic diagram of the structure of hCBD-eBAF-R 4 is shown. In order from the N-terminal side, hCBD that is a cellulose and / or chitin-binding domain, a linker into which a HRV-3C cleavage sequence that is a protease recognition sequence is introduced, and eBAF-R 4 that is a luminescence domain.
- the vertical axis represents the measured light emission intensity (RLU).
- the dried filter paper was stored at room temperature of 26 ° C. The storage period of 52 weeks indicates a storage period of one year.
- the procedure of the Example of a protease activity detection model system is shown.
- (a) (a-1) A chimeric protein aqueous solution is dropped onto a piece of filter paper and bound to the filter paper. This was dried (i) to produce a chimeric protein-bound filter paper piece (i).
- A-2) The reaction buffer (ii) was added and wetted sufficiently. Then, the buffer was removed, and a buffer solution (iii) containing protease was added again.
- A-3) The obtained sample was allowed to stand at 4 ° C.
- a model system mechanism is schematically shown.
- A-1 A state in which the chimeric protein is bound to the filter paper is schematically shown.
- the chimeric protein has, in order from the N-terminal side, hCBD that is a cellulose and / or chitin binding domain, a linker into which a HRV-3C cleavage sequence that is a protease recognition sequence is introduced, and BAF that is a luminescence domain.
- the HRV-3C cleavage sequence is LEVLFQ / GP (/ indicates the cleavage site).
- A-2) The chimeric protein is cleaved by the action of HRV-3C protease, and the BAF moiety is released to the aqueous layer.
- (B) The chemiluminescence measurement result of the collected supernatant (aqueous layer) is shown.
- the vertical axis represents the measured relative light emission intensity (RLU).
- the aqueous layer of the sample (+) to which the cleaving enzyme (HRV-3C protease) was added measured 3000 times the luminescence compared to the aqueous layer of the sample ( ⁇ ) to which the cleaving enzyme was not added.
- (C) shows the result of SDS-PAGE electrophoresis.
- Samples from the left are the aqueous layer of the sample ( ⁇ ) without the addition of the cleavage enzyme (protease), the aqueous layer of the sample (+) with the addition of the cleavage enzyme, and the purified hCBD-HRV3Cs-eBAF-Ym3 sample (hCBD-BAF, Control).
- the detected bands indicate (i) hCBD-BAF, (ii) the BAF portion that has been cut and released from the filter paper into the aqueous layer, and (iii) the HRV-3C enzyme, respectively.
- D shows the GFP fluorescence of BAF in the aqueous layer.
- the left is an aqueous layer of a sample to which no cleavage enzyme (protease) is added
- the right is an aqueous layer of a sample to which a cleavage enzyme is added.
- An example of the sequence of the chitin binding domain is shown.
- A An example of the amino acid sequence of chitin-binding domain 2 (chBD2) derived from Pyrococcus furiosus and the base sequence encoding it are shown.
- (B) An example of the amino acid sequence of ChBD2 (TN) in which Glu (E279) is replaced with Thr (T) and Asp (D281) is replaced with Asn (N) in the amino acid sequence of chBD2, and the base sequence encoding it. Show.
- FIG. 9A shows regions where various chimeric proteins are applied.
- (B) (b-1) shows a bright field image of a fluorescent protein-chitin hybrid material stored for 3 days under a fluorescent lamp.
- (B-2) A fluorescence image of the hybrid material stored for 3 days by excitation light irradiation is shown.
- (B-3) shows a fluorescence image after storage for 10 months.
- FIG. 1 A schematic diagram of the structure of hCBD-RLuc is shown. In order from the N-terminal side, it has cellulose and / or chitin-binding domain hCBD, a linker into which a protease recognition sequence HRV-3C cleavage sequence has been introduced, and luminescence domain RLuc.
- the dried filter paper was stored at room temperature of 26 ° C.
- the emission spectrum of eBAF-Ym3 and the chemiluminescence activity of the hCBD-eBAF-Ym3 binding chitin material when luciferin (luminescent substrate) is added (green) are shown.
- a chitin material derived from the crab shell was used for the chitin.
- (A) shows the emission spectrum of eBAF-Ym3.
- the horizontal axis indicates the emission wavelength (Wavelength (nm)).
- the vertical axis represents the measured relative light emission intensity (Relative Intensity).
- (A) The emission spectrum of eBAF-R 3 is shown.
- the horizontal axis indicates the emission wavelength (Wavelength (nm)).
- the vertical axis represents the measured relative luminescence intensity (Relative Intensity).
- a chitin material derived from the crab shell was used for the chitin.
- the emission spectrum of eBAF-R 4 is shown. The horizontal axis indicates the emission wavelength (Wavelength (nm)). The vertical axis represents the measured relative luminescence intensity (Relative Intensity).
- B About an example of a hCBD-eBAF-R 4 bond chitin hybrid material, (b-1) a bright field image, (b-2) a chemiluminescence image, and (b-3) a superimposed image are shown. Luminescence of hCBD-eBAF-Ym3 binding chitin hybrid material. Cicada shell was used as the chitin material.
- A Comparison of hCBD-eBAF-Ym3 binding material and control.
- A-1 shows a bright field image and (a-2) a chemiluminescence image.
- the left is a hCBD-eBAF-Ym3 binding chitin material, and the right is a container (control) containing only buffer.
- B An overall view of a semi-shelled shell bound with hCBD-eBAF-Ym3.
- B-1) shows a bright field image and (b-2) a chemiluminescence image.
- the chimeric protein of the present invention is a protein having a domain that binds to cellulose and / or chitin (cellulose / chitin-binding domain) and a luminescent domain.
- the cellulose / chitin-binding domain is either a domain that can bind to cellulose (cellulose-binding domain), a domain that can bind to chitin (chitin-binding domain), or a domain that can bind to both cellulose and chitin. May be.
- cellulose binding domain examples include a domain possessed by cellulase. Many cellulose-binding domains derived from various organisms such as microorganisms, plants, and animals are known, and these known cellulose-binding domains can be widely used.
- chitin binding domain there is a domain possessed by chitinase.
- Many chitin binding domains derived from various organisms such as microorganisms, plants and animals are known, and these known chitin binding domains can be widely used.
- chitin binding domains include chitin binding domains possessed by chitinases derived from thermostable bacteria.
- heat-resistant bacteria include bacteria belonging to the genus Thermococcus or Pyrococcus, and specific heat-resistant bacteria include Pyrococcus furiosus, Thermococcus litoralis, Pyrococcus sp.KOD1, Thermotoga ⁇ maritima.
- the amino acid sequence shown in SEQ ID NO: 10 is chitin-binding domain 2 (ChBD2) derived from Pyrococcus furiosus, which is one preferred embodiment. This region corresponds to the region from amino acid 258 to amino acid 352 of chitinase derived from Pyrococcus cofuriosus.
- examples of the cellulose / chitin binding domain include a cellulose / chitin binding domain derived from a heat-resistant bacterium as disclosed in JP 2007-075046 A. Specifically, a heat-resistant cellulose / chitin-binding domain obtained by introducing a mutation into the heat-resistant chitin-binding domain derived from the above-mentioned heat-resistant bacteria can be mentioned.
- cellulose / chitin-binding domain in the amino acid sequence (SEQ ID NO: 10) of chitin-binding domain 2 (ChBD2) derived from Pyrococcus furiosus, an amino acid sequence in which two acidic amino acids (E279 and D281) are replaced with other amino acids.
- SEQ ID NO: 10 amino acid sequence in which two acidic amino acids (E279 and D281) are replaced with other amino acids.
- An amino acid sequence encoding a polypeptide having cellulose binding activity is exemplified.
- Examples of other amino acids in which acidic amino acids are substituted include neutral amino acids with low hydrophobicity, such as Gln, Asn, Ala, Ser, Thr, Cys, Met, etc., preferably Gln, Asn, Ala, Ser, Thr, Cys, more preferably Gln, Asn, Ala, Ser, Thr.
- Thr (T) is more preferable for substitution of Glu (E279)
- Asn (N) is more preferable for substitution of Asp (D281).
- ChBD2 (TN) sequence in which Glu (E279) is replaced with Thr (T) and Asp (D281) is replaced with Asn (N) in the amino acid sequence of ChBD2 (FIG. 8, SEQ ID NO: 11) .
- Luminescent domain examples include various luciferases, fluorescent proteins, and fusion proteins thereof (for example, BAF).
- Examples of the luciferase include various luciferases derived from fireflies, Iriomote fireflies, sea squirrels, railroad insects, red beetles, dinoflagellates, renilla mushrooms, and the like.
- Fluorescent proteins include GFP, YFP, BFP, CFP, OFP, DsRED, RFP etc. are mentioned.
- the luciferase or the fluorescent protein may be used alone as the luminescent domain, but preferably the luciferase and the fluorescent protein are bound directly or via a spacer of appropriate length, and the energy transfer between the luciferase and the fluorescent protein.
- a protein producing (BRET) that is, a BAF protein (or simply BAF) is preferred as the luminescent domain.
- a method for producing DNA encoding BAF is exemplified in Patent Document 2, for example, and can be obtained by binding a luciferase gene and a fluorescent protein gene via a DNA sequence corresponding to an appropriate second linker.
- a chimeric protein whose luminescent domain is BAF may be referred to as “CBD-BAF”.
- the chimeric protein of the present invention is a DNA encoding a chimeric protein in which a DNA encoding a cellulose / chitin binding domain and a DNA encoding a luminescent domain are linked directly or via a DNA sequence corresponding to the first linker. It can be obtained by introducing a gene construct or vector containing the above into a host cell (for example, E. coli) to give a transformant and culturing the transformant.
- a host cell for example, E. coli
- the first linker is composed of amino acids and is not particularly limited as long as the functions of the cellulose / chitin binding domain and the luminescent domain are not impaired.
- the number of amino acids in the first linker may be one or more, 2 to 100, for example, 4 to 80, preferably 5 to 60, more preferably about 6 to 40, and further preferably 7 to 30. And about 8 to 16 in particular.
- the second linker is not particularly limited as long as it is composed of an amino acid and does not hinder energy transfer from luciferase to the fluorescent protein.
- the number of amino acids in the second linker is usually 8 to 26, preferably 8 to 16, more preferably 10 to 14, especially 12. When the number of amino acids in the linker is 7 or less or 27 or more, the energy transfer efficiency is greatly reduced.
- a protease recognition sequence is introduced into the linker (first linker or second linker), the presence or absence of protease in the sample can be detected using the chimeric protein of the present invention.
- a sample containing such a linker-binding substance can be detected or quantified by introducing an amino acid sequence in which a substance in the sample binds to the linker and thereby changes the luminescence activity.
- protease recognition sequences, linker binding substances and the like are known and can be appropriately selected by those skilled in the art. Specific examples of the combination of protease and protease recognition sequence include, but are not limited to, HRV-3C protease and amino acid sequence LEVLFQ / GP (/: cleavage site).
- luciferase natural luciferase may be used as the luciferase, or luciferase with improved properties such as stability and luminescent properties may be used.
- the fluorescent protein a natural fluorescent protein may be used, or a fluorescent protein having improved properties such as stability and luminescent properties may be used.
- Renilla luciferase when used as the luminescent domain, a natural Renilla luciferase (e.g., Rluc) may be used as the Renilla luciferase, and a Renilla luciferase (e.g., having improved properties such as stability and luminescent properties) may be used. Rluc8, Rluc8 / A123S / D162E / I163L) may be used.
- luciferase includes both natural luciferase and any modified luciferase in which the properties of luciferase are changed.
- fluorescent protein includes both a natural fluorescent protein and any modified fluorescent protein in which the characteristics of the fluorescent protein are changed.
- the fluorescent fluorescent proteins are green fluorescent fluorescent protein (GFP), yellow fluorescent fluorescent protein (YFP), blue fluorescent fluorescent protein (BFP), cyan fluorescent fluorescent protein (CFP), orange fluorescent fluorescent protein (OFP), DsRED, red fluorescent Examples include photoprotein (RFP).
- GFP includes natural fluorescing proteins derived from jellyfish belonging to the genus Aequorea (for example, Aequorea victoria, Aequorea coerulescens, etc.) and the like, and various GFP derivatives such as EGFP. included.
- YFP amino acid-substituted mutants such as EYFP, Topaz, Venus, and Citrine are widely included.
- DsRED includes a natural fluorescent protein derived from the genus Discosoma genus, mutants with altered amino acid sequences (substitutions, additions, deletions, insertions, etc.), as well as multimeric natural DsRED
- the monomer type for example, mCherry etc.
- monomer type DsRED is preferable.
- RFP includes natural red fluorescent protein derived from sea anemone (for example, Entacmaea quadricolor), etc. (however, it is understood that DsRED that emits red color is not included) and mutants whose amino acid sequence is modified. Widely included (for example, TurboRFP).
- other fluorescent light-emitting proteins include naturally-occurring fluorescent light-emitting proteins and mutants in which amino acid sequences have been modified (substitution, addition, deletion, insertion, etc.).
- a fluorescent protein with RLU Relative light unit, relative emission intensity
- a fluorescence wavelength that varies depending on pH for example, YFP
- the chimeric protein of the present invention can be used as a pH indicator.
- substances such as GFP, whose RLU or wavelength does not change much depending on pH can be used for quantitative determination of substances such as the chimeric protein of the present invention or proteins labeled thereby without depending on pH.
- the DNA of the present invention is a DNA encoding the chimeric protein of the present invention.
- the chimeric protein of the present invention can be obtained by incorporating the gene of the present invention described later into an expression vector and expressing it in an appropriate host cell.
- host cells include animal cells including mammalian cells, plant cells, eukaryotic cells such as yeast, prokaryotic cells such as Escherichia coli, Bacillus subtilis, algae, and fungi, and plant cells. Good.
- E. coli and the like can be used as a preferred host cell.
- the chimeric protein of the present invention is that its luminescence activity is maintained for a long time when it is attached to or bonded to any material such as a sheet, film, particle or bead composed of cellulose or chitin and dried. There is to be. Therefore, the chimeric protein of the present invention is useful as a luminescent material.
- the chimeric protein of the present invention is also useful as a standard substance because its luminescence activity does not decrease during long-term storage.
- cellulose either natural cellulose or regenerated cellulose can be used.
- natural cellulose include refined pulp obtained from conifers and hardwoods, cellulose obtained from cotton linters and cotton lint, cellulose obtained from seaweeds such as valonia and falcon, cellulose obtained from sea squirts, and cellulose produced by bacteria.
- regenerated cellulose include those obtained by once dissolving natural cellulose fibers and then regenerating them into fibers while maintaining the cellulose composition.
- Chitin can be produced from crab shells, for example.
- the chitin used in the present invention treats the washed crab shell with an acid such as hydrochloric acid to remove inorganic substances (such as calcium), and then treats with caustic soda to remove organic substances (e.g., proteins), Further, chitin obtained by treating with alcohol to remove lipids and obtaining an insoluble residue can be used.
- the crab shell material may be pulverized into particles.
- semi-shelled shells can be used as chitin. Cicada shells can be used without treatment because chitin is exposed on the inner surface.
- CBD-BAF and “hCBD-BAF” are understood to be included in the “chimeric protein”.
- Example 1 Preparation of plasmid (1) pCII-CBD-eBAF-Ym3 and pCII-CBD (TN) -eBAF-Ym3
- a gene encoding CBD (wt) or CBD (TN) was amplified by PCR.
- the primers used for PCR are as follows: chBD2-F-NdeI, 5'-GGAATTCCATATGACTACCCCTGTCCCAGTCTC-3 '; chBD2-R-NdeI, 5'-CGATATCCATATGAATTACTTGTCCGTTTATTTCTAG-3 '.
- the PCR fragment was digested with NdeI and incorporated into the NdeI site of pCII-eBAF-Ym3 to construct pCII-CBD-eBAF-Ym3 and pCII-CBD (TN) -eBAF-Ym3.
- eBAF-Ym3 is described in Patent Document 1. This is a BAF protein in which mutations A123S, D162E, and I163L are introduced into the RLuc8 portion of eBAF-Y.
- the gene encoding CBD and CBD (TN) here is a sequence derived from the genome of a hyperthermophile.
- the base sequence of the linking portion is 5'-GGTACCGGGGGATCCCATATG-3 ', and hCBD and eBAF-Ym3 are linked in-frame with the amino acid sequence of GTGGSH (ATG in the NdeI site is the start Met of eBAF-Ym3 Equivalent).
- FIG. 3 shows the test results of the luminescence activity after adsorbing / binding to the chimeric protein filter paper expressed using pCII-hCBD-HRV3Cs-eBAF-Ym3 ⁇ NdeI and drying.
- pCII-hCBD-HRV3Cs-eBAF-R 3 and pCII-hCBD-HRV3Cs-eBAF-R 4 pCII-hCBD-HRV3Cs-eBAF- Ym3 ⁇ NdeI was removed by digesting ,, EBAF-YM3 moiety with NdeI and XbaI, was inserted BAF-R 3 or BAF-R 4 instead.
- BAF-R 3 and BAF-R 4 contain TurboRFP and mCherry as red fluorescent proteins, respectively.
- BAF-R 3 and BAF-R 4 were prepared according to the method described in Patent Document 1.
- Fig. 4 and Fig. 4 show test results of luminescence activity after adsorbing / binding to a chimeric protein filter paper expressed using pCII-hCBD-HRV3Cs-eBAF-R 3 and pCII-hCBD-HRV3Cs-eBAF-R 4 and drying. As shown in FIG.
- FIG. 10 shows the test results of the luminescence activity after adsorbing / binding to the chimeric protein filter paper expressed using the obtained pCII-hCBD-HRV3Cs-RLuc, drying, and storing at room temperature.
- Recombinant Protein was expressed as a His tag fusion protein in Escherichia coli BL21 strain by a cold shock inducible promoter system (TAKARA). Recombinant protein was purified using a Ni-NTA affinity column.
- TAKARA cold shock inducible promoter system
- FIG. 1 schematically shows an outline of the method.
- Chimeric proteins include CBD-eBAF-Ym3, hCBD-HRV3Cs-eBAF-Ym3 ⁇ NdeI (hereinafter sometimes referred to as “hCBD-eBAF-Ym3”), hCBD-HRV3Cs-eBAF-R 3 (hereinafter referred to as “hCBD”).
- hCBD hCBD-HRV3Cs-eBAF-R 3
- hCBD-HRV3Cs-eBAF-R 4 hereinafter sometimes referred to as" hCBD-eBAF-R 4
- hCBD-HRV3Cs-RLuc hereinafter referred to as" hCBD-HRV3R ").
- hCBD-RLuc sometimes referred to as “hCBD-RLuc”.
- Luminescence image observation of chimera protein binding filter paper was cut out with an apple punch, and CBD-eBAF-Ym3 was bound to the center. Thereafter, a luciferin solution was added to the washed filter paper pieces, and yellow-green light emission was visually observed. After confirming that no diffusion from the coated part was observed, a luminescence image was obtained by exposure for 4 seconds in the High Resolution mode (minimum sensitivity) with LAS-4000. The results are shown in FIG. 2A.
- CBD-eBAF-Ym3 having a CBD (chBD2 (TN) type, gene (base) sequence derived from a natural hyperthermophilic bacterium other than the mutagenized portion) as a cellulose / chitin binding domain. It was used. Except this example, all encoded the amino acid sequence of chBD2 (TN), but an artificially synthesized gene optimized for codon usage in E. coli was used (sometimes referred to as “hCBD”).
- Luminescence measurement after storage at room temperature of chimeric protein-bound filter paper CBD-eBAF-Ym3 protein-bound filter paper was placed in a plastic petri dish, capped, and stored in the dark at room temperature (26 ° C to 27 ° C).
- the dried filter paper piece was put in a measurement tube for luminometer (Nunc), and 200 ⁇ l of a luminescence reaction buffer (60 mM NaCl, 50 mM Tris-HCl, pH 8.0) was added and sufficiently wetted.
- 200 ⁇ l of 1 ⁇ M luciferin solution was added, and luminescence measurement was performed. The amount of luminescence was measured by integrating for 10 seconds using Luminescencer-PSN (Ato).
- FIG. 2B For hCBD-HRV3Cs-eBAF-Ym3 ⁇ NdeI, hCBD-eBAF-R 3 , hCBD-HRV3Cs-eBAF-R 4 and hCBD-HRV3Cs-RLuc, the amount of luminescence after storage was measured in the same manner. The results are shown in FIGS. 3 to 5 and FIG. 10, respectively.
- FIGS. 6 and 7A An outline of an implementation system of the protease activity detection system is schematically shown in FIGS. 6 and 7A.
- HCBD-HRV3Cs-eBAF-Ym3 was induced for expression using pCII-hCBD-HRV3Cs-eBAF-Ym3-containing E. coli, and then purified. Using the obtained hCBD-HRV3Cs-eBAF-Ym3, the protein-bound dried filter paper piece was prepared.
- the filter paper piece was placed in a 2.0 ⁇ m microcentrifuge tube, 100 ⁇ l of 1 ⁇ HRV-3C buffer (150 ⁇ mM NaCl, 50 ⁇ mM Tris-HCl, pH 7.5) was added and wetted sufficiently, and then the buffer was completely removed once.
- 1xHRV-3CHR buffer solution or 120 ⁇ l of 1xHRV-3C buffer solution containing 4U HRV-3C protease (Novagen) was added again and allowed to stand at 4 ° C for 64 hours. After the reaction, the microcentrifuge tube was centrifuged, and 40 ⁇ l of the supernatant was separated into another tube.
- FIG. 9B shows a bright field image under a fluorescent lamp, and (b-2) shows a fluorescent image.
- (B-1) and (b-2) in FIG. 9B are photographs taken from the same angle. The negative control and the uncoated part are green because the irradiated green light is reflected. Furthermore, the fluorescence image after storing the same sample at room temperature for 10 months is shown in (b-3) of FIG. 9B.
- HCBD-HRV3Cs-eBAF-Ym3 was applied directly to the semi-shell.
- the hybrid material was immersed in the above-described reaction buffer, a luciferin solution was added, and the state of luminescence was recorded with a digital camera. The results are shown in FIG.
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Abstract
Description
本出願は、2011年12月19日に出願された日本国出願第2011-277363号明細書および2012年1月27日に出願された日本国出願第2012-014817号明細書(それらの開示全体が参照により本明細書中に援用される)に基づく優先権を主張する。
項1:発光ドメインとセルロース及び/又はキチン結合ドメインを含むキメラ蛋白質であって、前記発光ドメインがルシフェラーゼ及び蛍光発光蛋白質からなる群から選ばれる少なくとも1種の発光蛋白質を含む、キメラ蛋白質。
項2:発光ドメインとセルロース及び/又はキチン結合ドメインが直接又は第1リンカーを介して結合されてなる、項1に記載のキメラ蛋白質。
項3:前記発光ドメインがルシフェラーゼ及び蛍光発光蛋白質を含み、ルシフェラーゼから蛍光発光蛋白質へのエネルギー移動(BRET)が生じ得るものである、項1又は2に記載のキメラ蛋白質。
項4:ルシフェラーゼと蛍光発光蛋白質が第2リンカーを介して結合されてなる、項3に記載のキメラ蛋白質。
項5:蛍光発光蛋白質が、GFP、YFP、BFP、CFP、OFP、DsREDまたはRFPである項1~4のいずれか1項に記載のキメラ蛋白質。
項6:蛍光発光蛋白質がYFPまたはRFPである、項5に記載のキメラ蛋白質。
項7:第1リンカー及び/又は第2リンカーがプロテアーゼ切断配列を含む、項1~6のいずれか1項に記載のキメラ蛋白質。
項8:項1~7のいずれかに記載のキメラ蛋白質をコードするDNAまたはその相補鎖。
項9:項1~7のいずれかに記載のキメラ蛋白質をセルロース又はキチンを含む粒子、ビーズ、シート又はフィルムに結合させてなる、発光材料。
セルロース/キチン結合ドメインは、セルロースに結合できるドメイン(セルロース結合ドメイン)、キチンに結合できるドメイン(キチン結合ドメイン)、セルロール及びキチンの両方に結合できるドメインのいずれであってもよい。
発光ドメインは、各種ルシフェラーゼ、蛍光発光蛋白質、あるいはこれらの融合蛋白質(例えばBAF)が挙げられる。ルシフェラーゼとしては、ホタル、イリオモテボタル、ウミボタル、鉄道虫、ヒカリコメツキムシ、渦鞭毛藻、ウミシイタケなどに由来する各種ルシフェラーゼが挙げられ、蛍光発光蛋白質としては、GFP、YFP、BFP、CFP、OFP、DsRED、RFPなどが挙げられる。
本発明のキメラ蛋白質は、セルロース/キチン結合ドメインをコードするDNAと発光ドメインをコードするDNAを直接又は第1リンカーに対応するDNA配列を介して連結したキメラ蛋白質をコードするDNAを含む遺伝子構築物またはベクターを宿主細胞(例えば大腸菌)に導入して形質転換体とし、この形質転換体を培養することにより得ることができる。
第1リンカーは、アミノ酸からなり、セルロース/キチン結合ドメインと発光ドメインの各々の機能を損なわない限り特に限定されない。第1リンカーのアミノ酸の数は、1個以上であればよく、2~100個、例えば4~80個、好ましくは5~60個、より好ましくは6~40個程度、さらに好ましくは7~30個、特に8~16個程度が挙げられる。
本明細書において、ルシフェラーゼは、天然のルシフェラーゼを使用してもよく、安定性や発光特性などの性質が改善されたルシフェラーゼを使用してもよい。
また、キチンとして、セミ類の抜け殻を用いることもできる。セミ類の抜け殻は、キチンが内表面に露出しているため、無処理で使用することができる。
プラスミドの作製
(1)pCII-CBD-eBAF-Ym3及びpCII-CBD(TN)-eBAF-Ym3
CBD-eBAF-Y発現ベクターを作製するために、CBD(wt)又はCBD(TN)をコードする遺伝子をPCRにより増幅した。PCRに用いられたプライマーは次の通りである:
chBD2-F-NdeI,5’-GGAATTCCATATGACTACCCCTGTCCCAGTCTC-3’;
chBD2-R-NdeI, 5’-CGATATCCATATGAATTACTTGTCCGTTTATTTCTAG-3’。
PCR断片をNdeIで消化し、pCII-eBAF-Ym3のNdeI部位へ組み込み、pCII-CBD-eBAF-Ym3及びpCII-CBD(TN)-eBAF-Ym3を構築した。
大腸菌での効率的な蛋白質発現のために、大腸菌でのコドン最適化を目的としたCBD遺伝子の人工合成を行った(CBD(TN)のみ)。当該人工合成CBD(TN)遺伝子(以下hCBDと呼称して区別するが、アミノ酸配列はCBD(TN)と同一である)を、上記pCII-CBD(TN)-eBAF-Ym3のCBD(TN)部分と置換したpCII-hCBD-eBAF-Ym3を構築した。またこの時、後々の組換えに備え、人工遺伝子設計に際し、hCBD配列の3’側にAsp718-BamHI-NdeI部位を付加した。その結果、連結部分の塩基配列は、5’-GGTACCGGGGGATCCCATATG-3’となり、G-T-G-G-S-Hのアミノ酸配列でhCBDとeBAF-Ym3をインフレームに連結することになる(NdeI部位のATGはeBAF-Ym3の開始Metに相当)。
pCII-hCBD-eBAF-Ym3のAsp718-BamHI部位に、AspHRV3CsBam-Sens:5’-GTACCGGTGGTTCCGCGGGTCTGGAAGTTCTGTTCCAGGGGCCCTCCGCGGGTtccggtg-3’とAspHRV3CsBam-Anti:GATCCACCGGAACCCGCGGAGGGCCCCTGGAACAGAACTTCCAGACCCGCGGAACCACCGからなる合成2重鎖DNAを挿入し、HRV-3Cプロテアーゼの切断配列を挿入した。Asp718部位からBamHI部位までに対応するアミノ酸配列は、G-T-G-G-S-A-G-L-E-V-L-F-Q-G-P-S-A-G-S-G-G-Sであり、中央のLEVLFQ/GPが当該プロテアーゼの切断配列である(/:切断部位)。
pCII-eBAF-Yで代表される各種BAFの大腸菌発現ベクターにおいて、2008年に開発済の400種類を超える各種BAFは全てNdeI-XbaI部位でクローニングされている。一方でpCII-hCBD-HRV3Cs-eBAF-Ym3は、hCBD部分がNdeI部位で挿入されており、BAFの置換体を作製するためにhCBD部分の5’側のNdeI部位が新たなBAF置換体作製の障害になる。そのため、hCBDdelNdeIoligo-Sens: 5’-TCATCATCATCATCAcATGACCACTCCGGTG-3’、hCBDdelNdeIoligo-Anti: 5’-CACCGGAGTGGTCATgTGATGATGATGATGA-3’を用いて、1塩基の塩基置換変異導入により、NdeI部位を破壊した、pCII-hCBD-HRV3Cs-eBAF-Ym3ΔNdeIを作製した。なお、この一塩基変異導入にはストラタジーン社のQuickExchenge systemを用いた。
pCII-hCBD-HRV3Cs-eBAF-Ym3ΔNdeIを用いて発現させたキメラ蛋白質濾紙に吸着/結合させて乾燥した後の発光活性の試験結果を図3に示す。
pCII-hCBD-HRV3Cs-eBAF-Ym3ΔNdeIをNdeI及びXbaIを用いて消化し、、eBAF-Ym3部分を除去して、その代わりにBAF-R3又はBAF-R4を挿入した。かくして、pCII-hCBD-HRV3Cs-eBAF-R3及びpCII-hCBD-HRV3Cs-eBAF-R4を作製した。なお、BAF-R3及びBAF-R4は、それぞれ赤色蛍光発光蛋白質として、TurboRFP及びmCherryを含んでいる。BAF-R3及びBAF-R4は、特許文献1に記載の方法に準じて作製した。
上記(5)と同様にして、pCII-hCBD-HRV3Cs-eBAF-Ym3ΔNdeIからeBAF-Ym3部分を除去して、その代わりにRLuc(ウミシイタケルシフェラーゼ)を挿入した。得られたpCII-hCBD-HRV3Cs-RLucを用いて発現させたキメラ蛋白質濾紙に吸着/結合させて乾燥させ、室温保存した後の発光活性の試験結果を図10に示す。
各キメラ蛋白質について、リコンビナント蛋白質をHisタグ融合蛋白質として、大腸菌BL21株において低温ショック誘導性プロモーターシステム(TAKARA)により発現させた。リコンビナント蛋白質は、Ni-NTAアフィニティーカラムを用いて精製した。
穴あけパンチで作製した直径6mmの丸形濾紙(ADVANTEC)片をパラフィルム上に置き、当該濾紙片にHisタグ精製した各種キメラ蛋白質の高濃度水溶液を数μlずつ滴下、次いで乾燥の工程を繰り返した。十分量のキメラ蛋白質を結合後、大量の精製水にて、当該濾紙片を洗浄し、未結合のCBD-BAFを除去した。洗浄後の濾紙片をパラフィルム上で風乾することで、各種キメラ蛋白質結合濾紙を作製した。図1に、方法の概要を模式的に示す。キメラ蛋白質としては、CBD-eBAF-Ym3、hCBD-HRV3Cs-eBAF-Ym3ΔNdeI(以下、「hCBD-eBAF-Ym3」と記載する場合がある。)、hCBD-HRV3Cs-eBAF-R3(以下、「hCBD-eBAF-R3」と記載する場合がある。)、hCBD-HRV3Cs-eBAF-R4(以下、「hCBD-eBAF-R4」と記載する場合がある。)及びhCBD-HRV3Cs-RLuc(以下、「hCBD-RLuc」と記載する場合がある。)を用いた。
リンゴ型パンチで切り抜き、中央部にCBD-eBAF-Ym3を結合させた。その後、洗浄した濾紙片に対し、ルシフェリン溶液を添加して、黄緑色発光を視認観察した。塗布部からの拡散が見られないことを確認後、LAS-4000にて、High Resolution mode(感度最低)で4秒間露光により、発光画像を取得した。結果を、図2Aに示す。
なお、本実施例のみ、セルロース/キチン結合ドメインとしてCBD(chBD2(TN)型で、変異導入部分以外は天然型の超好熱性細菌由来の遺伝子(塩基)配列)を有する、CBD-eBAF-Ym3を使用した。本実施例以外は、全てchBD2(TN)のアミノ酸配列をコードするが、大腸菌でのコドン使用に最適化した人工合成遺伝子を使用した(「hCBD」と呼ぶ場合がある。)。
CBD-eBAF-Ym3蛋白質結合濾紙をプラスチックペトリディッシュに入れ、フタをした後、室温(26℃~27℃)暗所にて保存した。測定直前に、当該乾燥濾紙片をルミノメータ用測定チューブ(Nunc)に入れ、発光反応バッファー(60 mM NaCl, 50 mM Tris-HCl, pH8.0)200 μlを加えて、十分湿潤させた。当該チューブに、1 μMルシフェリン溶液200 μlを添加し、発光測定を行った。発光量はLuminescencer-PSN (アトー)を用いて、10秒間の積算により測定した。結果を図2Bに示す。
hCBD-HRV3Cs-eBAF-Ym3ΔNdeI、hCBD-eBAF-R3、hCBD-HRV3Cs-eBAF-R4及びhCBD-HRV3Cs-RLucについても、同様に保存後の発光量を測定した。結果を、図3~5及び図10にそれぞれ示す。
系の概要を、図6及び図7Aに模式的に示す。
カニ甲羅を塩酸処理(脱カルシウム)、NaOH処理(除タンパク)、次いでアルコール処理(除脂質)を順次施し、キチン素材(カニ甲羅キチン素材)を得た。得られたキチン素材に、3種のhCBD-BAF蛋白質(hCBD-HRV3Cs-eBAF-Ym3、hCBD-HRV3Cs-eBAF-R3及びhCBD-HRV3Cs-eBAF-R4)を、領域を分けて塗布(図9A)して、これを乾燥した。室温にて3日間保存した後、505nmの緑色LEDイルミネータを照射し、オレンジ色フィルターを透過する光をデジカメで撮影した。図9Bの(b-1)は蛍光灯下での明視野像、(b-2)は蛍光像をそれぞれ示す。図9Bの(b-1)及び(b-2)は、同一アングルから撮影した写真である。陰性対照と未塗布部分が緑色なのは、照射緑色光が反射しているためである。さらに、同一試料を室温にて10か月乾燥保存した後の蛍光像を、図9Bの(b-3)に示す。
hCBD-BAFに用いた各種BAFのスペクトル測定
3種のhCBD-BAF蛋白質(hCBD-HRV3Cs-eBAF-Ym3、hCBD-HRV3Cs-eBAF-R3及びhCBD-HRV3Cs-eBAF-R4)に用いた各種BAF蛋白質単体(eBAF-Ym3、eBAF-R3及びeBAF-R4)について、特許文献2に記載の方法に準じてスペクトル測定を行なった。結果を、図11~13に示す。
セミ抜け殻へのCBD-BAF蛋白質の結合及び発光観察
キチン素材として、セミ抜け殻を用いた。セミ抜け殻に直接hCBD-HRV3Cs-eBAF-Ym3を塗布した。当該ハイブリッド材料を前述の反応バッファーに浸し、ルシフェリン溶液を添加した後、発光の様子をデジタルカメラで記録した。結果を図14に示す。
Claims (9)
- 発光ドメインとセルロース及び/又はキチン結合ドメインを含むキメラ蛋白質であって、前記発光ドメインがルシフェラーゼ及び蛍光発光蛋白質からなる群から選ばれる少なくとも1種の発光蛋白質を含む、キメラ蛋白質。
- 発光ドメインとセルロース及び/又はキチン結合ドメインが直接又は第1リンカーを介して結合されてなる、請求項1に記載のキメラ蛋白質。
- 前記発光ドメインがルシフェラーゼ及び蛍光発光蛋白質を含み、ルシフェラーゼから蛍光発光蛋白質へのエネルギー移動(BRET)が生じ得るものである、請求項1又は2に記載のキメラ蛋白質。
- ルシフェラーゼと蛍光発光蛋白質が第2リンカーを介して結合されてなる、請求項3に記載のキメラ蛋白質。
- 蛍光発光蛋白質が、GFP、YFP、BFP、CFP、OFP、DsREDまたはRFPである請求項1~4のいずれか1項に記載のキメラ蛋白質。
- 蛍光発光蛋白質がYFPまたはRFPである、請求項5に記載のキメラ蛋白質。
- 第1リンカー及び/又は第2リンカーがプロテアーゼ切断配列を含む、請求項1~6のいずれか1項に記載のキメラ蛋白質。
- 請求項1~7のいずれかに記載のキメラ蛋白質をコードするDNAまたはその相補鎖。
- 請求項1~7のいずれかに記載のキメラ蛋白質をセルロース又はキチンを含む粒子、ビーズ、シート又はフィルムに結合させてなる、発光材料。
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JP2015029472A (ja) * | 2013-08-02 | 2015-02-16 | 東洋ビーネット株式会社 | プロテアーゼ活性測定法 |
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