WO2019054506A1 - Procédé de fabrication de fibrilles - Google Patents

Procédé de fabrication de fibrilles Download PDF

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WO2019054506A1
WO2019054506A1 PCT/JP2018/034275 JP2018034275W WO2019054506A1 WO 2019054506 A1 WO2019054506 A1 WO 2019054506A1 JP 2018034275 W JP2018034275 W JP 2018034275W WO 2019054506 A1 WO2019054506 A1 WO 2019054506A1
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fibroin
amino acid
concentration
fibrils
solution
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PCT/JP2018/034275
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English (en)
Japanese (ja)
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裕生 上久保
有吾 林
健大 佐藤
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Ｓｐｉｂｅｒ株式会社
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Priority to JP2019542323A priority Critical patent/JP7303550B2/ja
Publication of WO2019054506A1 publication Critical patent/WO2019054506A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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

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  • the present invention relates to a method of producing fibrils.
  • Nanostructures using metal molecules are in practical use or in a state close to practical use, such as dye-sensitized solar cells (titanium oxide) and conductive inks (silver nanowires).
  • Nanostructures are also attracting attention in the field of biotechnology, and protein nanofibers are scaffold sheets for cells designed as desired for mechanical properties, biomolecular devices, cell engineering devices, regenerative medicine, tissue engineering, biosensors, etc. It is expected to be used as an actuator and as materials for lightweight and high strength materials, green nano hybrids, environmental purification materials, self-healing materials, filters, spinning, coatings, and high precision precision devices related to structure and physical property analysis.
  • Non-patent Document 1 protein nanofibers have not been put to practical use. If the length of the nanostructure can be controlled and made uniform, it becomes possible to produce a stable structure with controlled physical properties and functions, for example, strength, elongation, pore size, specific surface area, water retention, reaction site It can be used as a tool that can be controlled, and can also be used to build a model that predicts physical properties (Non-Patent Document 2). Thus, there is a need for a method of producing proteinaceous fibrils of uniform length.
  • An object of the present invention is to provide a method for producing fibrils of uniform length comprising fibroin.
  • a method for producing fibrils which comprises the following steps (A) to (C): (A) Solubilizing fibroin containing a domain sequence represented by the formula [(A) n motif-REP] m using a solubilizing agent to obtain a solubilization solution, (B) forming a suspension containing fibrils by diluting the solubilization solution until the concentration of the solubilizer reaches a concentration at which the fibroin aggregates are generated, and maintaining the concentration; C) A step of recovering fibrils produced in the step (B).
  • (A) n motif represents an amino acid sequence composed of 4 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in the n motif is 65% or more.
  • REP represents an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • the plurality of (A) n motifs may be identical to each other or different from each other.
  • the plurality of REPs may be identical amino acid sequences to each other or different amino acid sequences.
  • [2] The process according to [1], wherein the amount of fibroin in the aggregate produced in the step (B) is 0.1 to 10% by weight of the amount of fibroin used in the step (A).
  • step (C) is a step of centrifuging the suspension and collecting a supernatant.
  • step (C) is a step of centrifuging the suspension and collecting a supernatant.
  • solubilizer is a chaotropic agent.
  • the chaotropic agent is selected from the group consisting of urea, guanidinium hydrochloride, sodium iodide and perchlorate.
  • a method of producing fibrils of uniform length comprising fibroin can be provided.
  • FIG. 1 It is a schematic diagram which shows the change of the structure of fibroin.
  • (A) shows fibroin solubilized by a solubilizer, and (b) shows fibroin in which columnar fibrils are formed.
  • BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram of CD spectrum which shows the secondary structure of a general protein, and the relationship of a wavelength. It is a figure of UV spectrum of fibroin in each urea solution. 1 is 23 mg fibroin / ml 6 M urea solubilizing solution, 2 is a 2-fold dilution of 1, 3 is a 3-fold dilution of 1, 4 is a 4-fold dilution of 1 and 5 is a 5-fold dilution of 1.
  • FIG. 5 is a histogram of fibril length obtained from dilutions with fibroin concentrations of 1 mg / mL and 2.5 mg / mL. It is a figure of the histogram of the height of the fibril by SPM observation of 2 times dilution liquid.
  • the method for producing fibrils is characterized by including the following steps (A) to (C).
  • C A step of recovering fibrils produced in the step (B).
  • the fibroin according to the present invention is a protein comprising a domain sequence represented by the formula [(A) n motif-REP] m .
  • domain sequence refers to a crystal region specific to fibroin (typically corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically the REP of the amino acid sequence).
  • the corresponding amino acid sequence is represented by the formula: [(A) n Motif-REP] m .
  • n motif is amino acid sequence composed of 4-27 amino acid residues, and (A) alanine residues to total amino acid residues in the n motif than 65% .
  • the number of alanine residues relative to the total number of amino acid residues in the n motif is preferably 70% or more, more preferably 75% or more, still more preferably 80% or more, and 85% or more More preferably, it is 90% or more, still more preferably 95% or more, and particularly preferably 100% (meaning it is composed of only alanine residues). It is preferable that at least seven (A) n motifs in the domain sequence are composed of only alanine residues.
  • the (A) n motif is (A) n (A represents an alanine residue, n is an integer of 4 to 27, preferably an integer of 4 to 20, more preferably It means having an amino acid sequence represented by 4) to 16).
  • REP represents an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • the plurality of (A) n motifs may be identical to each other or different from each other.
  • the plurality of REPs may be identical amino acid sequences to each other or different amino acid sequences.
  • modified fibroin refers to fibroin whose domain sequence differs from that of naturally occurring fibroin.
  • modified fibroin is one having the amino acid sequence specified in the present invention, which is derived from naturally occurring fibroin and modified in its amino acid sequence (eg, in the amino acid sequence of cloned naturally occurring fibroin)
  • the amino acid sequence may be modified by substitution, deletion, insertion and / or addition of one or more amino acid residues), or may be artificially designed and synthesized regardless of naturally occurring fibroin (For example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).
  • Substitutions, deletions, insertions and / or additions of amino acid residues can be carried out by methods known to those skilled in the art such as partial directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983) and the like.
  • Naturally occurring fibroin is a protein comprising a domain sequence represented by the formula [(A) n motif-REP] m .
  • the amino acid sequence may be added to the N-terminal side and / or the C-terminal side of the domain sequence, but when producing homogeneous fibrils, the amino acid sequence (N-terminal sequence and C-terminal sequence) on either one or both It is preferable that is not added.
  • Naturally occurring fibroin include fibroin produced by, for example, insects or spiders.
  • fibroin produced by insects include Bombyx mori (Bombyx mori), Quwaco (Bombyx mandarina), pemphigus (Antheraea yamamai), moth (Anteraea pernyi), moth (Eriogyna pyretorum), moth (Pilosamia Cynthia ricini) ), Silkworms produced by silkworms such as silkworms (Samia cynthia), cricketworms (Caligura japonica), tusser moths (Antheraea mylitta), and muga moths (Antheraea assama), hornets ejected by the larvae of the hornets (Vespa simillima xanthoptera) Silk protein is mentioned.
  • fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA 27840.1 (amino acid sequence)) and the like.
  • the fibroins produced by the spiders include, for example, spiders belonging to the genus Araneus such as spider spiders, spider spiders, spider spiders, blue spider spiders, and spider spiders, spider spiders (genus Neoscona) such as spider spiders, spider spiders, spider spiders and spiders , Spiders belonging to the genus Pronus (Pronus), such as Torino Fundamas, spiders belonging to the genus Torino Fundama (Cyrtarachne) such as Torino Fundamas, and Otorino Fundames, such as spiders such as Togegumo and Tibusegumo Spiders belonging to the genus Gasteracantha, spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Gasteracantha and those belonging to the genus Ordgarius A spider belonging to the genus Angiope (Argiope), a spider belonging to the genus Angiope, a spider
  • fibroin produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 (derived from Nephila clavipes) (genbank accession number AAC 04504 (amino acid sequence), U37520 (base sequence)), major angu11ate spidroin 1 [La rodectus hesperus] (GenBank Accession No.
  • ABR68856 amino acid sequence
  • EF 595246 base sequence
  • dragline silk protein spidroin 2 [from Nephila clavata] (GenBank Accession No. AAL 32472 (amino acid sequence), AF 44 1 245 (base sequence)), major anpullate spidroin 1 [from Euprosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ 973 155 (base sequence)), and major ampullate spidroin 2 [Euprosthenops australis] (GenB nk accession number CAM 32249.1 (amino acid sequence), AM 490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC 14589.
  • Naturally derived fibroin further include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidroin, ampullate, fibroin, “silk and polypeptide”, or “silk and protein” are described as keywords among sequences including INV as DIVISION among sequence information registered in NCBI GenBank.
  • the sequence can be confirmed by extracting a specified product string from CDS, and a described sequence of a specific string from SOURCE to TISSUE TYPE.
  • a specific sequence of fibroin is a partial sequence derived from ADF-3 (Araneus diadematus Fibroin-3), which constitutes a dragline of Araneus diadematus shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, There may be mentioned fibroin comprising the amino acid sequence shown by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • fibril refers to a fibrous material having a diameter of 1 nm to 100 nm and a length greater than the diameter, for example, a length of 10 times or more of the diameter. Fibrils are sometimes referred to as nanofibers or nanorods.
  • the fibrils composed of fibroin mean fibrils in which the component of the fibrous material is fibroin.
  • the fibrils composed of fibroin are preferably cylindrical fibrils as shown in FIG. 1 (b).
  • the length of the fibrils depends on the number m of repeats of the domain sequence represented by the formula: [(A) n Motif-REP] m , and fibrils longer as m becomes larger can be obtained.
  • the length of fibrils can be lengthened depending on the molecular weight and concentration of fibroin, but may be, for example, 30-150 nm for a dose of about 50 kDa and a concentration of about 1 mg / ml, a concentration of about 2.5 mg / ml It may be 50 to 250 nm in ml.
  • the diameter (height) of the fibrils may be, for example, 2 to 5 nm. According to the invention, fibrils of uniform length are obtained.
  • “uniform” means that 70% is included in the range of ⁇ 50% with respect to the length showing the maximum value on the histogram.
  • the step (A) is a step of solubilizing fibroin containing [(A) n motif-REP] m domain sequence using a solubilizing agent to obtain a solubilization solution.
  • a solubilizing agent can be used as long as it can solubilize fibroin, but a chaotropic agent (hereinafter also referred to as "chaotrope agent”) is preferable.
  • chaotropic agent include urea, guanidinium hydrochloride (GuHCl), guanidine thiocyanate (GTC), sodium iodide (NaI), perchlorate and the like, with preference given to urea and guanidinium hydrochloride.
  • Fibroin can be solubilized, for example, by dissolving it in water or a buffer containing a solubilizing agent.
  • a buffer for example, Tris-HCl buffer (trishydroxymethylaminomethane and hydrochloric acid), phosphate buffer (phosphate and sodium phosphate), acetate buffer (acetic acid and sodium acetate), citrate buffer (citrate) And sodium citrate), borate buffer, MES (2-morpholinoethanesulfonic acid) buffer, Pipes (piperazine-1,4-bis (2-ethanesulfonic acid)) buffer, MOPS (3-morpholinopropane sulfone) Examples include acid) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid) buffer, tricine buffer and CAPS (3-cyclohexylaminopropane sulfonic acid) buffer.
  • HEPES 4- (2-hydroxyethyl) -1-piperaz
  • the form of fibroin to be solubilized may be powder or liquid.
  • urea As a dissolution method in the case of using urea as a solubilizing agent, for example, the following methods can be mentioned. 200 to 300 ⁇ l of urea buffer (6 to 8 M urea, 10 mM Tris HCl, pH 7.0) is added to 3 to 7 mg of fibroin powder sample and shaken (1800 rpm) for 5 minutes to dissolve fibroin. Sonication (20-30%, 10 seconds, 4-5 times, interval 5-10 minutes) may be performed to completely dissolve fibroin. Whether fibroin is completely dissolved in the buffer containing the solubilizing agent can be confirmed by ultraviolet / visible absorption measurement described later, or the like.
  • the step (A) may further include filtering the obtained solubilized solution. It is preferable to use what removed the impurity by filter filtration, when performing the below-mentioned solubility and structural analysis.
  • filter filtration for example, filtration using a filter (Ultrafree-MC-GV, Durapore PVDF 0.22 ⁇ m) can be mentioned.
  • the concentration of the solubilizer means the concentration of the solubilizer in the solubilization solution obtained by dissolving fibroin.
  • the dilution of the solubilization solution can be carried out by diluting the prepared solubilization solution with water or buffer containing no solubilizer.
  • the buffer for example, those described above can be used, and it is preferable to carry out dilution using a Tris buffer. Dilution of the solubilization solution reduces the concentration of solubilizer.
  • a method of diluting the solubilization solution there can be mentioned, for example, a method of gradually diluting, for example, a method of dialysis, and a method of serially diluting 2 times, 3 times, 4 times and 5 times.
  • dilution solution The solution in which the concentration of the solubilizer in the solubilization solution is reduced by dilution is hereinafter also referred to as "dilution solution".
  • the solubilizing solution is diluted to reduce the concentration of the solubilizing agent to a concentration at which fibroin aggregates are produced (hereinafter also referred to as "equilibrium concentration"). Aggregates are formed. A suspension is formed by the formation of the aggregates.
  • the structure of fibroin present in the solubilization part of the suspension is changed to form fibrils of uniform length. It is presumed that this is because the dissolution and aggregation of fibroin are in an equilibrium state by maintaining a concentration at which all fibroins do not aggregate, thereby efficiently obtaining fibrils of uniform length.
  • the state in which the dissolution and aggregation are in equilibrium means that the fibroin molecule moves to a solution or moves to an aggregate. The state of dissolution of fibroin and structural changes can be confirmed by analysis according to the method described later.
  • Whether aggregates of fibroin are formed can be confirmed visually, but can also be confirmed by measuring the protein (fibroin) content of the supernatant and the precipitate fraction described later.
  • the diluted solution can be centrifuged (20 ° C., 14500 rpm, 30 minutes) using a centrifuge (KUBOTA 3740, manufactured by Kubota Mfg. Co., Ltd.), and the presence or absence of a precipitate can be confirmed.
  • KUBOTA 3740 manufactured by Kubota Mfg. Co., Ltd.
  • the diluted solution is suspended can be confirmed visually, but can also be confirmed, for example, by measuring the absorbance of the diluted solution.
  • the absorbance of the supernatant after centrifugation of the diluted solution is measured, and the amount of protein present in the supernatant is the amount of protein present in the solubilized solution before dilution and If the concentration is reduced by 10% or more, or the absorbance of the solution in which the precipitate is dissolved is measured, the amount of protein present in the solution in which the precipitate is dissolved is 10% of the amount of protein present in the solubilized solution before dilution In the case of the above, it can be judged that the diluted solution is suspended, that is, an aggregate is formed.
  • the concentration at which the aggregates form can be maintained by stopping the dilution, for example by settling.
  • the time for maintaining the concentration at which precipitation occurs may be, for example, 1 hour or more and 120 hours or less.
  • the lower limit of the time to maintain the concentration at which the aggregates form may be, for example, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours.
  • concentration which an aggregate produces you may be 120 hours, 96 hours, 72 hours, 48 hours, and 24 hours, for example.
  • fibroins are partially in contact with each other (portion shown by a circle of broken line in FIG. 1A).
  • fibroin involved in the present invention is self-assembled upon gradual dilution to form cylindrical fibrils as shown in FIG. 1 (b).
  • FIG. 1 (b) In order to increase the fibril productivity, it is preferable to bring as much fibroin as possible to FIG. 1 (b) from the state of FIG. 1 (a).
  • industrial-level production it can be confirmed by the formation state of aggregates as one of the indexes.
  • concentration of solubilizer By maintaining the concentration of solubilizer at a stage where the amount of aggregates formed is small, more fibrils can be obtained.
  • the fibrils can be obtained in a high yield by maintaining the amount of aggregates with a higher concentration of dilution solution and forming less fibrils, but in view of the time until the acquisition, solubilization of a preferable concentration in industrial production
  • the solution may be set.
  • Fibrils can be obtained more efficiently by suppressing the amount of aggregate formation to 10% by weight or less of the total amount of fibroin in the aggregate (A). That is, the amount of fibroin in the aggregate is 0.1 to 10% by weight, preferably 0.1 to 8% by weight, and more preferably 0.1 to 6% by weight of the total amount of fibroin used in step (A). is there.
  • the concentration in the equilibrium state may be determined based on the formation of aggregates depending on the fibroin used, for example, 1.5 to 10.0 times, based on the concentration of the solubilizer in the solubilization solution. It can be diluted 8-fold to 8.0-fold or 2.0-fold to 6.0-fold.
  • concentration of urea in the diluent is 1.5 to 4.5 M, preferably 2.
  • a concentration of 0 to 4.0 M, more preferably 2.5 to 3.5 M can be raised.
  • Step (C) is a step of recovering fibrils produced in step (B).
  • the step (C) can be a step of centrifuging the suspension containing fibrils generated in the step (B) and recovering the supernatant containing fibrils.
  • conditions for centrifugation KUBOTA 3740, manufactured by Kubota Mfg. Co., Ltd.
  • conditions for centrifugation KUBOTA 3740, manufactured by Kubota Mfg. Co., Ltd.
  • conditions for centrifugation KUBOTA 3740, manufactured by Kubota Mfg. Co., Ltd.
  • the occurrence of precipitation can be confirmed by measuring the protein (fibroin) content of the supernatant and the precipitate fraction.
  • the protein content of the precipitate fraction can be determined by resolubilizing the precipitate fraction using a solubilizer and measuring with NanoDrop®.
  • ATR-FTIR Total reflection attenuation-secondary structure evaluation by Fourier transform infrared spectroscopy
  • ATR-FTIR measurement can be performed using FTIR-6100 (manufactured by JASCO Corporation), ATR PRO ONE (manufactured by JASCO Corporation) or the like.
  • the measurement conditions may be performed according to the manual attached to the device.
  • FTIR-6100 manufactured by JASCO Corporation
  • ATR PRO ONE manufactured by JASCO Corporation
  • the measurement conditions may be performed according to the manual attached to the device.
  • ThT is added to the fibroin solubilization solution, and the fluorescence intensity is measured.
  • the addition amount of ThT may be 0.05 to 0.2 mg / ml.
  • spectral measurements can be made.
  • the fluorometer include JASCO FP-8200 (manufactured by JASCO Corporation) and the like. The measurement may be performed according to the manual attached to the device.
  • a plate reader can follow the change in fluorescence intensity over time.
  • a plate reader for example, SYNERGY HTX (manufactured by Biotech Co., Ltd.) or the like can be used. The measurement may be performed according to the manual attached to the device. Based on the formation of unidirectional ⁇ -sheet structure by solubilizing fibroin with urea and diluting undiluted solubilization solution (hereinafter also referred to as “fibroin urea solubilization stock solution”) to an appropriate concentration An increase in the fluorescence intensity of Thioflavin T can be confirmed by a fluorometer. In addition, it can be traced by the plate reader that the ⁇ sheet structure aligned in one direction is formed over time. Furthermore, this analysis can also determine optimal dilution conditions.
  • Example 1 To 5.1 mg of fibroin powder sample, add 222 ⁇ l of urea buffer (6 M urea, 10 mM tris HCl, pH 7.0), shake for 5 minutes (1800 rpm), then sonicate (20%, 10 seconds, 4 times, interval 10 minutes) Done to completely solubilize fibroin.
  • the solubilized solution was filtered using a filter (Ultrafree-MC-GV, Durapore PVDF 0.22 ⁇ m) to remove impurities. At that time, 50 ⁇ l was flowed so as not to clog the filter.
  • the UV-visible absorption of the filter-filtered solubilization solution was measured with NanoDrop®.
  • the solubilization solution exhibited an ultraviolet-visible absorption spectrum with a maximum at 280 nm, and no significant scattering was observed, confirming that fibroin was completely dissolved (spectrum 1 in FIG. 3).
  • fibroin having the amino acid sequence as shown in SEQ ID NO: 1 it is suitable to prepare 2-3 times diluted solubilizing solution, ie maintaining a concentration of 2-3 M urea, for uniform length fibrils was confirmed by structural analysis of The protein content in the precipitate of the 2-3 fold diluted solubilization solution was 7-9% of the total protein content (see FIG. 4).
  • FIG. 6 shows a difference CD spectrum (dotted line) of the urea solubilized stock solution (solid line) of FIG. 5 and a 2-fold diluted solution. As shown in FIG. 6, from the fact that a CD spectrum showing a negative maximum around 220 nm was obtained, it was estimated that fibroin in the 2-fold diluted solution forms a ⁇ -sheet structure.
  • ATR-FTIR Total reflection attenuation-secondary structure evaluation by Fourier transform infrared spectroscopy
  • FTIR-6100 JASCO Corporation
  • ATR PRO ONE JASCO Corporation
  • the prism used for the measurement of ATR was a diamond, and the incident angle was 45 °.
  • ATR measurement was performed under the following conditions. Number of integrations: background-512, sample-128 Decomposition: 4 cm -1 , Interferometer Speed: 4 mm / sec, Detector: MCT Sensitivity: ⁇ 1, Filter: 10 kHz, Measurement range: 700-7800 cm -1 Aperture: 1.8 mm, apodization: cosine Interval measurement: 5 minutes, interval: 100 seconds Sample mounting amount: 9 ⁇ l
  • ThT The fluorescent dye thioflavin T (ThT), which strongly reacts to the ⁇ -sheet structure, was used for analysis by fluorescence intensity spectrum.
  • JASCO FP-8200 JASCO Corporation
  • SYNERGY HTX Biotech Corporation
  • the measurement by a fluorometer was performed under the following conditions. Measuring range 440 to 600 nm Excitation wavelength 450 nm Measurement concentration 0.1 mg / ml Scan speed Medium Number of measurements 3 times for each sample
  • Image analysis was performed using ImageJ software to measure the length and diameter. As a result, a histogram regarding the length of the formed nanofibers is shown in FIG. From this histogram, it was found that the length of the formed nanofibers was an integral multiple of about 50 nm, and nanofibers having different repetition numbers were formed. Furthermore, when the histogram of the length of the nanofiber obtained from the dilution liquid in which fibroin concentration differs is compared (FIG. 12), it has confirmed that the one where fibroin concentration is large has become large [repetition number]. Image analysis was performed using software attached to SPM-9700, and height (diameter) was measured. As a result, the height of the formed nanofibers was uniformly about 3 nm (FIG. 13).
  • the produced nanofiber has a cylindrical structure with uniform length and diameter as a basic unit.
  • the length and diameter of the nanofibers as the basic unit can be adjusted by the arrangement and molecular weight of fibroin used.
  • Natural cotton, silk, wool and the like are aggregates of nanostructures controlled with high precision. On the other hand, it is expected to artificially produce fibroin having a highly controlled structure according to the present invention on an industrial scale. In addition, fibrils produced by the present invention are also expected to be applied to cell sheets, biomolecular devices, filters, spinning, cosmetics and the like.

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Abstract

La présente invention concerne un procédé de fabrication de fibrilles qui comprend : une étape (A) dans laquelle une fibroïne contenant une séquence de domaine représentée par la formule [(A)nmotif-REP] m est solubilisé avec un agent de solubilisation pour obtenir une solution de solubilisation ; une étape (B) dans laquelle la solution de solubilisation est diluée jusqu'à ce que la concentration du solubilisant atteigne la concentration à laquelle des agrégats de la fibroïne sont formés, et ladite concentration est maintenue pour produire ainsi une suspension contenant des fibrilles ; et une étape (C) pour récupérer les fibrilles produites à l'étape (B).
PCT/JP2018/034275 2017-09-15 2018-09-14 Procédé de fabrication de fibrilles WO2019054506A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020241769A1 (fr) * 2019-05-29 2020-12-03 Ｓｐｉｂｅｒ株式会社 Microcorps de protéine structurale et son procédé de production, procédé de production de nanofibre et procédé de production de structure de protéine
US20220119463A1 (en) * 2019-01-09 2022-04-21 Spiber Inc. Modified fibroin
WO2022260147A1 (fr) * 2021-06-10 2022-12-15 Ｓｐｉｂｅｒ株式会社 Additif pour composition cosmétique

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0790182A (ja) * 1993-09-22 1995-04-04 Kanebo Ltd 保存安定性に優れた絹フィブロイン及びその製造法
WO2002102845A1 (fr) * 2001-06-14 2002-12-27 National Institute Of Agrobiological Sciences Procede de production d'un polypeptide fonctionnel provenant de fibroine de soie, et son utilisation
JP2008169171A (ja) * 2007-01-15 2008-07-24 Luc Sangyo Kk 絹フィブロイン含有組成物及びその製造法
WO2009119596A1 (fr) * 2008-03-26 2009-10-01 国立大学法人岐阜大学 Procédé de classification de protéines, procédé de dissolution de protéines, procédé de fractionnement et de collecte de fibres non animales et de protéines provenant d'une fibre animale
JP2011196001A (ja) * 2010-03-23 2011-10-06 Shinshu Univ 水生昆虫由来のシルクナノファイバー及びシルク複合ナノファイバー、並びにその製造方法
WO2017030197A1 (fr) * 2015-08-20 2017-02-23 国立研究開発法人理化学研究所 Procédé de fabrication de composition polypeptidique présentant une structure de type fibroïne

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103757729B (zh) * 2013-12-28 2015-10-28 复旦大学 一种丝蛋白纳米微纤及其制备方法
CN106729981B (zh) * 2016-11-28 2019-08-06 武汉纺织大学 一种丝素蛋白纳米纤维及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0790182A (ja) * 1993-09-22 1995-04-04 Kanebo Ltd 保存安定性に優れた絹フィブロイン及びその製造法
WO2002102845A1 (fr) * 2001-06-14 2002-12-27 National Institute Of Agrobiological Sciences Procede de production d'un polypeptide fonctionnel provenant de fibroine de soie, et son utilisation
JP2008169171A (ja) * 2007-01-15 2008-07-24 Luc Sangyo Kk 絹フィブロイン含有組成物及びその製造法
WO2009119596A1 (fr) * 2008-03-26 2009-10-01 国立大学法人岐阜大学 Procédé de classification de protéines, procédé de dissolution de protéines, procédé de fractionnement et de collecte de fibres non animales et de protéines provenant d'une fibre animale
JP2011196001A (ja) * 2010-03-23 2011-10-06 Shinshu Univ 水生昆虫由来のシルクナノファイバー及びシルク複合ナノファイバー、並びにその製造方法
WO2017030197A1 (fr) * 2015-08-20 2017-02-23 国立研究開発法人理化学研究所 Procédé de fabrication de composition polypeptidique présentant une structure de type fibroïne

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SAI SUKEMOTO ET AL.: "Purification of antifungal active substances from wild Japanese silk moth fibroin powder", no. 28, 2003, pages 25 *
TSENG, P. ET AL.: "Directed assembly of bio-inspired hierarchical materials with controlled nanofibrillar architectures", NATURE NANOTECHNOLOGY, vol. 12, no. 5, May 2017 (2017-05-01), pages 474 - 480, XP055582811, ISSN: 1748-3387, DOI: 10.1038/nnano.2017.4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220119463A1 (en) * 2019-01-09 2022-04-21 Spiber Inc. Modified fibroin
WO2020241769A1 (fr) * 2019-05-29 2020-12-03 Ｓｐｉｂｅｒ株式会社 Microcorps de protéine structurale et son procédé de production, procédé de production de nanofibre et procédé de production de structure de protéine
JPWO2020241769A1 (ja) * 2019-05-29 2021-12-16 Ｓｐｉｂｅｒ株式会社 構造タンパク質微小体及びその製造方法、ナノファイバーの製造方法、並びにタンパク質構造体の製造方法
JP7007008B2 (ja) 2019-05-29 2022-02-10 Ｓｐｉｂｅｒ株式会社 構造タンパク質微小体及びその製造方法、ナノファイバーの製造方法、並びにタンパク質構造体の製造方法
CN114206910A (zh) * 2019-05-29 2022-03-18 丝芭博株式会社 结构蛋白微体及其制备方法、纳米纤维的制备方法和蛋白质结构体的制备方法
WO2022260147A1 (fr) * 2021-06-10 2022-12-15 Ｓｐｉｂｅｒ株式会社 Additif pour composition cosmétique

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