JP2021063143A - Molded body and molded-body manufacturing method - Google Patents

Abstract

To provide a biodegradable molding that sufficiently resists strength reduction due to ultraviolet degradation and a method of producing the same.SOLUTION: Provided are: a molded body of a composition containing polypeptide and carbon black; and a molded-body manufacturing method including a mixing step for mixing polypeptide and carbon black to obtain a composition and a molding step for molding the composition by heating and pressurizing the composition.SELECTED DRAWING: None

Description

The present invention relates to a molded product and a method for producing a molded product.

In recent years, bioplastics have been attracting attention as an alternative to petroleum-derived plastics due to growing awareness of environmental conservation. For example, Patent Document 1 describes that a molded product obtained by molding a polypeptide derived from a natural spider silk protein has a high flexural modulus.

International Publication No. 2017/047504

Substitutes for petroleum-derived plastics are required to have characteristics according to their applications. For example, bioplastics used for long-term outdoor exposure are required to have weather resistance.

Therefore, an object of the present invention is to provide a biodegradable molded product capable of sufficiently suppressing a decrease in strength due to deterioration of ultraviolet rays and a method for producing the same.

One aspect of the present invention relates to a molded article of a composition containing a polypeptide and carbon black.

The molded product is obtained by molding a composition containing a polypeptide and carbon black. With such a configuration, the molded product is suppressed from deterioration by ultraviolet rays, and the strength is reduced due to the deterioration by ultraviolet rays ( In particular, the decrease in bending strength) is remarkably suppressed. Therefore, the molded product can maintain high strength (particularly high bending strength) even when exposed to the outdoors for a long time, for example.

In one embodiment, the polypeptide may be a structural protein.

In the above embodiment, the structural protein may contain at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.

Another aspect of the present invention relates to a method for producing a molded product of a composition containing a polypeptide and carbon black. This production method includes a mixing step of mixing the above-mentioned polypeptide and the above-mentioned carbon black to obtain a composition, and a molding step of molding the above-mentioned composition by heating and pressurizing.

According to the above-mentioned production method, a biodegradable molded product capable of sufficiently suppressing a decrease in strength due to deterioration by ultraviolet rays can be easily obtained.

In one embodiment, the polypeptide may be a structural protein.

In the above embodiment, the structural protein may contain at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.

In one aspect, the mixing step may be a step of mixing the polypeptide powder and the carbon powder, and at this time, the composition may be a mixed powder containing the polypeptide powder and the carbon powder.

According to the present invention, there is provided a biodegradable molded product capable of sufficiently suppressing a decrease in strength due to deterioration of ultraviolet rays, and a method for producing the same.

It is a schematic cross-sectional view which shows the pressure molding machine. (A) is a schematic cross-sectional view showing a pressure molding machine before introducing the composition, (b) is a schematic cross-sectional view showing a pressure molding machine immediately after introducing the composition, and (c) is a schematic cross-sectional view showing the composition by heating. It is a schematic cross-sectional view which shows the pressure molding machine in the state of being pressurized.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. It should be noted that the drawings are partially exaggerated for ease of understanding, and the dimensional ratios and the like are not limited to those described in the drawings.

The molded product according to the present embodiment is obtained by introducing a composition containing a polypeptide and carbon black into a mold (mold) and molding the product.

The polypeptide is preferably a structural protein. Structural proteins are proteins that have a role in constructing biological structures, and are different from functional proteins such as enzymes, hormones, and antibodies. Examples of the structural protein include naturally occurring structural proteins such as fibroin, collagen, resilin, elastin and keratin. As naturally occurring fibroin, fibroin produced by insects and arachnids is known. In the present embodiment, the structural protein preferably contains at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, tussah, and tussah. ), Silk moth (Samia cinthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama) Silk protein can be mentioned.

More specific examples of fibroin produced by insects include, for example, silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

There are up to 7 types of silk glands in spiders, each of which produces fibroin (spider silk protein) with different properties. Spider silk proteins are, according to their source organs, a large bottle of spider protein with high toughness (major amplifier spider protein, MaSp), a small bottle of spider protein with high extensibility (minor amplifier spider protein, MiSp), and a whip. It is named spider silk protein in the form (Flagelliform (Flag)), tubular (tubulariform), aggregate (aggregate), grape-like (aciniform) and pear-like (pyriform).

Examples of fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne), spiders belonging to the genus Trinofundamashi, and spiders belonging to the genus Cyrtarachne. Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachunura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ) Spiders, spiders, spiders belonging to the genus Cyclosa, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Tetragnatha, such as Yasagata spider, Harabiroashidaka spider, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha, spiders such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the family Spiders (Tetragnathidae) such as spiders belonging to the genus Euprostenops Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of fibroin produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)), fibroin-. 4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number A) U37520 (nucleic acid sequence)), major angu11ate spidroin 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein spiroin 2 (Amino acid sequence), AF441245 (Nucleotide sequence)), major operlate spidroin 1 [derived from Euprosthenops australis] (GenBank accession number CAJ00428 (nucleic acid sequence), AJ973155 (nucleic acid sequence)), and major amplifier Accession No. CAM3224.9.1 (Amino Acid Sequence), AM490169 (Nucleotide Sequence)), minor amplifier silk fibroin 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (Nucleotide Sequence)), minor complete silk fibroin (GenBank Accession No. AAC14591.1 (Amino Acid Sequence)), minor amplify spidroin-like product [Nephilengies cruisentata] (GenBank Accession No. ABR) 3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally occurring fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The structural protein may be a polypeptide derived from the above-mentioned natural structural protein, that is, a recombinant polypeptide. For example, recombinant fibroin is produced in several heterologous protein production systems, and transgenic goats, transgenic silk moths, or transgenic plant or mammalian cells are used as methods for producing the recombinant fibroin (Science, Science, Inc.). 2002, 295, pp. 472-476).

Recombinant fibroin can be obtained, for example, by deleting one or more of the sequences encoding the _{(A) n motif from the cloned naturally occurring fibroin gene sequence.} Further, for example, it is obtained by designing an amino acid sequence corresponding to the deletion of _{one or more (A) n} motifs from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. You can also do it. _{In each case, in addition to the modification corresponding to the deletion of (A) n} motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to the above may be modified. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).

The recombinant polypeptide of the large spitting tube bookmarking protein is, for example, a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _{m (here, (A) n in formula 1).} The motif shows an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues is 80% or more of the total number of amino acid residues in the _{n motif. REP is 10 to 200 amino acid residues.} Indicates an amino acid sequence composed of. M represents an integer of 8 to 300. Multiple existing (A) _n motifs may be the same amino acid sequence or different amino acid sequences. Multiple REPs may be mutually present. It may be expressed as the same amino acid sequence or different amino acid sequences.). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 12 can be mentioned.

As a recombinant polypeptide of collagen, for example, _{a protein containing a domain sequence represented by the formula 2: [REP2] o} (where, in formula 2, o represents an integer of 5 to 300. REP2 is Gly-X. 1 Indicates an amino acid sequence composed of Y, where X and Y indicate arbitrary amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.) Can be done. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 13 can be mentioned. The amino acid sequence shown in SEQ ID NO: 13 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI Genbank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 5 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a recombinant polypeptide of resilin, for example, _{a protein containing a domain sequence represented by the formula 3: [REP3] p} (where, in formula 3, p represents an integer of 4 to 300. REP3 is Ser-J1. The amino acid sequence composed of J-Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. Indicates an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. Multiple REP3s may have the same amino acid sequence or different amino acid sequences. Good.) Can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 14 can be mentioned.
The amino acid sequence shown in SEQ ID NO: 14 replaces Thr at residue 87 with Ser and Asn at residue 95 in the amino acid sequence of recillin (NcBI Genbank accession number NP 61157, Gl: 24654243). The amino acid sequence (tag sequence) shown by SEQ ID NO: 17 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.

Examples of the recombinant polypeptide of elastin include proteins having amino acid sequences such as NCBI Genbank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 15 can be mentioned. The amino acid sequence shown by SEQ ID NO: 15 is the amino acid sequence shown by SEQ ID NO: 5 at the N-terminal of the amino acid sequence from the 121st residue to the 390th residue of the amino acid sequence of the accession number AAC98395 of Genbank of NCBI. And the hinge arrangement) are added.

Examples of the recombinant polypeptide of keratin include Type I keratin of Capra hilcus. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 16 (amino acid sequence of NCBI Genbank accession number ACY30466) can be mentioned.

The recombinant polypeptide is 90% or more of (i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10, or (ii) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10. It may be a recombinant fibroin containing an amino acid sequence having the sequence identity of.

(I) Recombinant fibroin containing the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 will be described. The amino acid sequence shown in SEQ ID NO: 2 is from the amino acid sequence shown in SEQ ID NO: 1 (corresponding to naturally occurring fibroin) corresponding to naturally occurring fibroin to every other two from the N-terminal side to the C-terminal side (corresponding to naturally occurring fibroin) A) The _n- motif is deleted, and one [(A) _n- motif-REP] is inserted before the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 10, _{two alanine residues are inserted on the C-terminal side of each (A) n} motif of the amino acid sequence shown in SEQ ID NO: 4, and a part of glutamine (Q) residue is further added. It is substituted with a serine (S) residue and a part of the amino acid on the N-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 1.

(Ii) A recombinant fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 will be described. (Ii) The recombinant fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10. (Ii) Recombinant fibroin is also a protein containing a domain sequence represented by _{formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The recombinant fibroin described above may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of recombinant fibroin.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and since it has the property of specifically binding to a metal ion such as nickel, it is a single recombinant fibroin obtained by metal chelating chromatography. It can be used separately. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence containing a His tag).

In addition, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. By utilizing the epitope tag, recombinant fibroin can be easily purified with high specificity.

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. Recombinant fibroin from which the tag sequence has been separated can also be recovered by treating the protein adsorbed via the tag sequence with a protease.

As a more specific example of the recombinant fibroin containing the tag sequence, (iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or (iv) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 Examples thereof include recombinant fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.

The recombinant polypeptide is 90% or more of the amino acid sequence set forth in (iii) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or the amino acid sequence set forth in (iv) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. It may be a recombinant fibroin containing an amino acid sequence having the sequence identity of.

The amino acid sequences shown by SEQ ID NOs: 6, 7, 8, 9 and 11 are the amino acid sequences shown by SEQ ID NO: 5 (His tag) at the N-terminal of the amino acid sequences shown by SEQ ID NOs: 1, 2, 3, 4 and 10, respectively. ) Is added. (Iv) Recombinant fibroin is also a protein containing a domain sequence represented by _{formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The structural protein preferably contains a recombinant polypeptide. By including the recombinant polypeptide as the structural protein, it is possible to adjust the flexural modulus, bending strength and hardness of the obtained molded product to desired values.

[Recombinant cells expressing structural proteins]
The method for producing the recombinant polypeptide will be described in detail below. The recombinant polypeptide of interest is prepared, for example, by a host transformed with an expression vector having a gene sequence encoding a structural protein and one or more regulatory sequences operably linked to the gene sequence. It can be produced by expressing a gene.

The method for producing the gene encoding the recombinant polypeptide of interest is not particularly limited. For example, a gene encoding a natural structural protein can be used to amplify and clone it by a polymerase chain reaction (PCR) or the like, or the gene can be produced by chemical synthesis. The chemical synthesis method of the gene is also not particularly limited. For example, based on the amino acid sequence information of the structural protein obtained from the NCBI web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized in (1) by PCR or the like. At this time, in order to facilitate purification and confirmation of the protein, a gene encoding a polypeptide having an amino acid sequence consisting of a start codon and a His10 tag added to the N-terminal of the above amino acid sequence may be synthesized.

The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the protein of interest may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. The expression vector preferably contains a promoter at a position capable of autonomous replication in the host cell, integration into the host chromosome, and transcription of the gene encoding the recombinant polypeptide of interest. Used for.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Preferred examples of prokaryotes include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.

Examples of the vector into which the gene encoding the recombinant polypeptide of interest is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (specially manufactured). Kai 2002-238569) and the like can be mentioned.

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.).

Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

Examples of the vector include YEP13 (ATCC37115) and YEp24 (ATCC37051).

As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like can be mentioned.

As a method for expressing a gene by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. ..

The target recombinant polypeptide is, for example, obtained by culturing a host transformed with the expression vector according to the present embodiment in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. Can be manufactured by. The method for culturing the host according to the present embodiment in the culture medium can be carried out according to the method usually used for culturing the host.

When the host according to this embodiment is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the host can be used as a culture medium for the host according to this embodiment. As long as the medium contains the above and can efficiently culture the host, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digested products thereof can be used.

As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culturing is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The recombinant polypeptide according to this embodiment can be isolated and purified by a method usually used for isolating and purifying a protein. For example, when the recombinant polypeptide is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, and then an ultrasonic crusher or a French press. , Mantongaulin homogenizer, dynomil and the like to disrupt host cells to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting out method using ammonium sulfate or the like, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

When the recombinant polypeptide is expressed by forming an insoluble matter in the cell, the host cell is similarly recovered, crushed, and centrifuged to obtain an insoluble matter of the recombinant polypeptide as a precipitate fraction. To collect. The insoluble form of the recovered recombinant polypeptide can be solubilized with a protein denaturing agent. After the operation, a purified preparation of the recombinant polypeptide can be obtained by the same isolation and purification method as described above.

When the recombinant polypeptide is secreted extracellularly, the recombinant polypeptide can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

In the present embodiment, the shape of the polypeptide blended in the composition is not particularly limited, and may have, for example, a powdery (lyophilized powder or the like) or a fibrous (spun or the like) shape. The molded product according to the present embodiment can be a fused product of a composition containing a polypeptide having the above-mentioned shape.

In a preferred embodiment, the polypeptide blended in the composition may be in powder form (polypeptide powder). The average particle size d ₁ of the polypeptide powder may be, for example, 0.1 μm or more, preferably 1 μm or more. The average particle size d ₁ of the polypeptide powder may be, for example, 200 μm or less, preferably 100 μm or less. In the present specification, the average particle size d ₁ of the polypeptide powder indicates a value measured by a particle size distribution measuring device.

In this embodiment, carbon black is not particularly limited. Examples of carbon black include hydrophilic carbon black and surface-modified carbon black. Further, carbon black for paint or ink can also be used.

In a preferred embodiment, the carbon black blended in the composition may be in powder form (carbon black powder). The average particle size d ₂ of the carbon black powder may be, for example, 1 nm or more, preferably 10 nm or more. The average particle size d ₂ of the carbon black powder may be, for example, 1000 nm or less, preferably 500 nm or less. In the present specification, the average particle size d ₂ of the carbon black powder indicates the arithmetic mean diameter obtained by observing with an electron microscope.

In addition, the polypeptide and carbon black blended in the composition may both be in the form of powder. That is, the molded product may be obtained by introducing a mixed powder containing a polypeptide powder and a carbon black powder into a mold and molding it.

The amount of carbon black blended in the composition may be, for example, 0.05 parts by mass or more, preferably 0.5 parts by mass or more, and more preferably 1.0 part by mass with respect to 100 parts by mass of the polypeptide. That is all. The amount of carbon black blended in the composition may be, for example, 20 parts by mass or less, preferably 10 parts by mass or less, and more preferably 5 parts by mass or less with respect to 100 parts by mass of the polypeptide.

[Manufacturing method of molded product]
The method for producing a molded article according to an embodiment includes a mixing step of mixing a polypeptide and carbon black to obtain a composition, and a molding step of introducing the composition into a mold and molding the composition. ..

The mixing step may be a step of adding carbon black to the composition containing the polypeptide, or a step of adding the polypeptide to the composition to which the carbon black is added, and the polypeptide and the carbon black may be added at the same time. It may be a step of adding and mixing.

The composition may further contain components other than the polypeptide and carbon black (eg, plasticizers, colorants, fillers, synthetic resins, etc.). The content of these other components is preferably 50 parts by mass or less with respect to 100 parts by mass of the polypeptide.

The amount of carbon black mixed in the mixing step may be, for example, 0.05 parts by mass or more, preferably 0.5 parts by mass or more, and more preferably 1.0 part by mass with respect to 100 parts by mass of the polypeptide. That is all. The amount of carbon black blended in the composition may be, for example, 20 parts by mass or less, preferably 10 parts by mass or less, and more preferably 5 parts by mass or less with respect to 100 parts by mass of the polypeptide.

The polypeptide mixed in the mixing step may have a powdery (lyophilized powder or the like) or fibrous (spun or the like) shape, and the molded product has a composition containing the polypeptide having such a shape. It can be a fusion of things.

In a preferred embodiment, the mixing step may be the step of mixing the polypeptide powder and the carbon powder. At this time, the composition obtained in the mixing step may be a mixed powder containing the polypeptide powder and the carbon powder.

The molding step is a step of introducing the composition obtained in the mixing step into a mold and molding the composition. In the molding step, the composition may be heated and / or pressurized during the molding process, and it is preferable to heat and pressurize.

In the molding step, for example, molding can be performed using a pressure molding machine. FIG. 1 is a schematic cross-sectional view showing a pressure molding machine that can be used for manufacturing a molded product. The pressure molding machine 10 shown in FIG. 1 includes a mold 2 having a through hole formed and capable of heating, and an upper pin 4 and a lower pin 6 capable of moving up and down in the through hole of the mold 2. Is. The composition is introduced into the gap created by inserting the upper pin 4 or the lower pin 6 into the mold 2, and the composition is used by the upper pin 4 and the lower pin 6 while heating the mold 2. A molded product can be obtained by compressing.

FIG. 2 shows a process diagram for obtaining a molded product, in which (a) is before the introduction of the composition, (b) is immediately after the introduction of the composition, and (c) is heating and pressurizing the composition. It is a schematic cross-sectional view which shows the pressure molding machine in a state. As shown in FIG. 2A, the composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the composition 8a before heating and pressurizing is formed through the through hole. Heat and pressurize inside. The upper pin 4 is lowered until a predetermined pressing force is reached, and heating and pressurization are continued until the composition reaches a predetermined temperature in the state shown in FIG. 2C, and the composition after heating and pressurizing. Obtain 8b. Then, the temperature of the mold 2 is lowered by using a cooler (for example, a spot cooler), and when the composition 8b reaches a predetermined temperature, the upper pin 4 or the lower pin 6 is removed from the mold 2 and the contents are removed. Take out. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lower pin 4 may be lowered and the lower pin 6 may be raised.

The heating is preferably performed at a mold temperature of 80 to 250 ° C, more preferably 100 to 200 ° C. The pressurization is preferably performed at a pressure of 20 MPa or more.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to the above embodiment.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the Examples.

(1) Preparation of Structural Protein Expressing Strain The nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes are obtained from the GenBank web database and then produced. Amino acids shown in SEQ ID NO: 12 are substituted, inserted and deleted for the purpose of improving sex, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 5 is added to the N-terminal. A recombinant fibroin having a sequence (hereinafter, also referred to as “PRT587”) was designed.

Next, the gene encoding PRT587 was outsourced for synthesis. As a result, a gene having an NdeI site immediately upstream of the 5'end and an EcoRI site immediately downstream of the 3'end was obtained. The gene was cloned into a cloning vector (pUC118), treated with restriction enzymes NdeI and EcoRI, and recombined into a protein expression vector pET-22b (+).

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT587 obtained above. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours, and then the same culture solution was added to _{100 mL of the seed culture medium shown in Table 1 so that the OD 600 was 0.005.} The temperature of the culture solution was kept at 30 ° C., and the cells were further cultured in a flask for about 15 hours until _{the OD 600 reached 5, to obtain a seed culture solution.}

The obtained seed culture solution was added to a jar fermenter to which 500 mL of the production medium shown in Table 2 was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., controlled to be constant at pH 6.9, and the culture was carried out so that the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. As the antifoaming agent, ADEKANOLG-295S (manufactured by ADEKA Corporation) was used.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, a 1 M aqueous solution of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the target protein. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broth before and after the addition of IPTG, and the expression of the target protein was confirmed by the appearance of a band of the target protein size depending on the addition of IPTG.

(3) Purification of Structural Protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM phenylmethylsulfonyl fluoride (PMSF), and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL at 60 ° C. Was stirred with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white agglutinated protein obtained after dialysis was recovered by centrifugation, water was removed by a lyophilizer, and the lyophilized powder was recovered.

<Making a molded product>
(Example 1)
3 parts by mass of carbon black (manufactured by Mitsubishi Chemical Corporation: # 650B) was added to 100 parts by mass of the freeze-dried powder, and the mixture was ground with an attritor mill at 1000 rpm for 25 minutes to obtain a composition (mixed powder). The obtained composition is passed through the mold 2 of the pressure molding machine 10 shown in FIG. 1 (the mold 2 is a cylindrical mold and has a circular through hole having a cross section of 20 mm in diameter). It was introduced into the hole. After that, the heating of the die 2 was started, and the composition was heated by inserting the upper pin 4 and the lower pin 6 into the through hole using a hot press machine. At this time, the pressurizing condition of the composition was controlled to be 30 MPa. Heating was stopped when the temperature of the composition reached 190 ° C., and after cooling, the contents were taken out from the mold to obtain a molded product.

(Comparative Example 1)
A molded product was produced in the same manner as in Example 1 except that carbon black was not blended.

<Evaluation>
^{The molded bodies of Examples and Comparative Examples were irradiated with light of 290 to 490 nm at 60 W / m 2} for a predetermined time (24 to 96 hours) in an atmosphere of a temperature of 50 ° C. and a humidity of 60% according to ISO4892-1. The molded product after irradiation was subjected to a bending test by an autograph, and the bending strength and strain at the time of breaking were measured. The results are shown in Tables 3 and 4.

As shown in Table 3, the molded product of Example 1 is suppressed from deterioration due to ultraviolet rays as compared with the molded product of Comparative Example 1, and high bending strength is maintained even when exposed to ultraviolet rays for a long time. It was confirmed that. Further, as shown in Table 4, it was confirmed that the molded product of Example 1 had a larger strain at break and was excellent in toughness as compared with the molded product of Comparative Example 1.

2 ... Mold, 4 ... Upper pin, 6 ... Lower pin, 8a ... Composition before heating and pressurizing, 8b ... Composition after heating and pressurizing, 10 ... Pressurizing molding machine.

Claims (7)

A molded product of a composition containing a polypeptide and carbon black. The molded article according to claim 1, wherein the polypeptide is a structural protein. The molded product according to claim 2, wherein the structural protein contains at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein. A method for producing a molded product of a composition containing a polypeptide and carbon black.
A mixing step of mixing the polypeptide and the carbon black to obtain a composition, and
A molding step of heating and pressurizing the composition, and
Manufacturing method, including. The production method according to claim 4, wherein the polypeptide is a structural protein. The production method according to claim 5, wherein the structural protein comprises at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein. The mixing step is a step of mixing the polypeptide powder and the carbon powder.
The production method according to any one of claims 4 to 6, wherein the composition is a mixed powder containing the polypeptide powder and the carbon powder.

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