WO2019131924A1 - Molded body and molded-body manufacturing method - Google Patents

Abstract

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.

Description

Molded article and method for producing molded article

The present invention relates to a molded body and a method of manufacturing the molded body.

BACKGROUND ART In recent years, bioplastics have attracted attention as substitutes for petroleum-derived plastics due to rising awareness of environmental conservation. For example, Patent Document 1 describes that a molded article obtained by molding a polypeptide derived from natural spider silk protein has a high flexural modulus.

International Publication No. 2017/047504

Alternatives to petroleum-derived plastics are required to have characteristics that are suited to their applications. For example, bioplastics used for long-term outdoor applications are required to have weatherability.

Then, an object of this invention is to provide the biodegradable molded object which can fully suppress the intensity | strength fall by ultraviolet-ray deterioration, and its manufacturing method.

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

The molded article is a molded article of a composition containing a polypeptide and carbon black. With such a configuration, the molded article is inhibited from deterioration by ultraviolet light, and its strength decreases due to deterioration by ultraviolet light ( In particular, the decrease in bending strength is significantly suppressed. Therefore, the molded article can maintain high strength (in particular, high bending strength) even when exposed to the outdoors for a long time, for example.

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

In the above embodiment, the structural protein may include 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 article of a composition containing a polypeptide and carbon black. This manufacturing method includes a mixing step of mixing the polypeptide and the carbon black to obtain a composition, and a forming step of forming the composition by heating and pressing.

According to the said manufacturing method, the biodegradable molded object which can fully suppress the intensity | strength fall by ultraviolet-ray deterioration can be obtained easily.

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

In the above embodiment, the structural protein may include 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, wherein the composition may be a mixed powder including the polypeptide powder and the carbon powder.

ADVANTAGE OF THE INVENTION According to this invention, the biodegradable molded object which can fully suppress the intensity | strength fall by ultraviolet-ray deterioration, and its manufacturing method are provided.

It is a schematic cross section which shows a pressure forming machine. (A) is a schematic cross section showing a pressure molding machine before introducing a composition, (b) is a schematic cross section showing a pressure molding machine immediately after introducing the composition, and (c) is heating the composition FIG. 2 is a schematic cross-sectional view showing a pressure molding machine in a state of being pressurized.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. The drawings are drawn with exaggeration in part for ease of understanding, and dimensional ratios and the like are not limited to those described in the drawings.

The molded article according to the present embodiment is obtained by introducing a composition containing a polypeptide and carbon black into a mold and processing it.

Preferably the polypeptide is a structural protein. A structural protein is a protein having a role in constructing a biological structure, and is different from functional proteins such as enzymes, hormones and antibodies. As structural proteins, naturally occurring structural proteins such as fibroin, collagen, resilin, elastin and keratin can be mentioned. Known naturally occurring fibroins are fibroins produced by insects and arachnids. In this embodiment, the structural protein preferably includes at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.

Examples of 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.

More specific examples of insect-produced fibroin include, for example, silkworm fibroin L chain (GenBank accession No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).

There are up to seven types of silk glands in spiders, each producing fibroin (spider silk protein) with different properties. Spider silk proteins are, according to their source organs, large tenacious spider proteins (major ampullate spider protein (MaSp), high elongating fissure-like spider proteins (minor ampullate spider protein (MiSp), It has been designated as a flagelliform (Flag), a tubular (tubuliform), an aggregate, a grapevine (aciniform) and a pearlike spider silk protein.

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 belonging to the genus Arachnura such as a black-toothed spider, a spider belonging to the genus Acusilas such as a spider, a spider spider belonging to the genus Acusilas Spiders belonging to the genus Cytophora, spiders belonging to the genus Pythogmo (Poltys), spider spiders belonging to the genus Poltys, spider spiders such as the spider spider belonging to the genus Cyclosa such as the spider spider such as the spider spider, the spider spider, the spider spider and the crow spider spider, and the spider spider belonging to the genus Cyclosa Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asinacea such as asinacea, assassinum, assassinum and assassinum Spiders belonging to the genus Gummo (Tetragnatha), spiders belonging to the genus Negrocarpus, such as the spiders belonging to the genus Necha Spiders belonging to the genus Azosoma (Menosira), spiders belonging to the genus Spermatoglyphus (Dyschiriognatha), such as the spider snail, spiders belonging to the genus Latrodectus such as the spider snail, the spider moth, the spider moth, and the spider moth Produced by spiders belonging to the family Agnagidae (Tetragnathidae) such as spiders belonging to the genus Prostenopus (Euprosthenops) The spider silk protein is mentioned. Examples of spider silk proteins include MaSp (MaSp1 and MaSp2), dragline proteins such as ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of 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. 1 (amino acid sequence)), minor ampullate silk protein 2 [Nephyla clavipes (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullalate spidroin-like protein [Nephilengys cruentata] (GenBank Accession No. ABR37278.1 (amino acid sequence)), and the like.

More specific examples of naturally derived fibroin further include fibroin whose sequence information is registered in NCBI GenBank. For example, 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.

The structural protein may be a polypeptide derived from the above-mentioned naturally occurring structural protein, ie a recombinant polypeptide. For example, recombinant fibroin is produced in several heterologous protein production systems, and transgenic goats, transgenic silkworms, or recombinant plants or mammalian cells are used as a method for producing them (Science, 2002, 295, pp. 472-476).

The recombinant fibroin can be obtained, for example, by deleting one or more of the sequences encoding the (A) _n motif from the cloned gene sequence of naturally occurring fibroin. Also, 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. It can also be done. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally derived 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. 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.

The recombinant polypeptide of the large nasogastric silkworm silk protein is, for example, a protein comprising a domain sequence represented by the formula 1: [(A) _n motif-REP] _m (wherein (A) _n The motif indicates an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in _n motif is 80% or more.REP is 10 to 200 amino acid residues. The m represents an integer of 8 to 300. A plurality of (A) _n motifs may be identical to each other or different from each other. The amino acid sequences may be the same or different. Specifically, a protein comprising the amino acid sequence shown by SEQ ID NO: 12 can be mentioned.

As a recombinant polypeptide of collagen, for example, a protein comprising a domain sequence represented by Formula 2: [REP2] _o (wherein, in Formula 2, o represents an integer of 5 to 300. REP2 is a Gly-X) An amino acid sequence consisting of a single Y is shown, and X and Y are any amino acid residues other than Gly, and the plurality of REP2 may be identical amino acid sequences to each other or may be different amino acid sequences. Can. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 13 can be mentioned. The amino acid sequence shown by SEQ ID NO: 13 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (Genbank accession numbers of NCBI: CAA56335.1, 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-terminus of the amino acid sequence from residue 301 to residue 540.

As a recombinant polypeptide of resilin, for example, a protein comprising a domain sequence represented by the formula 3: [REP3] _p (wherein, in the formula 3, p represents an integer of 4 to 300. REP3 is Ser-1J-1) J shows an amino acid sequence composed of J-Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr. Is any amino acid residue, preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.The plurality of REP3 may have the same amino acid sequence or different amino acid sequences. Good) can be mentioned. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 14 can be mentioned.
The amino acid sequence shown by SEQ ID NO: 14 is an amino acid sequence of Resilin (Genbank Accession Nos. NP 611 157, Gl: 24654243 of NCBI), wherein Thr at position 87 is substituted with Ser, and Asn at position 95 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 obtained by substituting Asp.

As a recombinant polypeptide of elastin, for example, a protein having an amino acid sequence of Accession Nos. AAC98395 (human), I47076 (sheep), NP786966 (bovine) of Genbank of NCBI can be mentioned. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 15 can be mentioned. The amino acid sequence set forth in SEQ ID NO: 15 is the amino acid sequence set forth in SEQ ID NO: 5 at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of accession number AAC 98395 of Genbank of Genbank (tag sequence And hinge arrangement) are added.

As a recombinant polypeptide of keratin, for example, type I keratin of Capra hircus etc. can be mentioned. Specifically, a protein comprising the amino acid sequence shown in SEQ ID NO: 16 (the amino acid sequence of Genbank Accession No. ACY30466 of NCBI) can be mentioned.

The recombinant polypeptide comprises 90% or more of (i) the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10, or (ii) the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 It may be a recombinant fibroin comprising an amino acid sequence having the sequence identity of

(I) A recombinant fibroin comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 is described. The amino acid sequence represented by SEQ ID NO: 2 is an alternate sequence from the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally occurring fibroin (corresponding to naturally occurring fibroin) from the N terminal side to the C terminal side A) _n motif is deleted and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 4 is one in which all GGX in the REP of the amino acid sequence shown by SEQ ID NO: 2 is replaced with GQX. The amino acid sequence shown by SEQ ID NO: 10 inserts two alanine residues at the C-terminal side of each (A) _n motif of the amino acid sequence shown by SEQ ID NO: 4 and further contains some glutamine (Q) residues. It is one in which a serine (S) residue is substituted and a portion of the amino acid at the N-terminal side is deleted so as to be approximately the same as the molecular weight of SEQ ID NO: 4. The amino acid sequence shown by SEQ ID NO: 3 is one in which all GGX in the REP of the amino acid sequence shown by SEQ ID NO: 1 is replaced with GQX.

(Ii) A recombinant fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 is described. (Ii) The recombinant fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10. (Ii) Recombinant fibroin is also a protein comprising a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m . The above sequence identity is preferably 95% or more.

The above-mentioned recombinant fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This makes it possible to isolate, immobilize, detect and visualize the recombinant fibroin.

As a tag sequence, for example, an affinity tag utilizing specific affinity (binding, affinity) with another molecule can be mentioned. A histidine tag (His tag) can be mentioned as a specific example of an affinity tag. The His tag is a short peptide consisting of 4 to 10 histidine residues, and has the property of specifically binding to a metal ion such as nickel, so that a single peptide of recombinant fibroin by metalating metal chromatography can be obtained. It can be used for separation. As a specific example of the tag sequence, for example, the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence containing His tag) can be mentioned.

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, "epitope tags" utilizing antigen-antibody reactions can also be used. By adding a peptide (epitope) showing 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 using an epitope tag, recombinant fibroin can be easily purified with high specificity.

Furthermore, those in which the tag sequence can be separated by a specific protease can also be used. It is also possible to recover the recombinant fibroin from which the tag sequence has been released by subjecting the protein adsorbed via the tag sequence to a protease treatment.

As more specific examples of the recombinant fibroin containing the tag sequence, (iii) the amino acid sequence shown 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 And a recombinant fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in

The recombinant polypeptide comprises (iii) an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or (iv) an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 90 by more than 90%. It may be a recombinant fibroin comprising an amino acid sequence having the sequence identity of

The amino acid sequences shown in SEQ ID NOs: 6, 7, 8, 9 and 11 are the amino acid sequences shown in SEQ ID NO: 5 (His tag) at the N-terminus of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 and 10, respectively. (Inclusive) is added. (Iv) Recombinant fibroin is also a protein comprising a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m . The above sequence identity is preferably 95% or more.

Preferably, the structural protein comprises a recombinant polypeptide. By including a recombinant polypeptide as a structural protein, it is possible to adjust the flexural modulus, flexural strength and hardness of the resulting molded article to desired values.

[Recombinant cells that express structural proteins]
The method for producing the recombinant polypeptide is described in detail below. The recombinant polypeptide of interest is, for example, 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.

There are no particular restrictions on the method for producing the gene encoding the target recombinant polypeptide. For example, a gene encoding a natural structural protein can be used to produce a gene by amplification and cloning by polymerase chain reaction (PCR) or the like, or by chemical synthesis. The chemical synthesis method of the gene is not particularly limited, and, for example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. based on the amino acid sequence information of the structural protein obtained from the NCBI web database etc. The gene can be chemically synthesized by a method of ligating the oligonucleotide synthesized at step S 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 at the N-terminus of the above amino acid sequence may be synthesized.

The regulatory sequence is a sequence that controls the expression of a recombinant protein in a 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 a promoter, an inducible promoter which functions in a host cell and is capable of inducing expression of a target protein may be used. An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as temperature, osmotic pressure or an increase or decrease in pH value.

The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector and the like. As the expression vector, a vector capable of autonomous replication in a host cell or capable of being integrated into the host chromosome and containing a promoter at a position capable of transcribing a gene encoding a target recombinant polypeptide is preferable. Used for

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

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

Examples of vectors for introducing a gene encoding a target recombinant polypeptide include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (specifically, JP2002-238569 gazette) etc. can be mentioned.

Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like).

As a yeast, the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

Examples of vectors include YEP13 (ATCC 37115), YEp24 (ATCC 37051) and the like.

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

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

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

When the host according to the present embodiment is a prokaryote such as E. coli or a eukaryote such as yeast, as a culture medium for the host according to the present embodiment, carbon sources, nitrogen sources, inorganic salts, etc. As long as it is a medium that can efficiently culture the host, either a natural medium or a synthetic medium may be used.

The carbon source may be any as long as the above-mentioned transformed microorganism can assimilate, for example, glucose, fructose, sucrose and molasses containing them, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid etc. Organic acids and alcohols such as ethanol and propanol can be used.

Nitrogen sources include, for example, ammonium, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.

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

The culture of a prokaryote such as E. coli or a eukaryote such as yeast can be performed under aerobic conditions such as shake culture or submerged aeration culture, for example. The culture temperature is, for example, 15 to 40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be carried out using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like.

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

The recombinant polypeptide according to the present embodiment can be isolated and purified by methods commonly used for protein isolation and purification. For example, when the recombinant polypeptide is expressed in a dissolved state in cells, after completion of culture, host cells are recovered by centrifugation, suspended in an aqueous buffer, and then sonicated, French press The host cells are disrupted using a Manton Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. Methods commonly used for isolation and purification of proteins from supernatant obtained by centrifuging the cell-free extract, ie solvent extraction method, salting out method with ammonium sulfate etc., desalting method, organic solvent Precipitation method, anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Co., Ltd.), or the like using resin such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, hydrophobic chromatography using a resin such as butyl sepharose or phenyl sepharose, gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing Etc. alone or in combination And use, it is possible to obtain a purified product.

Also, when the recombinant polypeptide forms an insoluble form in cells and is expressed, the host cell is similarly recovered and then disrupted and centrifuged to obtain an insoluble form of the recombinant polypeptide as a precipitate fraction. Recover. The insoluble form of the recovered recombinant polypeptide can be solubilized with a protein denaturant. 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 culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

In the present embodiment, the shape of the polypeptide to be added to the composition is not particularly limited, and may be, for example, powdery (lyophilized powder etc.) or fibrous (spinned etc.) shape. The molded article according to the present embodiment may be a fused body of a composition containing a polypeptide of the shape as described above.

In a preferred embodiment, the polypeptide incorporated into the composition may be in the form of powder (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. In addition, 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 the present embodiment, carbon black is not particularly limited. Examples of carbon black include hydrophilic carbon black, surface modified carbon black and the like. In addition, carbon black for paint or ink can also be used.

In a preferred embodiment, the carbon black blended into the composition may be in the form of powder (carbon black powder). The average particle size _{d 2} of the carbon black powder may be for example 1nm or more, preferably 10nm or more. The average particle size _{d 2} of the carbon black powder may be for example 1000nm or less, preferably 500nm or less. In the present specification, a carbon black powder having an average particle size d ₂ of the show the arithmetic mean diameter was determined by electron microscopic observation.

In addition, both the polypeptide and the carbon black to be blended into the composition may be in the form of powder. That is, the molded body may be obtained by introducing a mixed powder containing the polypeptide powder and the carbon black powder into a mold, and processing 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 parts by mass with respect to 100 parts by mass of the polypeptide. It is above. In addition, 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, more preferably 5 parts by mass or less, with respect to 100 parts by mass of the polypeptide.

[Method of producing molded article]
The method for producing a molded article according to one 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 same. .

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

The composition may further contain other components than the polypeptide and carbon black (eg, plasticizer, colorant, filler, synthetic resin, 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, more preferably 1.0 parts by mass with respect to 100 parts by mass of the polypeptide. It is above. In addition, 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, more preferably 5 parts by mass or less, with respect to 100 parts by mass of the polypeptide.

The polypeptide to be mixed in the mixing step may have a powdery (lyophilized powder etc.) or fibrous (spinned etc.) shape, and the molded body has a composition containing the polypeptide of such shape It may be a fused body of matter.

In a preferred embodiment, the mixing step may be a 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. 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 to manufacture a molded body. The press molding machine 10 shown in FIG. 1 is provided with a heatable mold 2 in which a through hole is formed, and an upper pin 4 and a lower pin 6 capable of moving up and down in the through hole of the mold 2. It is. The above composition is introduced into the space created by inserting the upper pin 4 or the lower pin 6 into the mold 2 and the composition is heated by the upper pin 4 and the lower pin 6 while the mold 2 is heated. By compacting, a molded article can be obtained.

FIG. 2 shows a process drawing for obtaining a molded article, wherein (a) is before introduction of the composition, (b) immediately after introduction of the composition, and (c) heating and pressing the composition. It is a schematic cross section which shows the pressure molding machine of a state. As shown in FIG. 2A, the composition is introduced into the through hole in a state where only the lower pin 6 is inserted into the through hole of the mold 2. Subsequently, as shown in FIG. 2 (b), the upper pin 4 is inserted into the through hole of the mold 2 and lowered, heating of the mold 2 is started, and the composition 8a before heating and pressurization is through hole Heat and pressure inside. The upper pin 4 is lowered to a predetermined pressing force, and heating and pressing are continued until the composition reaches a predetermined temperature in the state shown in FIG. Get 8b. Thereafter, the temperature of the mold 2 is decreased using a cooler (eg, 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 Take out. The pressurization may be performed by lowering the upper pin 4 with the lower pin 6 fixed, but both lowering of the upper pin 4 and raising of the lower pin 6 may be performed.

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

As mentioned above, although the suitable embodiment of the present invention was described, the present invention is not limited to the above-mentioned embodiment.

Hereinafter, the present invention will be more specifically described by way of examples, but the present invention is not limited to the examples.

(1) Preparation of Structural Protein-Expressing Strain Base sequences and amino acid sequences of fibroin (GenBank accession numbers: P46804.1, GI: 1174415) derived from Nephila clavipes are obtained from the GenBank web database and then produced. The amino acid residue shown in SEQ ID NO: 12 is added with an amino acid residue substitution, insertion and deletion for the purpose of improving sex, and the amino acid sequence shown in SEQ ID NO: 5 (tag sequence and hinge sequence) added to the N terminus A recombinant fibroin having a sequence (hereinafter also referred to as "PRT 587") was designed.

Next, the gene encoding PRT587 was synthetically commissioned. As a result, a gene having an NdeI site immediately upstream of the 5 'end of the gene 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 with NdeI and EcoRI, and recombined into a protein expression vector pET-22b (+).

(2) Protein expression E. coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT587 obtained above. After culturing the transformed E. coli in 2 mL of LB medium containing ampicillin for 15 hours, the same culture solution was added to 100 mL of a seed culture medium shown in Table 1 so as to have an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30 ° C., and the culture was further continued for about 15 hours in a flask 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 cultured so as to maintain the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Adecanol LG-295S (manufactured by ADEKA Co., Ltd.) was used as an antifoaming agent.

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 culture was controlled at a constant pH of 6.9. Further, the culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 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 expression of the target protein. Twenty hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. SDS-PAGE was performed using cells prepared from the culture solution before IPTG addition and after IPTG addition, and expression of a target protein was confirmed by appearance of a band of the target protein size depending on IPTG addition.

(3) Purification of Structural Protein Two hours after IPTG was added, the cells collected 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 disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) to high purity. The precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, 60 ° C. The solution was stirred for 30 minutes and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Pure Chemical Industries, Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed by a lyophilizer, and the lyophilized powder was recovered.

<Preparation of Molded Product>
Example 1
Three parts by mass of carbon black (Mitsubishi Chemical Co., Ltd .: # 650B) was added to 100 parts by mass of the above-mentioned freeze-dried powder, and ground with an attritor mill at 1000 rpm for 25 minutes to obtain a composition (mixed powder). The obtained composition is penetrated by a mold 2 (a mold 2 is a cylindrical mold having a circular through hole with a diameter of 20 mm) of a pressure molding machine 10 shown in FIG. It was introduced into the hole. Thereafter, heating of the mold 2 was started, and the composition was heated by inserting the upper pin 4 and the lower pin 6 into the through holes using a hot press. Under the present circumstances, it controlled so that the pressurization conditions of a composition might be 30 Mpa. Heating was stopped when the temperature of the composition reached 190 ° C., and after cooling, the contents were removed from the mold to obtain a molded article.

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

<Evaluation>
The molded articles of the 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 temperature 50 ° C. and humidity 60% according to ISO4892-1. The molded product after irradiation was subjected to a bending test with an autograph to measure bending strength and strain at break. The results are shown in Tables 3 and 4.

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

The molded article of the present invention is sufficiently suppressed in strength reduction due to ultraviolet ray deterioration, and is biodegradable. Therefore, the molded article of the present invention can be suitably used for applications where weather resistance is required.

DESCRIPTION OF SYMBOLS 2 ... Mold, 4 ... Upper pin, 6 ... Lower pin, 8a ... Composition before heating and pressure, 8b ... Composition after heating and pressure, 10 ... Pressure forming machine

Claims (7)

1. A molded article of a composition containing a polypeptide and carbon black.
2. The molded article according to claim 1, wherein the polypeptide is a structural protein.
3. The molded article according to claim 2, wherein the structural protein comprises at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.
4. A method for producing a molded article of a composition containing a polypeptide and carbon black, comprising:
   A mixing step of mixing the polypeptide and the carbon black to obtain a composition;
   A forming step of forming the composition by heating and pressing;
   Including the manufacturing method.
5. The method according to claim 4, wherein the polypeptide is a structural protein.
6. The method according to claim 5, wherein the structural protein comprises at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins.
7. The mixing step is a step of mixing the polypeptide powder and the carbon powder,
   The method according to any one of claims 4 to 6, wherein the composition is a mixed powder comprising the polypeptide powder and the carbon powder.

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