JP2020105231A - Molded article and method for producing molded article - Google Patents

Abstract

To provide a molded article having stable physical properties against environmental changes, and to provide a method for producing the same.SOLUTION: There is provided a molded article of a composition comprising a polypeptide, wherein the flexural strength of the molded article at room temperature measured after alternately exposing to a temperature environment of greater than 0°C and a temperature environment of 0°C or less. within a temperature range of -100 to 120°C is less than or equal to ± 20% of the flexural strength at room temperature measured prior to the aforementioned exposure.SELECTED DRAWING: None

Description

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

In recent years, bioplastics, which are non-petroleum materials, have been attracting attention due to increasing awareness of environmental protection. For example, "TERRAMAC" (registered trademark) having a polylactic acid ratio of 99.5% and a silk molded product obtained by resinizing silk powder are known. Among these bioplastics, "Terramac" (registered trademark) has a flexural modulus of 4.3 GPa, and the silk molding has a flexural modulus of 4.5 GPa, and each has sufficient strength. Is also known (for example, see Non-Patent Document 1).

Japanese Patent Laid-Open No. 6-156071

Shinji Hirai, Monthly Journal of Functional Materials, June 2014, "Environmentally friendly silk and wool resin using waste-derived animal protein"

However, the stability of physical properties of bioplastics against environmental temperature changes has not yet been clarified. Therefore, an object of the present invention is to provide a molded product having stable physical properties against environmental temperature changes and a method for producing the molded product.

The present invention relates to a molded article of a composition containing a polypeptide, which is measured within a temperature range of −100 to 120° C. after being alternately exposed to a temperature environment of higher than 0° C. and a temperature environment of 0° C. or lower. Provided is a molded article, which has a bending strength at room temperature of ±20% or less of the bending strength at room temperature measured before exposure.

The Young's modulus at room temperature, which was measured after the molded body was alternately exposed to a temperature environment exceeding 0° C. and a temperature environment below 0° C. within a temperature range of −100 to 120° C., was measured before the exposure. It may be a molded body having a Young's modulus of ±15% or less at room temperature.

The molded body preferably has transparency. When the above-mentioned molded product has transparency, it can be applied to, for example, window applications without any problem.

The molded body preferably has a bending elastic modulus of more than 4.5 GPa. Further, since the above-mentioned molded product exhibits a high flexural modulus of more than 4.5 GPa, when it is molded into a panel shape, it is possible to obtain a strength equal to or higher than that of a conventional synthetic resin with a thinner thickness. Greatly contributes to weight reduction. The specific gravity is also equivalent to that of synthetic resins such as acrylic resin and polycarbonate resin, and is much lower than that of inorganic glass. Therefore, in many applications including automobile applications, it is possible to reduce the environmental load through weight reduction. The flexural modulus is preferably 5.0 GPa or more, more preferably 5.2 GPa or more. Although the upper limit of the bending elastic modulus is not limited, for example, the bending elastic modulus can be set to 15.0 GPa or less, further 10.0 GPa or less, or 7.0 GPa or less.

The polypeptide preferably contains at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins (artificial polypeptides). Natural spider silk proteins and polypeptides derived from natural spider silk proteins have very good flexural modulus and flexural strength, and their inclusion as polypeptides enables further weight reduction and higher functionality. ..

The polypeptide is preferably produced by a genetically modified microorganism. A polypeptide produced by a genetically modified microorganism can be obtained at a lower cost than a naturally-occurring polypeptide and can be stably supplied in quality.

The polypeptide may further contain silk fibroin, soy protein, casein, keratin, collagen, whey protein. By using these in combination with a natural spider silk protein or a polypeptide derived from a natural spider silk protein, it becomes easy to design the performance of the molded article according to the purpose of use.

The molded body may be provided as a heat and pressure molded body. The term "molded product" refers to a product molded by a mold (mold) or the like. By heating and pressurizing, a molded product having a further excellent flexural modulus can be obtained.

The molded body can be produced by a method including a step of heating and pressing the composition containing the above-mentioned polypeptide.

According to the present invention, a molded product having stable physical properties against environmental temperature changes, and a method for manufacturing the molded product are provided.

It is a schematic cross section of a pressure molding machine. FIG. 4A is a schematic cross-sectional view of a pressure molding machine in which (a) is before introducing the composition, (b) is immediately after introducing the composition, and (c) is a state in which the composition is heated and pressurized. 3 is a photograph of the molded body of Example 1. 20 is a graph showing the flexural strength and Young's modulus change rate of the molded body of Example 25.

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the embodiments described below.

The molded article according to the embodiment is composed of a composition containing a polypeptide, and as the polypeptide, a natural spider silk protein and/or a polypeptide derived from the natural spider silk protein (hereinafter, these are combined together It may be collectively referred to as “spider silk polypeptide”.). Polypeptides can include polypeptides other than spider silk polypeptides, and such polypeptides include silk fibroin, soy protein, casein, keratin, collagen, whey protein.

The molded article can be obtained by introducing the above composition into a mold and performing a molding process, and can be heated and/or pressurized in the molding process. The composition is typically in the form of powder (freeze-dried powder, etc.) or fibrous (fibers obtained by spinning, etc.), and the molded body contains the polypeptide of such a shape. It may be a fusion product of the composition.

Examples of the natural spider silk protein used as the polypeptide constituting the molded body include large spittoon guideline thread protein and weft thread protein.

The large votive tube dragline protein is produced in the large bottle-shaped line of a spider, and has a characteristic of excellent toughness. Examples of the large vesicle guideline protein include large bottle-shaped line spidroins MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus.

The weft thread protein is one produced in the flagella form of the spider, and the weft thread protein includes, for example, a flagelliform silk silk protein derived from the Nephila clavipes.

Examples of the polypeptide derived from a natural spider silk protein include a recombinant spider silk protein, for example, a mutant, analog or derivative of a natural spider silk protein. As such a polypeptide, a recombinant spider silk protein which is a large vesicular dragline silk protein is particularly preferable.

As the polypeptide, silk fibroin, soybean protein, casein, keratin, collagen, and whey protein can be used in combination with the above-mentioned spider silk polypeptide. When used in combination, the amount of silk fibroin or the like is, for example, preferably 20 parts by mass or less, more preferably 10 parts by mass or less, and even more preferably 5 parts by mass or less, relative to 100 parts by mass of the spider silk polypeptide. When the amount of silk fibroin or the like is in the above range, the transparency of the molded body is more excellent.

As the silk fibroin, sericin-removed silk fibroin and/or sericin-unremoved silk fibroin can be used. Silk is a fiber obtained from cocoons produced by silkworms, which are larvae of Bombyx mori, and the cocoon thread is composed of two fibroins covered with an outer glue (sericin). Fibroin is composed of many fibrils, and the outside of fibroin is covered with four layers of sericin to form one cocoon thread. Practically, sericin on the outside is dissolved and removed by refining and used as a silk filament for clothing. The specific gravity of silk is 1.33, the average fineness is 3.3 decitex, and the fiber length is generally about 1300 to 1500 m.

Silk fibroin is a lyophilized powder of silk fibroin obtained by purifying silk fibroin from natural or silkworm cocoons or used or discarded silk fabric as raw material, removing sericin covering silk fibroin and other fats. Freeze-dried powder of sericin-removed silk fibroin) is preferred. As the silk fibroin, silk fibroin that does not remove sericin (sericin-unremoved silk fibroin) can be used.

The molded body may be a molded body of only the polypeptide, but an additive component (for example, a plasticizer, a crystal nucleating agent, an antioxidant, a colorant, a filler, water, a synthetic resin, etc.) is added to the polypeptide. It may be a molded product of the added one. When an additive component such as a plasticizer is used, it is preferably 50% by mass or less of the total amount of the polypeptide. In addition, a contaminant generated in the process of obtaining the polypeptide may be included.

A preferable molded article has transparency. Although the transparency can be visually determined, it is preferable that the transmittance is 50% or more when an integrated time of 0.1 seconds is used in a wavelength range of 220 to 800 nm using an optical transmittance measuring device. Is.

The molded body can be produced using a pressure molding machine. FIG. 1 is a schematic cross-sectional view of a pressure molding machine that can be used to manufacture a molded body. The pressure molding machine 10 shown in FIG. 1 includes a mold 2 having a through hole formed therein and capable of heating, and an upper pin 4 and a lower pin 6 capable of vertically moving in the through hole of the mold 2. The composition containing the spider silk polypeptide is introduced into the cavity formed by inserting the upper pin 4 or the lower pin 6 into the mold 2, and the mold 2 is heated while the upper pin 4 and A molded product can be obtained by compressing the composition with the lower pin 6.

2A and 2B are process diagrams for obtaining a molded article, where FIG. 2A is a state before the composition is introduced, FIG. 2B is a state immediately after the composition is introduced, and FIG. 2C is a state in which the composition is heated and pressurized. It is a schematic cross section of a pressure molding machine. As shown in FIG. 2( a ), the composition is introduced into the through hole of the mold 2 with only the lower pin 6 inserted into the through hole, and as shown in FIG. The upper pin 4 is inserted into the through hole and lowered, the heating of the mold 2 is started, and the composition 8a before heating and pressing is heated and pressed in the through hole. The upper pin 4 is lowered until reaching a predetermined pressing force, 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. 8b is obtained. 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 extracted from the mold 2 and the contents Is taken out to obtain a molded body. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lowering of the upper pin 4 and the lowering of the lower pin 6 may be performed.

The heating is preferably performed at 80 to 300°C, more preferably 100 to 180°C, further preferably 100 to 130°C. The pressurization is preferably performed at 5 kN or more, more preferably 10 kN or more, still more preferably 20 kN or more. In addition, after reaching the predetermined heating and pressurizing condition, the time for continuing the treatment under that condition (heat retention condition) is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, and further preferably 5 to 30 minutes.

Since the molded product of the composition containing the polypeptide is preferably prepared using the polypeptide derived from the natural spider silk protein, this production method will be described in detail below.

As the polypeptide derived from the large vesicle guideline protein, which is a raw material of the molded article, the unit of the amino acid sequence represented by the formula 1: REP1-REP2(1) is 2 or more, preferably 5 or more, and more preferably Include polypeptides containing 10 or more. Alternatively, the polypeptide derived from the large vesicle guideline protein comprises a unit of the amino acid sequence represented by formula 1: REP1-REP2(1), and the C-terminal sequence is shown in any one of SEQ ID NOS: 1 to 3. Polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1-3 or the amino acid sequence shown in any of SEQ ID NOs: 1 to 3. In addition, in the polypeptide derived from the large vesicle guideline protein, the units of the amino acid sequence represented by Formula 1: REP1-REP2(1) may be the same or different. In the case of carrying out recombinant protein production using a microorganism such as Escherichia coli as a host, the polypeptide derived from the large vesicle guideline protein preferably has a molecular weight of 500 kDa or less, more preferably 300 kDa, from the viewpoint of productivity. Or less, more preferably 200 kDa or less.

In Formula 1, REP1 means polyalanine. In REP1, it is preferable that the number of alanines arranged consecutively is 2 or more residues, more preferably 3 or more residues, further preferably 4 or more residues, particularly preferably 5 or more residues. is there. Further, in REP1, the number of consecutive alanines arranged is preferably 20 residues or less, more preferably 16 residues or less, further preferably 12 residues or less, and particularly preferably 10 residues. It is as follows. In Formula 1, REP2 is an amino acid sequence consisting of amino acids of 10 to 200 residues, and the total number of residues of glycine, serine, glutamine, and alanine contained in the amino acid sequence is 40% with respect to the total number of amino acid residues. Or more, preferably 60% or more, more preferably 70% or more.

In the large yarn ejection guide thread, REP1 corresponds to a crystalline region that forms a crystalline β sheet in the fiber, and REP2 corresponds to an amorphous region that is more flexible and largely lacks a regular structure in the fiber. Applicable [REP1-REP2] corresponds to a repetitive region (repetitive sequence) composed of a crystal region and an atypical region, and is a characteristic sequence of a bookmark thread protein.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence of the amino acid sequence of ADF3 (NCBI accession number: AAC47010, GI: 1263287) consisting of the amino acids of the 50 residues at the C-terminus, and is shown in SEQ ID NO: 2. The amino acid sequence is the same as the amino acid sequence shown in SEQ ID NO: 1 with 20 residues removed from the C-terminal, and the amino acid sequence shown in SEQ ID NO: 3 is from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence with 29 residues removed.

As the polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2(1), for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 7 can be used. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 has the amino acid sequence (NCBI act) of ADF3 in which an amino acid sequence (SEQ ID NO: 4) consisting of a start codon, a His10 tag, and a HRV3C protease (Humanrhinovirus 3C protease) recognition site is added to the N-terminus. Session number: AAC47010, GI:1263287), which was mutated so that the translation terminates at the 543rd amino acid residue.

Further, as a polypeptide containing two or more units of the amino acid sequence represented by formula 1: REP1-REP2(1), one or more amino acids in the amino acid sequence represented by SEQ ID NO: 7 are substituted, deleted, inserted and A protein having a repeating region consisting of a crystal region and an amorphous region, which consists of an added amino acid sequence and/or can be used. In the present invention, "1 or more" means, for example, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, Or, it means one or several. In the present invention, "1 or several" means 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 Means two or one.

Examples of the polypeptide containing two or more units of the amino acid sequence represented by formula 1: REP1-REP2(1) include recombinant protein derived from ADF4 having the amino acid sequence represented by SEQ ID NO:8. To be The amino acid sequence shown in SEQ ID NO:8 is a partial codon amino acid sequence of ADF4 (NCBI accession number: AAC47011, GI:1263289) obtained from the NCBI database at the N-terminus with a start codon, a His10 tag, and an HRV3C protease (Humanrhinovirus3C). An amino acid sequence (SEQ ID NO: 4) consisting of a protease recognition site is added. Further, as a polypeptide containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2(1), one or more amino acids in the amino acid sequence represented by SEQ ID NO: 8 are substituted, deleted, inserted and A polypeptide having a repeating region consisting of a crystalline region and an atypical region, which consists of an added amino acid sequence, may be used. Examples of the polypeptide containing two or more units of the amino acid sequence represented by formula 1: REP1-REP2(1) include MaSp2-derived recombinant protein having the amino acid sequence represented by SEQ ID NO:9. To be The amino acid sequence shown in SEQ ID NO: 9 is the initiation codon at the N-terminal of the partial sequence of MaSp2 (NCBI accession number: AAT75313, GI: 503633147) obtained from the NCBI database, His10 tag and HRV3C protease (Humanrhinovirus 3C protease. ) An amino acid sequence consisting of a recognition site is added. Further, as a polypeptide containing two or more units of the amino acid sequence represented by formula 1: REP1-REP2(1), one or more amino acids in the amino acid sequence represented by SEQ ID NO: 9 are substituted, deleted, inserted and A polypeptide having a repeating region consisting of a crystalline region and an atypical region, which consists of an added amino acid sequence, may be used.

Examples of the polypeptide derived from the weft thread protein include a polypeptide containing 10 or more, preferably 20 or more, and more preferably 30 or more units of the amino acid sequence represented by the formula 2: REP3(2). In the case of carrying out recombinant protein production using a microorganism such as Escherichia coli as a host, the polypeptide derived from the weft thread protein preferably has a molecular weight of 500 kDa or less, more preferably 300 kDa or less, and further preferably from the viewpoint of productivity. Is less than 200 kDa.

In the formula (2), REP3 means an amino acid sequence composed of Gly-Pro-Gly-Gly-X, and X means one amino acid selected from the group consisting of Ala, Ser, Tyr and Val.

In the spider silk, the weft thread has a major feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. It is presumed that the large spitting tube guide thread has high stress and elasticity because it has a repeating region consisting of a crystalline region and an amorphous region. On the other hand, the weft thread has a higher stretchability, although the stress is inferior to that of the large discharge tube guide thread. It is believed that this is because most of the weft threads are composed of amorphous regions.

Formula 2: Examples of the polypeptide containing 10 or more units of the amino acid sequence represented by REP3(2) include a recombinant protein derived from a flagellated silk protein having the amino acid sequence represented by SEQ ID NO: 10. The amino acid sequence shown in SEQ ID NO: 10 is 1220 residues from the N terminus corresponding to the repeat part and motif of the partial sequence (NCBI accession number: AAF36090, GI: 7106224) of the flagellar silk protein of the American Nephila spp. obtained from the NCBI database. The amino acid sequence from the base to the 1659th residue (referred to as PR1 sequence) and the C-terminus of the partial sequence (NCBI accession number: AAC38847, GI:28333649) of the flagellated silk protein of the American Nephila spider obtained from the NCBI database. To the amino acid sequence (SEQ ID NO: 4) consisting of a start codon, a His10 tag, and an HRV3C protease recognition site at the N-terminus of the combined sequence. It is an array. Further, as a polypeptide containing 10 or more units of the amino acid sequence represented by formula 2: REP3(2), one or more amino acids in the amino acid sequence represented by SEQ ID NO: 10 are substituted, deleted, inserted and/or You may use the polypeptide which has the repeating region which consists of an added amino acid sequence and an amorphous region.

The polypeptide can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, and the gene encoding the natural spider silk protein is amplified from a spider-derived cell by polymerase chain reaction (PCR) or the like and cloned, or chemically synthesized. The method for chemically synthesizing the gene is not particularly limited, and for example, based on the amino acid sequence information of the natural spider silk protein obtained from the NCBI web database or the like, AKTA oligopilotplus 10/100 (GE Healthcare Japan KK) It is possible to synthesize by ligating oligonucleotides automatically synthesized by, for example, PCR. At this time, in order to facilitate purification and confirmation of the protein, a gene encoding a protein having an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon and a His10 tag to the N-terminal of the above amino acid sequence may be synthesized. ..

As the expression vector, a plasmid, phage, virus or the like that can express a protein from a DNA sequence can be used. The plasmid-type expression vector is not particularly limited as long as it can express the target gene in the host cell and can amplify itself. For example, when Escherichia coli Rosetta (DE3) is used as a host, pET22b(+) plasmid vector, pCold plasmid vector or the like can be used. Among them, it is preferable to use the pET22b(+) plasmid vector from the viewpoint of protein productivity. As the host, for example, animal cells, plant cells, microorganisms, etc. can be used.

The polypeptide used in the present invention is preferably a polypeptide derived from ADF3, which is one of the two major dragline proteins of the Japanese spider, Araneus diadematus. This polypeptide basically has high elongation and toughness, and can be easily synthesized.

A molded body according to an embodiment of the present invention is alternately exposed to a temperature environment of -100°C or higher and 0°C or lower and a temperature environment of higher than 0°C and 120°C or lower for a predetermined time. The bending strength at room temperature measured after the heating is ±20% or less, preferably ±17% or less of the bending strength measured at room temperature before the exposure. With such a molded body, the bending strength does not change significantly due to the temperature change of the use environment, and the reliability of the physical properties of the molded body becomes higher.

A molded body according to an embodiment of the present invention is alternately exposed to a temperature environment of -100°C or higher and 0°C or lower and a temperature environment of higher than 0°C and 120°C or lower for a predetermined time. The Young's modulus at room temperature measured after that is ±15% or less, preferably ±12% or less of the Young's modulus measured at room temperature before the exposure. With such a molded product, the Young's modulus does not change significantly due to temperature changes in the use environment, and the reliability of the physical properties of the molded product is further improved.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples without departing from the technical idea of the present invention.

<Gene synthesis>
(1) Synthesis of ADF3Kai gene A partial amino acid sequence of ADF3 (NCBI accession number: AAC47010, GI:1263287), which is one of the two major dragline proteins of the Japanese spider, is acquired from the NCBI web database. A gene encoding an amino acid sequence (SEQ ID NO: 5) in which a start codon, a His10 tag, and a HRV3C protease (Humanrhinovirus 3C protease) recognition site (SEQ ID NO: 4) were added to the N-terminus of the same sequence was synthesized and commissioned to GenScript. .. As a result, a pUC57 vector (NdeI site immediately upstream of the 5'end and XbaI site immediately downstream of the 5'end of the gene) into which the ADF3Kai gene having the nucleotide sequence of SEQ ID NO: 6 was introduced was obtained. Then, the same gene was treated with Nde I and EcoR I by restriction enzymes and recombined into a pET22b(+) expression vector.

(2) Synthesis of ADF3Kai-noNR Gene By site-directed mutagenesis using PrimeStar Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) using the pDF22b(+) vector into which the ADF3Kai gene obtained above was introduced as a template, The codon GTG corresponding to the 543rd amino acid residue valine (Val) in the amino acid sequence of ADF3Kai (SEQ ID NO: 5) was mutated to the stop codon TAA, and the gene of ADF3Kai-noNR shown in SEQ ID NO: 11 was obtained on pET22b(+). Built on. The accuracy of introducing the mutation was confirmed by a sequence reaction using a 3130xl Genetic Analyzer (Applied Biosystems). The amino acid sequence of ADF3Kai-noNR is as shown in SEQ ID NO:7.

<Expression of protein>
The pET22b(+) expression vector containing the gene sequence of ADF3Kai-noNR obtained above was transformed into E. coli Rosetta (DE3). After culturing the obtained single colony in 2 mL of LB medium containing ampicillin for 15 hours, 1.4 mL of the same culture solution was added to 140 mL of LB medium containing ampicillin, and the culture solution was added at 37° C. and 200 rpm. The cells were cultured until their OD600 was 3.5. Next, the culture solution having an OD600 of 3.5 was added to 7 L of 2×YT medium containing ampicillin together with 140 mL of 50% glucose, and further cultured until the OD600 reached 4.0. Then, isopropyl-β-thiogalactopyranoside (IPTG) was added to the obtained culture solution having an OD600 of 4.0 to a final concentration of 0.5 mM to induce protein expression. Two hours after the addition of IPTG, the culture solution was centrifuged to collect the bacterial cells. When a protein solution prepared from a culture solution before and after the addition of IPTG was electrophoresed on a polyacrylamide gel, a band of a target size was observed depending on the addition of IPTG, and it was confirmed that the target protein was expressed. confirmed. Escherichia coli expressing the ADF3Kai-noNR protein was stored in a freezer (-20°C).

<Polypeptide preparation example>
(I) About 4.5 g of E. coli cells expressing ADF3Kai-noNR protein and 30 mL of buffer AI (20 mM Tris-HCl, pH 7.4) were added to a centrifuge tube (50 mL), and a mixer (GE) was used. After dispersing the bacterial cells with SI-0286 (level 10) manufactured by the same company, centrifuge (10,000 rpm, 10 minutes, room temperature) with a centrifuge (MX-305 manufactured by Tommy Seiko), and discard the supernatant. It was
(II) To the precipitate (bacteria) obtained by centrifugation, 30 mL of the buffer solution AI and 0.3 mL of 0.1 M PMSF (dissolved in isopropanol) were added, and the above mixer (level 10) manufactured by GE was added. And dispersed for 3 minutes. Then, the cells were disrupted using an ultrasonic disrupter (VCX500, manufactured by SONIC & MATERIALS INC) and centrifuged (10,000 rpm, 10 minutes, room temperature).
(III) 30 mL of the buffer solution AI was added to the precipitate obtained by centrifugation, and the mixture was dispersed for 3 minutes with a mixer (T18 Basic Ultra Turrax, level 2 manufactured by IKA), and then the centrifugal separator manufactured by Tommy Seiko. After centrifugation (10,000 rpm, 10 minutes, room temperature), the supernatant was removed.
(IV) 7.5 M urea buffer I (7.5 M urea, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) was added to the centrifuge tube in which the supernatant was discarded, and the above SMT company was used. The precipitate was well dispersed with an ultrasonic crusher (level 7) manufactured by K.K. Then, it was made to dissolve for 120 minutes with the shaker (200 rpm, 60 degreeC) by the above-mentioned Titec company. The protein solution after dissolution was centrifuged (11,000×g, 10 minutes, room temperature) with the above-described Tomy Seiko centrifuge, and the supernatant was passed through a dialysis tube (Sanko Junyaku Co., Ltd., cellulose tube 36/32). Dialysis was carried out with water. The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and freeze-dried powder was recovered. The degree of purification of the target protein ADF3Kai-noNR in the obtained lyophilized powder was confirmed by image analysis of the results of polyacrylamide gel electrophoresis (CBB staining) of the powder using Totallab (nonlinear dynamics lt.). As a result, the degree of purification of ADF3Kai-noNR was about 85%.

(Example 1)
<Method of creating molded body>
1.35 g of the freeze-dried powder (hereinafter referred to as "sample") obtained in the above "polypeptide preparation example" was weighed and this sample was used for the mold 2 of the pressure molding machine 10 shown in Fig. 1 (cylindrical metal mold). It is a mold, and has a rectangular through hole having a cross section of 35 mm×15 mm). At this time, samples were added so that the thickness was uniform. After introducing all the samples, the heating of the mold 2 is started, and the upper pin 4 and the lower pin 6 are placed in the through holes by using a hand press machine (NT-100H-V09 manufactured by NPa System Co., Ltd.). The sample was pressurized by inserting it into the. At this time, the pressurization condition of the sample was controlled to be 40 kN. When the temperature of the sample reached 200°C, the heating was stopped, the sample was cooled with a spot cooler (TS-25EP-1 manufactured by Trusco Nakayama Co., Ltd.), and when the temperature of the sample reached 50°C, it was taken out and deburred After that, a rectangular parallelepiped shaped molded body of 35 mm×15 mm×2 mm was obtained.

<Bending elastic modulus and bending strength measuring method>
The obtained molded product was allowed to stand for 1 day in a constant temperature and humidity chamber (LHL-113, manufactured by Espec) under the condition of 20° C./65% RH, and then the following measurements were performed.
That is, a three-point bending test was conducted using an autograph (AG-X plus manufactured by Shimadzu Corporation) using a basket jig. The load cell used was 50 kN. At this time, the distance between fulcrums for three-point bending was fixed at 27 mm, and the measurement speed was 1 mm/min. In addition, the size of the molded body was measured with a micro caliper and placed on a jig for measurement. The flexural modulus was determined from the displacement (strain) from 0.05 to 0.25%.

<Transparency measuring method 1>
The transparency of the obtained molded product in the thickness direction (2 mm thickness) was visually measured, and when the transparency of the entire molded product was high, it was ⊚, and there were a few transparent but translucent or opaque parts. The case was marked with ◯, and the case without transparency was marked with x. That is, the molded product was placed on a paper on which the logo of Siber Co. was printed, and the transparency was confirmed by whether the logo was visible.

(Comparative Examples 1-2)
Using polycarbonate (PC) and polymethylmethacrylate (PMMA), a rectangular parallelepiped shaped body of 35 mm×15 mm×2 mm was obtained in the same manner as in Example 1. About these molded bodies, after leaving still for 1 day in a constant temperature and humidity chamber (LHL-113, manufactured by Espec Co., Ltd.) under the condition of 20° C./65% RH, bending elastic modulus and bending strength were the same as in Example 1. And the transparency was measured.

The results of Examples and Comparative Examples are summarized in Table 1 below. FIG. 3 shows a photograph of the molded product when the transparency of Example 1 was evaluated.

(Examples 2 to 18)
In Example 1, heating was stopped when the temperature of the sample reached 200° C., the sample was cooled by a spot cooler, and taken out when the temperature of the sample reached 50° C. That is, the heating temperature (X) was 200° C. and the annealing time (Y) was 0 minutes because the rapid cooling was started immediately after the heating temperature was reached. A molded body was obtained in the same manner as in Example 1 except that the heating temperature (X) and the annealing time (Y) were changed as shown in Table 2 below and the pressurizing condition was 30 kN. The obtained molded product was evaluated for transparency in the same manner as in Example 1 and summarized in Table 2.

(Examples 19 to 24)
A molded body was obtained in the same manner as in Example 1 except that the heating temperature (X), the annealing time (Y) and the pressurizing conditions were as shown in Table 3 below. The obtained molded product was evaluated for transparency in the same manner as in Example 1 and summarized in Table 3.

The flexural modulus and the flexural strength of Examples 16 to 18 and 5 to 7 were measured in the same manner as in Example 1, and the transmittance was measured based on the following transparency measuring method 2. The results are shown in Table 4.
<Transparency measuring method 2>
Using a UV-visible near-infrared spectrophotometer (manufactured by Shimadzu Corporation, UV-2600), the transmittance was obtained in the wave number range of 220 to 800 nm with an integration time of 0.1 seconds.

<Stability against environmental temperature changes>
The sample was pressurized at 30 kN, heating was stopped when the temperature of the sample reached 130° C., and the temperature of the sample was cooled to 40° C. with a spot cooler (TS-25EP-1 manufactured by Trusco Nakayama Co., Ltd.). The flexural strength and Young's modulus of the molded article (Example 25) produced in the same manner as in Example 1 were measured. After the measurement, the mold-molded body was allowed to stand in the storage chamber 1 in which the room temperature was set to −80° C. for 10 minutes, and then transferred into the storage chamber 2 in which the room temperature was set to 100° C. within 30 seconds, It was allowed to stand in the storage chamber 2 for 10 minutes. This operation was repeated 30 cycles. After 10 cycles, 20 cycles, and 30 cycles, the molded body was returned to room temperature, and the bending strength and Young's modulus at room temperature were measured.
Bending strength and Young's modulus change rate (unit: %) were calculated from the obtained measured values based on the following equations.
Bending strength change rate=(Bending strength after exposure−Bending strength before exposure)/(Bending strength before exposure)×100
Change rate of Young's modulus=(Young's modulus after exposure−Young's modulus before exposure)/(Young's modulus before exposure)×100

The results are shown in Table 5 and FIG. In the molded product of Example 25, the rate of change in bending strength was ±20% or less and the rate of change in Young's modulus was ±15% or less even after the environmental temperature change was repeated 30 times.

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

Claims (7)

A molded article of a composition containing a polypeptide,
In the temperature range of −100 to 120° C., the bending strength at room temperature measured after alternately exposing to a temperature environment above 0° C. and a temperature environment below 0° C. A molded product having a bending strength of ±20% or less. A molded article of a composition containing a polypeptide,
Within the temperature range of −100 to 120° C., Young's modulus at room temperature measured after alternately exposing to a temperature environment exceeding 0° C. and a temperature environment below 0° C. A molded product having a Young's modulus of ±15% or less. The molded body according to claim 1, wherein the molded body has transparency. The molded body according to claim 1, wherein the molded body has a flexural modulus of more than 4.5 GPa. The molded product according to any one of claims 1 to 4, wherein the polypeptide contains at least one selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins. The molded body according to any one of claims 1 to 5, wherein the molded body is a heat and pressure molded body. The method for producing the molded article according to claim 1, comprising a step of heating and pressing a composition containing the polypeptide.