Recombinant spider silk protein, modified polyurethane and application thereof

Abstract

The invention relates to the technical field of biology, in particular to recombinant spider silk protein, modified polyurethane and application of the recombinant spider silk protein and the modified polyurethane. The recombinant spider silk protein provided by the invention comprises spider silk protein or spider silk-like protein; the spider silk protein or spider silk-like protein comprises at least one spider silk globulin NT, at least one spider silk protein repeat region and at least one spider silk globulin CT from the N end to the C end. Research finds that when the recombinant spider silk protein is applied to waterborne polyurethane, the mechanical strength of a polyurethane material is remarkably improved, the mechanical property of the polyurethane material is enhanced, and the polyurethane material is environmentally friendly and non-toxic, has the characteristics of no toxicity, high strength, high elasticity and the like, and keeps the original characteristics of low organic matter emission, no pollution, high elasticity, air permeability and the like at the same time. The recombinant spider silk protein can have a CBM structure, so that the mechanical property can be improved. Fluorescent protein and / or chromoprotein can be fused on the recombinant spider silk protein, polyurethane with different appearances can be obtained, and color combination is firm.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant spider silk protein and modified polyurethane, and their uses. Background Technology

[0002] Polyurethane (PU) is a type of polymer formed by the condensation reaction of polyols and polyisocyanates. Based on the dispersion medium, it is divided into solvent-based polyurethane and waterborne polyurethane. Due to the high toxicity, flammability, and explosiveness of the organic solvents used in solvent-based polyurethane, it is gradually being phased out of the market. Waterborne polyurethane, with its advantages of being non-toxic, non-polluting, and chemically stable, is more favored in the market and is gradually replacing traditional solvent-based polyurethane in various applications. However, waterborne polyurethane has a lower solids content due to the absence of organic solvents, resulting in lower material strength and toughness compared to those prepared by organic solvent methods. Furthermore, in the increasingly diverse demands of everyday products, waterborne polyurethane can also serve as a carrier for a range of functional materials to meet various specific functional needs.

[0003] To address this, researchers have modified waterborne polyurethane to enhance its mechanical and functional properties. Patent CN112250825A discloses a method for modifying waterborne polyurethane with organic fluorine, grafting fluorine onto the polyurethane molecular chain via a grafting reaction. This method only slightly improves tensile strength, resulting in poor mechanical properties. Furthermore, its effectiveness is limited by the grafting rate and is not suitable for large-scale production of modified polyurethane. Patent CN111117466B discloses a method for preparing flame-retardant polyurethane coatings using tannic acid, boron nitride, carbon nanotubes, and waterborne polyurethane as raw materials. This method ultimately reduces combustion smoke, but it does not improve the dispersion of carbon nanotubes in the polyurethane, leading to a decrease in the material's mechanical properties. Therefore, modification methods primarily using inorganic doping are unlikely to achieve a dual breakthrough in both the performance and functionality of polyurethane.

[0004] Protein-modified waterborne polyurethane has gradually become a popular method for modifying polyurethane due to its excellent performance and functionality. Silk fibroin (such as silkworm silk and spider silk) is a hydrophilic material with repeating regions. The repeating regions formed by the β-sheet structure have extremely high rigidity, and its good hydrophilicity lays the foundation for the formation of hydrogen bonds between it and waterborne polyurethane. Patent CN118620295A discloses a method for modifying waterborne polyurethane with silk fibroin. The composite material prepared by this method still has excellent tensile strength and elongation at relatively thin thicknesses. However, the silk fibroin processing is too complex, and the binding sites and number of hydrogen bonds between silk fibroin and waterborne polyurethane are uncontrollable, resulting in large fluctuations in material properties. To improve the binding efficiency between protein and polyurethane, patent CN105885525A discloses a method for modifying waterborne polyurethane with soybean protein. This method uses a grafting reaction to combine modified soybean protein with polyurethane to form a highly hydrophobic waterborne polyurethane. However, the protein modification process uses a large amount of toxic substances such as fluorine and aldehydes, which remain in the waterborne polyurethane along with the protein, posing a significant health risk. Therefore, improving the bonding efficiency between proteins and waterborne polyurethane in an environmentally friendly and efficient manner is key to enhancing its mechanical properties. Summary of the Invention

[0005] This invention provides a recombinant spider silk protein and a modified polyurethane, and their uses.

[0006] This invention provides a recombinant spider silk protein, comprising spider silk protein or spider silk-like protein; the spider silk protein or spider silk-like protein comprises at least one spider silk globulin NT, at least one spider silk protein repeat region, and at least one spider silk globulin CT from the N-terminus to the C-terminus.

[0007] In some embodiments, the spider silk globulin is selected from the NT end or CT end of natural spider silk; And / or, the spider silk repeating region is selected from repeating regions of any kind of spider silk protein, including but not limited to at least one of MaSp1, MaSp4, Flag, DmarAmSp, MiSp, AcSp, PySp or TuSp; Optionally, the amino acid sequence of MaSp1 is as shown in positions 138-215 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 412-645 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of MaSp4 is as shown in positions 230-377 of SEQ ID NO.5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1131 of SEQ ID NO.6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the Flag is as shown in positions 233-365 of SEQ ID NO.7 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 697-1095 of SEQ ID NO.8 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of DmarAmSp is as shown in positions 230-338 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1014 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the AcSp is as shown in positions 230-434 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1302 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the globulin NT is as shown in positions 1 to 134 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 402 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the globulin CT is as shown in positions 219-338 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it; its nucleotide sequence is as shown in positions 655-1014 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it.

[0008] In some embodiments, at least one end of the recombinant spider silk protein is fused with at least one fluorescent protein and / or chromoprotein.

[0009] In some embodiments, at least one of the recombinant spider silk proteins, namely NT and / or CT, is replaced with at least one fluorescent protein and / or chromoprotein.

[0010] In some embodiments, the fluorescent protein includes, but is not limited to, at least one of fluorescent protein GFP, fluorescent protein mScarlet, fluorescent protein mTagBFP2, and fluorescent protein fwYellow; And / or, the chromoprotein includes, but is not limited to, at least one of the following: gfaspurple, spisPink, amilCP, and orange. Optionally, the amino acid sequence of the GFP protein is as shown in positions 219-456 of SEQ ID NO.3 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 655-1368 of SEQ ID NO.4 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein mScarlet is as shown in positions 380-608 of SEQ ID NO. 5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1138-1824 of SEQ ID NO. 6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein mTagBFP2 is as shown in positions 341-577 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1021-1731 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein fwYellow is as shown in positions 437-672 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1309-2016 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein gfaspurple is as shown in positions 1 to 221 of SEQ ID NO. 5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 663 of SEQ ID NO. 6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein spisPink is as shown in positions 1-224 of SEQ ID NO.7 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-663 of SEQ ID NO.8 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein amilCP is as shown in positions 1-221 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-663 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it.

[0011] Optionally, the amino acid sequence of the color protein Orange is as shown in positions 1 to 221 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 663 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it.

[0012] In some embodiments, at least one end of the recombinant spider silk protein is fused with at least one carbohydrate-binding domain (CBM).

[0013] In some embodiments, the carbohydrate binding domain CBM includes, but is not limited to, at least one of CBM1, CBM2, CBM2e, CBM3, or CBM96; Optionally, the amino acid sequence of CBM3 is as shown in positions 1-159 of SEQ ID NO.13 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-477 of SEQ ID NO.14 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of CBM2e is as shown in positions 1-95 of SEQ ID NO.17 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-285 of SEQ ID NO.18 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it.

[0014] In some embodiments, at least one of the recombinant spider silk proteins, namely NT and / or CT, is replaced with at least one carbohydrate-binding domain, CBM.

[0015] In some embodiments, the structure of the recombinant spider silk protein from the N-terminus to the C-terminus includes, but is not limited to, NT-2Rep-CT, NT-2Rep-GFP, gfaspurple-D3R-mScarlet, spisPink-Flag-mScarlet, amilCP-Dmar-mTagBFP2, Orange-AcSp-fwYellow, CBM3-Dmar-CBM3, CBM3-D3R-CBM3, CBM2e-Dmar-CBM2e, and CBM2e-D3R-CBM2e.

[0016] In some embodiments, connecting peptides are provided between the domains of the recombinant spider silk protein; And / or, at least one end of the recombinant spider silk protein is also fused with a His tag.

[0017] Embodiments of the present invention provide biological materials, including any one of the following: 1) A nucleic acid molecule encoding the recombinant spider silk protein; optionally, the nucleic acid molecule is DNA or RNA; 2) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines that express the recombinant spider silk protein described above; 3) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines containing the nucleic acid molecules described in 1); 4) Recombinant vectors, recombinant microorganisms, or transgenic cell lines containing the expression cassettes described in 2) or 3); 5) Host cells containing the recombinant vector described in 2), 3), or 4).

[0018] In some embodiments, the host cell of the recombinant microorganism is selected from Escherichia coli of the Enterobacteriaceae family; Optionally, the host cell is selected from Escherichia coli; optionally, the Escherichia coli is Escherichia coli BL21(DE3).

[0019] The present invention provides a method for preparing the aforementioned recombinant spider silk protein, comprising: fermenting a recombinant microorganism expressing the recombinant spider silk protein.

[0020] The present invention provides the use of the recombinant spider silk protein, the biomaterial, or the recombinant spider silk protein prepared by the preparation method in the field of polyurethane materials; Optional use in the preparation of waterborne polyurethane materials.

[0021] This invention provides a modified polyurethane, comprising the following parts by weight of raw materials: 20 parts by weight of waterborne polyurethane; The recombinant spider silk protein or the recombinant spider silk protein prepared by the preparation method is 0.0005~0.1 parts by weight.

[0022] In some embodiments, a solvent is also included, such as 5-15 mM PBS, 20-50 mM Tris-HCl, or pure water; And / or, the solvent is 1 to 2 parts by weight.

[0023] This invention provides a method for preparing the modified polyurethane as described above, comprising the following steps: According to the formula, weigh out the recombinant spider silk protein, dissolve it, then mix it evenly with the formula amount of water-based polyurethane, let it stand, pour it into a mold, and dry it.

[0024] The technical solution of this invention has the following advantages: 1. This invention provides a recombinant spider silk protein, comprising spider silk protein or spider silk-like protein; wherein the spider silk protein or spider silk-like protein comprises at least one spider silk globulin NT, at least one spider silk protein repeat region, and at least one spider silk globulin CT from the N-terminus to the C-terminus. This invention has found that applying the above-mentioned recombinant spider silk protein to waterborne polyurethane significantly improves the mechanical strength of the polyurethane material and enhances its mechanical properties. Furthermore, the protein solution added during the modification process does not contain toxic substances, thus making it environmentally friendly and non-toxic. It possesses characteristics such as non-toxicity, high strength, and high elasticity, while simultaneously maintaining the original characteristics of low organic matter emissions, no pollution, high elasticity, and breathability.

[0025] 2. This invention provides a recombinant spider silk protein. By fusing a carbohydrate-binding domain (CBM) at one end of the spider silk protein, the recombinant spider silk protein can directionally bind to waterborne polyurethane, enhancing the binding effect between the spider silk protein and the waterborne polyurethane. Simultaneously, the repeating regions of the recombinant spider silk protein or similar structural proteins can form intermolecular β-sheet structures, enhancing the intermolecular forces of the polyurethane chains, thereby significantly improving mechanical properties. In summary, the carbohydrate-binding domain (CBM) protein imparts an interaction between the recombinant spider silk protein and the waterborne polyurethane, forming a spider silk protein-polyurethane composite material through physical cross-linking. This significantly improves the mechanical properties of the spider silk protein-waterborne polyurethane composite material, which is also environmentally friendly and non-toxic, possessing characteristics such as non-toxicity, high strength, and high elasticity, while maintaining the original characteristics of low organic matter emissions, no pollution, high elasticity, and breathability.

[0026] Furthermore, this invention utilizes a dual CBM structure, with the CBM protein domain located at the N-terminus and C-terminus of the functional spider silk protein, enhancing the binding ability of spider silk protein with the waterborne polyurethane system. This results in the preparation of a waterborne polyurethane composite material containing spider silk protein, thereby enhancing its mechanical properties. Materials mechanics testing shows that the waterborne polyurethane membrane material with added spider silk protein exhibits a significant increase in mechanical strength of 118.9% and an increase in elongation of 37.4%. This improvement in physical properties can meet the specific application requirements of waterborne polyurethane materials and expand their application scenarios.

[0027] Furthermore, the recombinant spider silk protein expressed by CBM and spider silk protein can enhance the mechanical properties of polyurethane materials. The functional spider silk protein repeating region contains at least two repeating regions, and both ends of the spider silk protein have CBM domains.

[0028] 3. The present invention provides a recombinant spider silk protein, which can be stably expressed simultaneously by fusing fluorescent protein at one end of the spider silk protein or by fusing fluorescent protein and chromoprotein at both ends of the spider silk protein, and prepares a fluorescent spider silk protein-waterborne polyurethane composite material. This provides new functions for modified waterborne polyurethane, gives modified waterborne polyurethane different appearances, and the color is firmly bound, greatly expanding its application scenarios.

[0029] 4. The recombinant spider silk protein provided by this invention can be prepared by high-density fermentation of Escherichia coli, enabling large-scale protein production. No toxic or harmful reagents are used in the protein production process, and the reaction process is mild and controllable.

[0030] 5. The modified waterborne polyurethane material prepared in this invention is formed by directly fusing recombinant spider silk protein solution with waterborne polyurethane emulsion. No other preparations such as modifiers need to be added during the preparation process, which saves raw materials and ensures the preparation efficiency of modified waterborne polyurethane. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is an SDS-PAGE electrophoresis image of the recombinant spider silk protein in this invention; Figure 2 This is a photograph of the eluent containing fluorescent recombinant spider silk protein collected in this invention. Figure 3 This is a photograph of the fluorescent spider silk protein-waterborne polyurethane composite material under natural light in this invention. Figure 4 This is a physical image of the fluorescent spider silk protein-waterborne polyurethane composite material under excitation light in this invention; Figure 5 This is a stretching curve of samples of different recombinant spider silk proteins in this invention; Figure 6 This is a graph showing the significant differences in strength among different recombinant spider silk proteins in this invention. Figure 7 This is a graph showing the significant differences in elongation rates of different recombinant spider silk proteins in this invention. Figure 8 This is a stretching curve of samples of recombinant spider silk protein No. 019 at different concentrations in this invention. Figure 9This is a graph showing the significant differences in intensity of recombinant spider silk protein No. 019 at different concentrations in this invention. Figure 10 This is a graph showing the significant differences in elongation rates of recombinant spider silk protein No. 019 at different concentrations in this invention. Detailed Implementation

[0033] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0034] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] Example 1: Design and Expression of Recombinant Spider Silk Protein Recombinant spider silk protein No. 010: Contains the 2Rep spider silk protein repeat region with N-terminal and C-terminal sequences of spider silk protein at both ends, named NT-2Rep-CT, and its amino acid sequence is shown in SEQ ID NO. 1: MSHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDDMSTIAQSMVQSIQSLAAQGRTSPNKLQALNM AFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQTTGVVNQPFINEITQLVSMFAQAGMNDVSAGNSGRGQGGY GQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAGSGQGGYGGQGQGGYGQSGNSVT SGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASGVSSNEALIQ ALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVGKLHHHHHH * In the above sequence, the double-underlined regions are the N-terminus (positions 1-134) and C-terminus (positions 219-338) of the spider silk protein, respectively; the thick-underlined region is the recombinant spider silk protein 2Rep (positions 138-215); the wavy underline is the His tag (positions 341-346); and the single underline is the linker between the regions (positions 135-137, 216-218, and 339-340).

[0036] The DNA sequence of NT-2Rep-CT is shown in SEQ ID NO.2: Recombinant spider silk protein No. 011: Contains the 2Rep spider silk protein repeat region with the N-terminal sequence of spider silk protein and the fluorescent protein GFP at both ends, named NT-2Rep-GFP, and its amino acid sequence is shown in SEQ ID NO. 3: MSHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDDMSTIAQSMVQSIQSLAAQGRTSPNKLQALNM AFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQTTGVVNQPFINEITQLVSMFAQAGMNDVSAGNSGRGQGGY GQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAGSGQGGYGGQGQGGYGQSGNSMR KGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHM KQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA DKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGIT HGMDELYKKLHHHHHH * In the above sequence, the double-underlined regions are the N-terminus of the spider silk protein (positions 1-134) and the fluorescent protein GFP (positions 219-456), respectively. The thick-underlined region is the recombinant spider silk protein 2Rep (positions 138-215), the wavy underline is the His tag (positions 459-464), and the single underline is the linker between the regions (positions 134-137, 216-218, and 457-458).

[0037] The DNA sequence of NT-2Rep-GFP is shown in SEQ ID NO.4: Recombinant spider silk protein No. 012: Contains the D3R spider silk protein repeat region with gfaspurple and mScarlet linked at both ends, respectively, and is named gfaspurple-D3R-mScarlet. Its amino acid sequence is shown in SEQ ID NO. 5. MSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTVTKGGPLPFAWDILSPQSQYGSIPF TKYPEDIPDYVKQSFPEGYTWERIMNFEDGAVCTVSNDSSIQGNCFIYHVKFSGLNFPPNGPVMQKKTQGWEPNTE RLFARDGMLIGNNFMALKLEGGGHYLCEFKSTYKAKKPVKMPGYHYVDRKLDVTNHNKDYTSVEQCEISIARKSVV AGSSSGSSSGSPQSPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGPGGVSVVSATVSGPGPQGPSGPGPQ GPYGPGPQGPGPQGPGPQLPGPQGPSGPGPQGPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGPGGVSVV SATVSGSMDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGS RAFIKHPADIPDYWKQSFPEGFKWERVMIFEDGGTVSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEA STERLYPEDVVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARH STGGSGGSHHHHHH * In the above sequence, the double-underlined regions are the chromoprotein gfaspurple (positions 1-221) and the fluorescent protein mScarlet (positions 380-608), the thick-underlined regions are the recombinant spider silk protein D3R (positions 230-377), the wavy underline is the His tag (positions 609-614), and the single underline is the linker between the regions (positions 222-229 and 378-379).

[0038] The DNA sequence of gfaspurple-D3R-mScarlet is shown in SEQ ID NO. 6: Recombinant spider silk protein No. 013: Contains the Flag spider silk protein repeat region with the chromoprotein spisPink and the fluorescent protein mScarlet linked to its two ends, respectively, and is named spisPink-Flag-mScarlet. Its amino acid sequence is shown in SEQ ID NO. 7. MSHSKQALADTMKMTWLMEGSVNGHAFTIEGEGTGKPYEGKQSGTFRVTKGGPLPFAFDIVAPTLKYG FKCFMKYPADIPDYFKLAFPEGLTYDRKIAFEDGGCATATVEMSLKGNTLVHKTNFQGGNFPIDGPVMQKRTLGWE PTSEKMTPCDGIIKGDTIMYLMVEGGKTLKCRYENNYRANKPVLMPPSHFVDLRLTRTNLDKEGLAFKLEEYAVAR VLEVGSSSGSSSVTVSSTVSVGGAGGPGAGGVGLGGAGAGGVGPGGFGGPGGFGGRGGPGGPGGPGGAGGGAGGAG GLYGPGGLYGPGGLYGPGGAGVPGAPGAPGAPGRPGGIGGGAGAGGVGPGGLSGGVGGSGVSVTESVTVGSMDSTE AVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPD YWKQSFPEGFKWERVMIFEDGGTVSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEASTERLYPEDVVL KGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGGSGGSHHHH HH * In the above sequence, the double-underlined regions are the chromoproteins spisPink (positions 1-224) and mScarlet (positions 368-596), the thick-underlined regions are the recombinant spider silk protein Flag (positions 233-365), the wavy underline is the His tag (positions 597-602), and the single underline is the linker between the regions (positions 225-232 and 366-367).

[0039] The DNA sequence of spisPink-Flag-mScarlet is shown in SEQ ID NO.8: Recombinant spider silk protein No. 014: Contains the DmarAmSp spider silk protein repeat region with the chromoprotein amilCP and the fluorescent protein mTagBFP2 linked at both ends, named amilCP-Dmar-mTagBFP2, and its amino acid sequence is shown in SEQ ID NO. 9: MSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTVTKGGPLPFAWDILSPQCQYGSIPF TKYPEDIPDYVKQSFPEGYTWERIMNFEDGAVCTVSNDSSIQGNCFIYHVKFSGLNFPPNGPVMQKKTQGWEPNTE RLFARDGMLLGNNFMALKLEGGGHYLCEFKTTYKAKKPVKMPGYHYVDRKLDVTNHNKDYTSVEQCEISIARKPVV AGSSSGSSSGNSGAGEGAGGYRRYGGYGDGGAAAAAAAAAAAAAAAGGTGGYYYEAGGAGAAAAGAAAASAAGAAA GGARRAGGYGYGAGGAAGAAAAAGAAGGAGTRNTAYNSVGNSGSMVSKGEELIKENMHMKLYMEGTVDNHHFKCTS EGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTYRS KKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLNHHHHHH * In the above sequence, the double-underlined regions are the chromoprotein amilCP (positions 1-221) and the fluorescent protein mTagBFP2 (positions 341-577), the thick-underlined regions are the recombinant spider silk protein DmarAmSp (positions 230-338), the wavy underline is the His tag (positions 578-583), and the single underline is the linker between the regions (positions 222-229 and 339-340).

[0040] The DNA sequence of amilCP-Dmar-mTagBFP2 is shown in SEQ ID NO.10: Recombinant spider silk protein No. 015: Contains the AcSp spider silk protein repeat region and is linked at both ends to the chromoprotein Orange and the fluorescent protein fwYellow, respectively, and is named Orange-AcSp-fwYellow. Its amino acid sequence is shown in SEQ ID NO. 11: MSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTVTKGGPLPFAWDILSPQVGYGSIPF TKYPEDIPDYVKQSFPEGYTWERIMNFEDGAVCTVSNDSSIQGNCFIYHVKFSGLNFPPNGPVMQKKTQGWEPNTE RLFARDGMLLGNNFMALKLEGGGHYLCEFKTTYKAKKPVKMPGYHYVDRKLDVTNHNKDYTSVEQCEISIARKPVV AGSSSGSSSGSGAQGGFGPTTGAQSALISRIANALANTSTLRSVLRTGVSQQTASSVVQRTIQTLASNLGIDGNNL SRIALQAISQVPAGSDTASAYAQAFSTALVTGGVLNANNVDTLGSQVLSAVLNGVSSAAQGLGINVDTGSVQSDIRS SSSSLSTSSSASFSQTSGAASTTGFTGAGGYPGGAGPLGGGVGSLTGQTSFGQTSGFTRSGGSMTALTEGAKLFE KEIPYITELEGDVEGMKFIIKGEGTGDASVGKVDAQFICTTGDVPVPWSTLVTTLTYGAQCFAKYPRHIADFFKSC MPEGYVQERTITFEGDGVFKTRAEVTFENGSVYNRVKLNGQGFKKDGHVLGKNLEFNFTPHCLYIWGDQANHGLKS AFKIMHEITGSKEDFIVADHTQMNTPIGGGPVHVPEYHHITYHVTLSKDVTDHRDHLNIVEVIKAVDLETYRHHHH HH * In the above sequence, the double-underlined regions are the chromoprotein Orange (positions 1-221) and the fluorescent protein fwYellow (positions 437-672), the thick-underlined region is the recombinant spider silk protein AcSp (positions 230-434), the wavy underline is the His tag (positions 673-678), and the single underline is the linker between the regions (positions 222-229 and 435-436).

[0041] The DNA sequence of Orange-AcSp-fwYellow is shown in SEQ ID NO.12: Recombinant spider silk protein No. 016: Contains the DmarAmSp spider silk protein repeat region and is linked to CBM3 at both ends, named CBM3-Dmar-CBM3, and its amino acid sequence is shown in SEQ ID NO. 13: MVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSN GSYNGVTSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQ VTAYLNGVLVWGKEPGNSGAGEGAGGYRRYGGYGDGGAAAAAAAAAAAAAAGGTGGYYYEAGGGAGAAAAGAAAAS AAGAAAAGGARRAGGYGYGAGAGAAAAAGAAGGAGTRNTAYNSVGNSVSGNLKVEFYNSNPSDTTNSINPQFKVT NTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGVTSNVKGTFVKMSSSTNNADTYLEISFTGGTL EPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGKLHHHHHH * In the above sequence, the double-underlined region is the binding domain CBM3 (positions 1-159 and 268-427), the thick-underlined region is the recombinant spider silk protein DmarAmSp (positions 160-267), the wavy underline is the His tag (positions 430-435), and the single underline is the linker between the regions (positions 428-429).

[0042] The DNA sequence of CBM3-Dmar-CBM3 is shown in SEQ ID NO.14: Recombinant spider silk protein No. 017: Contains the D3R spider silk protein repeat region and is linked to CBM3 at both ends, named CBM3-D3R-CBM3, and its amino acid sequence is shown in SEQ ID NO. 15: MVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSN GSYNGVTSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQ VTAYLNGVLVWGKEPGSPQSPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGPGGVSVVSATVSGPGPQGP SGPGPQGPYGPGPQGPGPQGPGPQLPGPQGPSGPGPQGPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGP GGVSVVSATVSGSVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAI IGSNGSYNGVTSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFV EWDQVTAYLNGVLVWGKEPGKLHHHHHH * In the above sequence, the double-underlined region is the binding domain CBM3 (positions 1-159 and 310-468), the thick-underlined region is the recombinant spider silk protein D3R (positions 160-309), the wavy underline is the His tag (positions 471-476), and the single underline is the linker between the regions (positions 469-470).

[0043] The DNA sequence of CBM3-D3R-CBM3 is shown in SEQ ID NO.16: Recombinant spider silk protein No. 018: Contains the DmarAmSp spider silk protein repeat region and is linked to CBM2e at both ends, named CBM2e-Dmar-CBM2e, and its amino acid sequence is shown in SEQ ID NO. 17: MATFSVTSNWGSGYNFSIVIKNSGTTPIKNWKLEFDYNGNLTQVWDSKISSKINNHYVITNAGWNGEI PPGGSITIGGAGTGTPAELVNASISENGSGNSGAGEGAGGYRRYGGYGDGGAAAAAAAAAAAAAAAGGTGGYYYEA GGAGAAAAGAAAASAAGAAAGGARRAGGYGYGAGGAAGAAAAAGAAGGAGTRNTAYNSVGNSGSATFSVTSNWGSG YNFSIVIKNSGTTPIKNWKLEFDYNGNLTQVWDSKISSKINNHYVITNAGWNGEIPPGGSITIGGAGTGTPAELVN ASISENKLHHHHHH * In the above sequence, the double-underlined region is the binding domain CBM2e (positions 1-95 and 209-302), the thick-underlined region is the recombinant spider silk protein DmarAmSp (positions 98-206), the wavy underline is the His tag (positions 305-310), and the single underline is the linker between the regions (positions 96-97 and 303-304).

[0044] The DNA sequence of CBM2e-Dmar-CBM2e is shown in SEQ ID NO.18: ATGGCCACCTTCTCTGTTACTTCCAATTGGGGTTCTGGTTACAACTTTTCTATCGTTATTAAAAATTCTGGCACTACGCCGATCAAAAACTGGAAACTGGAGTTCGATTACAACGGTAACCTGACCCAGGTCTGGGACTCCAAAATCTCTTCCAAAATTAACAATCACTATGTGATTACCAATGCGGGCTGGAACGGCGAAATTCCACCTGGCGGCAGCATCACTATCGGCGGTGCAGGCACTGGTACTCCGGCAGAGCTGGTGAACGCTAGCATTTCCGAAAACGGCTCCGGTAATAGTGGTGCAGGCGAAGGTGCCGGCGGATATCGCCGTTATGGTGGATACGGTGATGGAGGCGCCGCTGCCGCGGCGGCAGCGGCAGCAGCAGCCGCGGCGGCGGCTGGCGGTACTGGAGGATACTATTATGAAGCGGGTGGCGCCGGCGCAGCAGCGGCGGGTGCCGCTGCGGCATCCGCGGCCGGTGCAGCGGCAGGCGGTGCCCGCCGCGCCGGTGGTTACGGCTATGGGGCAGGAGGTGCCGCAGGCGCGGCGGCCGCAGCGGGTGCCGCCGGCGGTGCTGGCACCCGTAACACCGCGTATAATAGCGTGGGCAACTCGGGATCCGCTACGTTCTCCGTGACTAGCAACTGGGGTTCCGGTTATAACTTCAGCATCGTAATCAAAAACTCTGGTACCACCCCGATCAAGAATTGGAAGCTGGAATTTGACTATAACGGCAACCTGACTCAGGTCTGGGATAGCAAAATCAGCTCTAAAATCAACAACCACTATGTAATCACTAACGCCGGCTGGAACGGTGAAATCCCACCGGGTGGTTCTATCACTATCGGTGGTGCAGGCACTGGCACGCCGGCAGAACTGGTTAACGCATCCATCTCTGAAAACAAACTGCACCACCACCACCACCACTGA Recombinant spider silk protein No. 019: Contains the D3R spider silk protein repeat region and is linked to CBM2e at both ends, named CBM2e-D3R-CBM2e, and its amino acid sequence is shown in SEQ ID NO. 19: MATFSVTSNWGSGYNFSIVIKNSGTTPIKNWKLEFDYNGNLTQVWDSKISSKINNHYVITNAGWNGEI PPGGSITIGGAGTGTPAELVNASISENGSGSPQSPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGPGGVS VVSATVSGPGPQGPSGPGPQGPYGPGPQGPGPQGPGPQLPGPQGPSGPGPQGPYGPGPQGPGPQGPGPQGPSGPGP QRPQGPGPQGPYGPGGVSVVSATVSGSGSATFSVTSNWGSGYNFSIVIKNSGTTPIKNWKLEFDYNGNLTQVWDSK ISSKINNHYVITNAGWNGEIPPGGSITIGGAGTGTPAELVNASISENKLHHHHHH * In the above sequence, the double-underlined region is the binding domain CBM2e (positions 1-95 and 250-343), the thick-underlined region is the recombinant spider silk protein D3R (positions 98-247), the wavy underline is the His tag (positions 346-351), and the single underline is the linker between the regions (positions 96-97 and 344-345).

[0045] The DNA sequence of CBM2e-D3R-CBM2e is shown in SEQ ID NO.20: The above sequences were synthesized by CoreSurf Technology (Suzhou). All the synthesized DNA sequences were inserted into the commercial pET28a plasmid vector, with the insertion site between BamHI and XhoI. The expression vector was transformed into Escherichia coli BL21(DE3) strain by chemical transformation.

[0046] The expression strain constructed above underwent fermentation culture, protein extraction and purification, and freeze-drying to obtain recombinant protein powder. The specific steps are as follows: 1. Shake-flask fermentation culture of bacterial cells The constructed expression strain was streaked onto LB agar (containing 50 μg / mL kanamycin) solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB agar (containing 50 μg / mL kanamycin) liquid medium and incubated overnight at 37°C and 220 rpm. Finally, a 1% inoculum was added to sterilized Erlenmeyer flasks containing 400 mL of LB agar and incubated at 37°C and 200 rpm until OD (exponential growth) was reached. 600 =0.8-1.0% Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the mixture was induced at 20℃ for 12-16 h. The fermentation broth was collected, centrifuged at 8000 rpm for 10 min, and the cells were collected.

[0047] 2. Protein extraction and purification Collect the bacterial cells and reconstitute them with 400 mL of 20 mM Tris-HCl (pH=8) to form a concentrated bacterial solution. OD 600 The concentration was 20-30; sonication was performed at 25 kHz, power 60%, with a 4-second sonication followed by a 6-second pause, for a total of 30-60 minutes, until the solution changed from turbid to clear. Centrifugation was carried out at 12000 rpm for 10 minutes, and the supernatant was collected. The supernatant was repeatedly passed through a nickel column (Sangon Biotech, product number NO. A600657) packed with pretreated packing material 2-3 times, collecting the throughflow. The column packing was washed 3-5 times with 20 mM Tris-HCl (1-3 column volumes each time) until the eluent was clear. The column packing was washed at least 3 times with 1-2 column volumes of 200 mM imidazole solution, collecting the eluent sequentially. The remaining 200 mM imidazole solution was washed away with 5 column volumes of 20 mM Tris-HCl, and finally stored in 20% ethanol. The washed column packing could be reused. The collected protein eluents were mixed and stored. A photograph of the collected eluent containing fluorescent recombinant spider silk protein is shown below. Figure 2 As shown.

[0048] 3. SDS-PAGE electrophoresis (1). Prepare 10% or 15% concentration SDS-PAGE gel (Yamei, product number: PG212 / PG214). (2). Sample preparation and detection: 10 μL of the sample from the protein purification process (the supernatant obtained by centrifugation after cell disruption collected in "2. Protein Extraction and Purification") was added to 2.5 μL of loading buffer, and the protein was denatured at 98℃ for 10 min, followed by SDS-PAGE electrophoresis detection. The detection results are as follows: Figure 1 As shown in the figure, the precipitate corresponds to the precipitate obtained by centrifugation after bacterial cell disruption in "2. Protein Extraction and Purification".

[0049] 3. Protein dialysis and lyophilization The purified protein solution was transferred to a dialysis bag and dialyzed with 10 volumes of 20 mM Tris-HCl overnight. The dialysate was changed three times the following day, with each dialyze lasting 2 hours. The dialyzed protein solution was then placed at -80°C for 3-4 hours until completely solidified, and then transferred to a lyophilizer for freeze-drying. The freeze-drying conditions were: freezing temperature -55°C and vacuum degree 5 Pa. The freeze-dried protein was collected and weighed to obtain the purified recombinant spider silk protein powder.

[0050] In summary, the expression of fluorescent spider silk proteins is as follows: Figure 1 As shown, the supernatant has the following color: Figure 2 As shown, all fluorescent spider silk proteins can be expressed in the fermentation lysate supernatant, and the successful expression of the fluorescent protein can be directly judged from the expression color of the supernatant.

[0051] Example 2: A method for preparing a recombinant spider silk protein-waterborne polyurethane composite membrane material This embodiment relates to a method for preparing a recombinant spider silk protein-waterborne polyurethane composite membrane material that enhances the properties of polyurethane materials. The specific preparation method is as follows: (1) Preparation of spider silk protein solution Weigh 1 mg of 010 protein lyophilized powder and dissolve it in 1 g of 20 mM Tris-HCl. Use a shaker to shake until the protein powder is completely dissolved to obtain a spider silk protein solution.

[0052] (2) Preparation of recombinant spider silk protein-waterborne polyurethane membrane composite material Weigh 20 g of waterborne polyurethane emulsion (purchased from Maclean, catalog number 909856) into a clean beaker, add the prepared No. 010 spider silk protein solution, and gently stir to mix the spider silk protein solution and polyurethane emulsion evenly. Let it stand for 10 min. Then pour it evenly into a mold, transfer it to a 37℃ incubator and dry it overnight. Remove the film-forming recombinant spider silk protein-waterborne polyurethane composite material from the mold to obtain the polyurethane composite film material containing spider silk protein.

[0053] Composite membrane test samples were prepared using a manual hydraulic press, with the samples in barbell shape, and 5-10 samples were prepared for each group. Subsequently, the composite membrane samples were subjected to mechanical testing using an electric tensile testing machine (purchased from Dongguan Zhiqu Precision Instrument Co., Ltd.), and their tensile strength, elongation at break and other properties were tested in accordance with GB / T10802-2023.

[0054] Comparative Example 1 The only difference between this comparative example and Example 2 is that, in the process of preparing recombinant spider silk composite polyurethane, recombinant spider silk protein solution is not added, but the same mass of 20 mM Tris-HCl is added. The other preparation processes and testing methods are exactly the same as in Example 2.

[0055] Table 1 Mechanical data of Example 2 and Comparative Example 1

[0056] Note: The data in the table is the average of the test results of 5 parallel samples in each group. The corresponding tensile curve in the attached figure is the test process response curve of one sample in each group. Therefore, there is a certain range of deviation between the values ​​in the table and the values ​​in the curve.

[0057] The mechanical data for Example 2 and Comparative Example 1 are shown in Table 1. Compared with the polyurethane membrane material without recombinant spider silk protein, the mechanical properties of the recombinant spider silk protein-waterborne polyurethane composite material are improved, with the strength increasing by 27.17% and the elongation also showing a certain enhancement effect. This indicates that the waterborne polyurethane material containing spider silk protein has certain performance characteristics.

[0058] Example 3: A method for preparing a fluorescent spider silk protein-waterborne polyurethane composite membrane material This embodiment relates to a method for preparing a recombinant spider silk protein-waterborne polyurethane composite membrane material to enhance the properties of polyurethane materials. The specific preparation method is as follows: (1) Preparation of spider silk protein solution Weigh 100 mg of lyophilized protein powder (011, 012, 013, 014, or 015) and dissolve it in 2 g of 20 mM Tris-HCl. Use a shaker to shake until the protein powder is completely dissolved to obtain a spider silk protein solution.

[0059] (2) Preparation of recombinant spider silk protein-waterborne polyurethane membrane composite material Weigh 20 g of aqueous polyurethane emulsion (purchased from Maclean, catalog number 909856) into a clean beaker, add the prepared spider silk protein solutions No. 011 to No. 015, and gently stir to mix the spider silk protein solutions and polyurethane emulsion evenly. Let stand for 10 min. Then pour it evenly into a mold, transfer it to a 37℃ incubator and dry it overnight. Remove the film-forming recombinant spider silk protein-aqueous polyurethane composite material from the mold to obtain the polyurethane composite film containing spider silk protein.

[0060] A physical image of the fluorescent spider silk protein composite polyurethane membrane is shown below. Figure 3 and Figure 4 As shown in the image (white light represents natural light; ultraviolet light conditions are: a high-intensity flashlight for jade identification is used as the light source, specifically the brand Yidaofu, product number SJ-002. This flashlight contains 6 light sources: white light, yellow light, red light, blue light, 365 violet light, and 395 violet light. For different fiber fluorescence excitation light, the specific light sources selected are: 011 recombinant spider silk protein, using violet light; 012 recombinant spider silk protein, using yellow light; 013 recombinant spider silk protein, using yellow light; 014 recombinant spider silk protein, using violet light; and 015 recombinant spider silk protein, using violet light.) Figure 3 The first horizontal row, from left to right, corresponds to the negative control (no spider silk protein added), recombinant spider silk protein samples 011 and 012, respectively. The second horizontal row, from left to right, corresponds to recombinant spider silk protein samples 013, 014, and 015, respectively. Figure 4 The first row in the middle, from left to right, corresponds to the negative control (no spider silk protein added), recombinant spider silk protein samples 011 and 012, respectively. Figure 4 The second row in the middle corresponds to recombinant spider silk proteins 013, 014, and 015 from left to right.

[0061] By adding fluorescent spider silk protein to polyurethane materials, the polyurethane membrane material was successfully made to exhibit a specific color during the preparation process, and it also has excellent stability and color fastness. This shows that spider silk protein can provide different functions under specific requirements by changing the binding domains at both ends.

[0062] Example 4: A method for preparing an enhanced recombinant spider silk protein-waterborne polyurethane composite membrane material This embodiment relates to a method for preparing a recombinant spider silk protein-waterborne polyurethane composite membrane material that enhances the properties of polyurethane materials. The specific preparation method is as follows: (1) Preparation of spider silk protein solution Weigh 1 mg of lyophilized protein powders No. 016, 017, 018 and 019 and dissolve them in 2 g of 20 mM Tris-HCl. Use a shaker to shake until the protein powders are completely dissolved to obtain a spider silk protein solution.

[0063] (2) Preparation of recombinant spider silk protein-waterborne polyurethane membrane composite material Weigh 20 g of aqueous polyurethane emulsion (purchased from Maclean, catalog number 909856) into a clean beaker, add the prepared spider silk protein solutions No. 016, 017, 018 and 019, and gently stir to mix the spider silk protein solutions with the polyurethane emulsion evenly. Let stand for 10 min. Then pour it evenly into a mold, transfer it to a 37℃ incubator to dry overnight, and remove the film-forming recombinant spider silk protein-aqueous polyurethane composite material from the mold to obtain the polyurethane composite film material containing spider silk protein.

[0064] Composite membrane test samples were prepared using a manual hydraulic press, with the samples in barbell shape, and 5-10 samples were prepared for each group. Subsequently, the composite membrane samples were subjected to mechanical testing using an electric tensile testing machine (purchased from Dongguan Zhiqu Precision Instrument Co., Ltd.), and their tensile strength, elongation at break and other properties were tested in accordance with GB / T10802-2023.

[0065] Table 2 Mechanical data of Example 4 and Comparative Example 1

[0066] The mechanical data for Example 4 and Comparative Example 1 are shown in Table 2, and the mechanical tensile curves are shown in the figure. Figure 5 As shown, the analysis of significant differences is as follows: Figure 6 (*** indicates p < 0.001) and Figure 7 As shown (* indicates p < 0.05), compared with polyurethane membrane materials without added spider silk protein, the mechanical properties of membrane materials with added recombinant spider silk proteins 016 and 017 were not significantly improved; in fact, the addition of recombinant spider silk protein 016 even led to a slight decrease in the mechanical properties of the composite polyurethane membrane material. Conversely, the addition of recombinant spider silk proteins 018 and 019 significantly enhanced the mechanical strength and elongation of the composite polyurethane membrane material. The different binding domains at both ends of the spider silk proteins are the main reason for the performance differences in polyurethane composite membrane materials. CBM3 binding domain proteins do not have the ability to bind to polyurethane materials, while CBM2e binding domain proteins can promote the binding of spider silk proteins to polyurethane materials. In addition, different spider silk proteins fuse to the same binding domain, and recombinant spider silk protein 019 endows the polyurethane material with superior mechanical properties.

[0067] Example 5: A method for preparing an optimized recombinant spider silk protein-waterborne polyurethane composite membrane material This embodiment relates to a method for preparing a recombinant spider silk protein-waterborne polyurethane composite membrane material that enhances polyurethane materials. The specific preparation method is as follows: 1. Preparation of spider silk protein solution: The preparation of recombinant spider silk protein solution is the same as in Example 4.

[0068] 2. Preparation of spider silk protein-waterborne polyurethane membrane composite material: The preparation method was the same as in Example 4, except that the amount of recombinant spider silk protein No. 019 added to 20 g of waterborne polyurethane raw material was controlled to be 0.5 mg, 1 mg, 5 mg, 10 mg, and 100 mg. Membrane test samples were prepared using a manual hydraulic press, with the samples in a barbell shape. Subsequently, the composite membrane samples were subjected to mechanical testing using an electric tensile testing machine (purchased from Dongguan Zhiqu Precision Instruments Co., Ltd.). Its tensile strength, elongation at break, and other properties were tested according to GB / T10802—2023.

[0069] Table 3 Mechanical data of Example 5 and Comparative Example 1

[0070] The mechanical data of Example 5 and Comparative Example 1 are shown in Table 3, and the mechanical tensile curves are shown in the figure. Figure 8 As shown, after adding different concentrations of spider silk protein No. 019, the strength and elongation of the waterborne polyurethane membrane material were improved to varying degrees. The strength increased by at least 31.6% and at most 118.9%, while the elongation increased by at least 4.2% and at most 37.4%. This indicates that the concentration of recombinant spider silk protein added in this invention has a significant correlation with the mechanical properties of the polyurethane material. The significant difference between Example 5 and Comparative Example 1 is shown in the bar chart below. Figure 9 (*** indicates p < 0.001) and Figure 10 (* indicates p < 0.05) As shown, the addition of recombinant spider silk protein in the range of 0.5 mg to 5 mg significantly enhances the mechanical strength of polyurethane materials. Within the same addition range, the elongation of the recombinant spider silk protein-waterborne polyurethane composite material also shows a significant increase. This indicates that the optimal addition ratio of recombinant spider silk protein is found within this range.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant spider silk protein, characterized in that, It includes spider silk protein or spider silk-like protein; the spider silk protein or spider silk-like protein includes at least one spider silk globulin NT, at least one spider silk protein repeat region, and at least one spider silk globulin CT from the N-terminus to the C-terminus.

2. The recombinant spider silk protein according to claim 1, characterized in that, The spider silk globulin is selected from the NT or CT end of natural spider silk; And / or, the spider silk repeating region is selected from repeating regions of any kind of spider silk protein, including but not limited to at least one of MaSp1, MaSp4, Flag, DmarAmSp, MiSp, AcSp, PySp or TuSp; Optionally, the amino acid sequence of MaSp1 is as shown in positions 138-215 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 412-645 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of MaSp4 is as shown in positions 230-377 of SEQ ID NO.5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1131 of SEQ ID NO.6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the Flag is as shown in positions 233-365 of SEQ ID NO.7 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 697-1095 of SEQ ID NO.8 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of DmarAmSp is as shown in positions 230-338 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1014 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the AcSp is as shown in positions 230-434 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 688-1302 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the globulin NT is as shown in positions 1 to 134 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 402 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the globulin CT is as shown in positions 219-338 of SEQ ID NO.1 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it; its nucleotide sequence is as shown in positions 655-1014 of SEQ ID NO.2 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it.

3. The recombinant spider silk protein according to claim 1 or 2, characterized in that, At least one end of the recombinant spider silk protein is fused with at least one fluorescent protein and / or chromoprotein; Alternatively, at least one of the recombinant spider silk proteins, namely NT and / or CT, is replaced with at least one fluorescent protein and / or chromoprotein. Optionally, the fluorescent protein includes, but is not limited to, at least one of fluorescent protein GFP, fluorescent protein mScarlet, fluorescent protein mTagBFP2, and fluorescent protein fwYellow; Optionally, the chromoprotein includes, but is not limited to, at least one of the following: gfaspurple, spisPink, amilCP, and orange. Optionally, the amino acid sequence of the GFP protein is as shown in positions 219-456 of SEQ ID NO.3 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 655-1368 of SEQ ID NO.4 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein mScarlet is as shown in positions 380-608 of SEQ ID NO. 5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1138-1824 of SEQ ID NO. 6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein mTagBFP2 is as shown in positions 341-577 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1021-1731 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the fluorescent protein fwYellow is as shown in positions 437-672 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1309-2016 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein gfaspurple is as shown in positions 1 to 221 of SEQ ID NO. 5 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 663 of SEQ ID NO. 6 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein spisPink is as shown in positions 1-224 of SEQ ID NO.7 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-663 of SEQ ID NO.8 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the chromoprotein amilCP is as shown in positions 1 to 221 of SEQ ID NO.9 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 663 of SEQ ID NO.10 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of the color protein Orange is as shown in positions 1 to 221 of SEQ ID NO.11 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it; its nucleotide sequence is as shown in positions 1 to 663 of SEQ ID NO.12 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more or 100% identity with it.

4. The recombinant spider silk protein according to any one of claims 1-3, characterized in that, The recombinant spider silk protein has at least one carbohydrate-binding domain (CBM) fused to at least one end; Optionally, the carbohydrate binding domain CBM includes, but is not limited to, at least one of CBM1, CBM2, CBM2e, CBM3, or CBM96; Optionally, the amino acid sequence of CBM3 is as shown in positions 1-159 of SEQ ID NO.13 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-477 of SEQ ID NO.14 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it. Optionally, the amino acid sequence of CBM2e is as shown in positions 1-95 of SEQ ID NO.17 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it; its nucleotide sequence is as shown in positions 1-285 of SEQ ID NO.18 or has at least 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more, or 100% identity with it.

5. The recombinant spider silk protein according to any one of claims 1-4, characterized in that, At least one of the recombinant spider silk proteins, namely spider globulin NT and / or spider globulin CT, is replaced with at least one carbohydrate-binding domain, CBM.

6. The recombinant spider silk protein according to any one of claims 1-5, characterized in that, The recombinant spider silk protein structures from the N-terminus to the C-terminus include, but are not limited to, NT-2Rep-CT, NT-2Rep-GFP, gfaspurple-D3R-mScarlet, spisPink-Flag-mScarlet, amilCP-Dmar-mTagBFP2, Orange-AcSp-fwYellow, CBM3-Dmar-CBM3, CBM3-D3R-CBM3, CBM2e-Dmar-CBM2e, and CBM2e-D3R-CBM2e.

7. The recombinant spider silk protein according to any one of claims 1-6, characterized in that, Connecting peptides are provided between the domains of the recombinant spider silk protein; And / or, at least one end of the recombinant spider silk protein is also fused with a His tag.

8. A biomaterial, characterized in that, Includes any one of the following: 1) A nucleic acid molecule encoding the recombinant spider silk protein according to any one of claims 1-7; optionally, the nucleic acid molecule is DNA or RNA; 2) An expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line expressing the nucleic acid molecule of the recombinant spider silk protein as described in any one of claims 1-7; 3) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines containing the nucleic acid molecules described in 1); 4) Recombinant vectors, recombinant microorganisms, or transgenic cell lines containing the expression cassettes described in 2) or 3); 5) Host cells containing the recombinant vector described in 2), 3), or 4).

9. The biomaterial according to claim 8, characterized in that, The host cells of the recombinant microorganisms are selected from Escherichia coli of the Enterobacteriaceae family; Optionally, the host cell is selected from Escherichia coli; optionally, the Escherichia coli is Escherichia coli BL21(DE3).

10. A method for preparing recombinant spider silk protein as described in any one of claims 1-7, characterized in that, include: The recombinant microorganism expressing the recombinant spider silk protein according to any one of claims 1-7 is fermented.

11. Use of the recombinant spider silk protein according to any one of claims 1-7, the biomaterial according to claim 8 or 9, or the recombinant spider silk protein prepared by the preparation method according to claim 10 in the field of polyurethane materials; Optional use in the preparation of waterborne polyurethane materials.

12. A modified polyurethane, characterized in that, It is made from the following raw materials in parts by weight: 20 parts by weight of waterborne polyurethane; 0.0005 to 0.1 parts by weight of the recombinant spider silk protein according to any one of claims 1-7 or the recombinant spider silk protein prepared by the preparation method according to claim 10.

13. The modified polyurethane according to claim 12, characterized in that, It also includes a solvent, wherein the solvent is at least one of 5-15 mM PBS, 20-50 mM Tris-HCl, and pure water; And / or, the solvent is 1 to 2 parts by weight.

14. A method for preparing modified polyurethane as described in claim 12 or 13, characterized in that, Includes the following steps: According to the formula, weigh out the recombinant spider silk protein, dissolve it, then mix it evenly with the formula amount of water-based polyurethane, let it stand, pour it into a mold, and dry it.

Images