A recombinant spider silk protein fiber based on spiCE-DS8 protein and a preparation method thereof

Abstract

The application discloses a kind of recombinant spider silk protein fibers based on SpiCE-DS8 protein and preparation method thereof, belong to polymer fiber material technical field.The application discloses the key functional domain SpiCE-DS8-S (KGGLDILGGLLKLSKAGKDGKNFF) of SpiCE-DS8 protein, which can promote the self-assembly and fiber formation of silk protein.By SpiCE-DS8 protein as the raw material of spinning solution, the recombinant spider silk protein fiber prepared has significant improvement in strength, toughness and elongation, which provides the possibility for improving the mechanical properties of in vitro recombinant silk, and the method of the application simplifies the spinning process, continuous spinning can be realized, which is beneficial to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of polymer fiber material technology, specifically relating to a recombinant spider silk protein fiber based on SpiCE-DS8 protein and its preparation method. Background Technology

[0002] Spider silk, as one of the strongest biomaterials, possesses superior toughness compared to most synthetic materials due to its combination of high strength and good ductility. Preparing high-concentration protein spinning solutions is a crucial prerequisite for biomimetic spinning of recombinant spider silk proteins. Spiders in the superfamily Orb-weavers are all web-building spiders, and their silk possesses exceptional comprehensive mechanical properties, making its application prospects very broad. However, due to spiders' inherently aggressive, self-protective, and territorial nature, large-scale collection of spider silk cannot be achieved through large-scale farming methods like silkworm silk harvesting. Therefore, spiders cannot produce silk in large quantities and utilize it in cocoon form like silkworms.

[0003] Reconstituted spider silk still holds great promise for applications due to its superior properties. Various experimental methods (such as wet and dry spinning) have been used to mimic the natural spinning process of silkworms to produce silk fibers from regenerated fibroin (RSF), some of which induce excellent mechanical properties. However, most of these methods do not allow for continuous production because the yarn typically requires several hours of treatment in a coagulation bath after spinning, or the fibers must be stretched by hand. Therefore, these methods are not suitable for industrial production and can only provide small quantities of fiber samples for laboratory use. Even in the few wet spinning methods that can establish a continuous spinning process for RSF, the mechanical properties of the silk fibers are significantly different from those of natural silk. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a recombinant spider silk protein fiber based on SpiCE-DS8 protein and its preparation method, so as to solve the technical problems of continuous spinning and poor mechanical properties of silk fibers in wet spinning.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide a SpiCE-DS8 protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] The present invention also discloses a gene encoding the above-mentioned SpiCE-DS8 protein, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0007] The present invention also discloses recombinant plasmids containing the above-mentioned genes.

[0008] This invention also discloses a method for preparing recombinant spider silk protein fibers, comprising the following steps:

[0009] S1. Cut the silkworm cocoons into small pieces, then boil them in an alkaline solution for 20-40 minutes. Remove the silk, rinse, and dry to obtain crude silk fibroin. Alkaline conditions can effectively promote the swelling, dissolution, and hydrolysis of sericin, and remove some of the wax through saponification.

[0010] S2. Dissolve the crude silk fibroin, heat at 50-60℃ for 3-5 hours, then dialyze for 46-50 hours, and finally freeze-dry to obtain silk fibroin.

[0011] S3. Dissolve silk fibroin in a polar solvent, then add SpiCE-DS8 protein and incubate for 10-14 hours to obtain recombinant silk fibroin spinning solution; the ratio of silk fibroin, polar solvent and SpiCE-DS8 protein is 0.3-0.4g: 8-12mL: 0.03-0.07g.

[0012] S4. Wet spinning is used to extrude the recombinant silk protein spinning solution into the coagulation solution, and the wet-spun fibers are collected by traction at a speed of 5-7 cm / s to finally obtain recombinant spider silk protein fibers.

[0013] Based on the above technical solution, the present invention can be further improved as follows:

[0014] Furthermore, the alkaline solution is a sodium carbonate, sodium bicarbonate, or urea solution.

[0015] Furthermore, the concentration of the alkaline solution is 0.01-0.03M.

[0016] Furthermore, the drying temperature is 55-65℃, and the time is 8-12 hours.

[0017] Furthermore, the solvent used for dissolution in S2 is LiBr or LiSCN solution. Cellulose can be partially dissolved by LiBr or LiSCN solution to form cellulose nanofibers, thereby preparing porous silk fibroin / cellulose sponge material with micro-nano hierarchical structure.

[0018] Furthermore, the concentration of the solvent used for dissolving in S2 is 9-10 M.

[0019] Furthermore, the molecular weight cutoff of the dialysis membrane used in dialysis is 13,000-15,000 Daltons.

[0020] Furthermore, the freeze-drying temperature is -30 to -10°C, and the time is 22-26 hours.

[0021] Furthermore, the polar solvent is hexafluoroisopropanol (HFIP). Hexafluoroisopropanol (HFIP) is a strongly polar solvent that can induce conformational changes within protein molecules, thereby enhancing their solubility. It is used to dissolve proteins containing a high content of β-structures.

[0022] Furthermore, the extrusion speed of the recombinant silk protein spinning solution is 45-55 μL / min.

[0023] Furthermore, the coagulation solution comprises 75-85 vol% EtOH solution, 0.5-0.7 M ammonium acetate, and 0.5-1.5 wt% PEG4000, with a pH of 5-6.

[0024] The present invention also discloses recombinant spider silk protein fibers prepared by the above preparation method.

[0025] The beneficial effects of this invention are as follows: The SpiCE-DS8 protein disclosed in this invention exists both in the ampulla of Vater of spiders and in spider traction silk, which is related to the superior comprehensive mechanical properties of spiders in the superfamily Orbhaviridae. SpiCE-DS8 protein can promote the mechanical properties of silk fibers spun in vitro, significantly improving their strain and toughness. Furthermore, the key functional domain SpiCE-DS8-S (KGGLDILGGLLKLSKAGKDGKNFF) of the SpiCE-DS8 protein can promote the self-assembly of silk proteins and fiber formation. By using SpiCE-DS8 protein as a raw material for the spinning solution, the recombinant spider silk protein fibers obtained show significant improvements in strength, toughness, and elongation, providing a possibility for improving the mechanical properties of in vitro recombinant silk. Moreover, the method of this invention simplifies the spinning process, enabling continuous spinning, which is beneficial for industrial mass production. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method of the present invention;

[0027] Figure 2 Stress-strain curves of the practical silk fibroin species (Chuanshan × Shushui);

[0028] Figure 3 This is a stress-strain curve of the transgenic spider silkworm (SP2-KI). Detailed Implementation

[0029] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0030] The amino acid sequence of the SpiCE-DS8 protein (SEQ ID NO: 1) is as follows:

[0031] MLTCRNLCVVLVLTVVLLSAIPCDAKGGLDILGGLLKLSKAGKDGKNFFQDLGKGLK DEFKGFFKRI.

[0032] The nucleotide sequence (SEQ ID NO: 2) of the gene encoding the SpiCE-DS8 protein is as follows:

[0033] ATGTTGACTTGCAGAAATTTATGTGTAGTATTGGTACTGACCGTTGTTCTCCTGTCAGCGATTCCATGTGATGCAAAAGGTGGTTTAGATATATTAGGTGGACTTCTAAAATTGTCGAAAGCCGGTAAAGATGGCAAAAACTTTTTTCAAGATTTGGGCAAGGGATTGAAAGACGAATTTAAAGGATTCTTTAAAAGGATTTGA.

[0034] Example

[0035] A method for preparing recombinant spider silk protein fibers, the process is as follows: Figure 1 As shown, it includes the following steps:

[0036] S1. Remove the signal peptide and synthesize SpiCE-DS8-L protein (KGGLDILGGLLKLSKAGKDGKNFFQDLGKGLKDEFKGFFKRI, SEQ ID NO: 3) and SpiCE-DS8-S protein (KGGLDILGGLLKLSKA GKDGKNFF, SEQ ID NO: 4). The peptides are synthesized using a peptide synthesizer and purified to obtain SpiCE-DS8 protein with a purity greater than 95%.

[0037] S2. Take 5g of silkworm cocoons, cut them into pieces, and then boil them in a 0.02M Na2CO3 solution for 30 minutes. Take out the degummed silk, wash it three times with ultrapure water, and dry it at 60℃ for 10 hours to obtain crude silk fibroin.

[0038] S3. Dissolve 3g of crude silk fibroin in 300mL of 9.3M LiBr solution and heat in a water bath at 55℃ for 4h to completely dissolve the silk fibroin. Then dialyze using a dialysis membrane with a water cutoff of 14000 Daltons for 48h until the conductivity is below 10μS. Freeze-dry the desalted crude silk fibroin at -20℃ for 24h to obtain silk fibroin with impurities removed.

[0039] S4. Dissolve 0.35g of silk fibroin in 10mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), then add 0.05g of SpiCE-DS8 protein and incubate for 12h to obtain a pale yellow, viscous, and transparent recombinant silk fibroin spinning solution.

[0040] S5. Using wet spinning, the recombinant silk protein spinning solution is extruded into the coagulation solution (80 vol% EtOH solution, 0.6 M ammonium acetate and 1 wt% PEG4000, pH 5.3, temperature 23℃) at a speed of 50 μL / min. After the spinning solution comes into contact with the coagulation solution, clearly visible fibers are immediately formed. The fibers are placed on the spinning wheel and collected by pulling at a speed of 6 cm / s, finally obtaining recombinant spider silk protein fibers.

[0041] Experimental Example

[0042] Using domesticated silkworm fibroin strains (Chuanshan × Shushui) and transgenic spider silkworms (SP2-KI, whose silk contains 80% spider protein Masp2) as control groups, and two different types of silkworm cocoons as sources of crude fibroin in step S2, recombinant spider silk protein fibers were prepared according to the method of Example 1. The mechanical properties of the silk fiber samples were determined.

[0043] (1) Observe and measure the diameter of different parts (n=3) in the fiber sample by microscope, and calculate the cross-sectional area of ​​adjacent sections of the fiber.

[0044] (2) At 25℃ and 48% relative humidity, a dynamic mechanical analyzer (DMA Q800) and a 1N load cell were used at a strain rate of 10mm / min (0.033s). -1 The tensile properties of the fibers were measured under the following conditions: each fiber was attached to a rectangular cardboard with a pore size of 1 cm using epoxy resin.

[0045] (3) Test 30 fibers for each sample and take the average value.

[0046] The measurement results are shown in Table 1. Figure 2 and Figure 3 As shown.

[0047] Table 1 Mechanical Performance Data

[0048]

[0049] For materials that do not yield during stretching, the elongation at break and the tensile strain at break are the same, and the average strain is the average tensile strain at break. As shown in Table 1, the addition of SpiCE-DS8-L protein improved the elongation and toughness of silk fibers from both the practical silkworm fibroin strain (Chuanshan × Shushui) and the transgenic spider silkworm (SP2-KI). The addition of SpiCE-DS8-S protein improved elongation, toughness, and breaking strength, especially in toughness, with an improvement of over 120% for the practical strain and nearly 20% for the transgenic spider silkworm. This indicates that the addition of SpiCE-DS8-L protein can improve the mechanical properties of wet spinning in vitro, which is beneficial to the industrial production of wet spinning. The effect of SpiCE-DS8-S protein is even better, indicating that SpiCE-DS8-S protein belongs to a key protein functional domain. With the same mass of protein added, the truncated SpiCE-DS8-S protein contains more protein functional domains, thus having a greater promoting effect on fiber bundling during filament formation, resulting in stronger mechanical properties.

[0050] Figure 2 Stress-strain curves for the domesticated silkworm fibroin-producing strain (Chuanshan × Shushui). Figure 3 The figure shows the stress-strain curves of the transgenic spider silkworm (SP2-KI). It can be seen from the figure that the strain increased significantly after the addition of SpiCE-DS8-L protein. The strain and stress also increased after the addition of SpiCE-DS8-S protein. Similar effects were observed in the transgenic spider silkworm SP2-KI.

Claims

1. A method for preparing recombinant spider silk protein fibers, characterized in that, Includes the following steps: S1. Cut the silkworm cocoons into pieces, then boil them in an alkaline solution for 20-40 minutes. Remove the silk, rinse, and dry to obtain crude silk fibroin. S2. Dissolve crude silk fibroin in LiBr solution, heat at 50-60℃ for 3-5 hours, then dialyze for 46-50 hours, and finally freeze dry to obtain silk fibroin. S3. Dissolve silk fibroin in 1,1,1,3,3,3-hexafluoro-2-propanol, then add SpiCE-DS8 protein and incubate for 10-14 h to obtain recombinant silk fibroin spinning solution; the amino acid sequence of the SpiCE-DS8 protein is shown in SEQ ID NO: 1, and the amino acid sequence of the key functional domain SpiCE-DS8-S protein is shown in SEQ ID NO: 4; S4. Wet spinning is used to extrude the recombinant silk protein spinning solution into the coagulation solution, and the wet-spun fibers are collected by traction at a speed of 5-7 cm / s to finally obtain recombinant spider silk protein fibers; the coagulation solution is composed of 80 vol% EtOH solution, 0.6 M ammonium acetate and 1 wt% PEG4000, and the pH is 5.

3.

2. The method for preparing recombinant spider silk protein fibers according to claim 1, characterized in that, The dialysis membrane used in the dialysis has a molecular weight cutoff of 13,000-15,000 Daltons.

3. The method for preparing recombinant spider silk protein fibers according to claim 1, characterized in that, The freeze-drying temperature is -30~-10℃, and the time is 22-26h.

4. The method for preparing recombinant spider silk protein fibers according to claim 1, characterized in that, The extrusion speed of the recombinant silk protein spinning solution is 45-55 μL / min.

5. A recombinant spider silk protein fiber, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

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