A biomimetic spider silk fusion protein, artificial protein fiber and preparation method thereof

Abstract

The application relates to a kind of biomimetic spider silk fusion proteins, artificial protein fibers and preparation method thereof.Fusion protein includes spider silk protein N-terminal domain and core repeat region.Elastin-like block is arranged after core repeat region, and further connected with spider silk protein C-terminal domain;Wherein, core repeat region includes multiple continuously arranged RP units, and elastin-like block includes multiple continuously arranged VPGKG units.The module connection relationship makes the fusion protein suitable for wet spinning, softening and post-drawing to obtain artificial protein fibers.The artificial protein fibers have good breaking strength and breaking elongation without adding aldehyde-based crosslinking agent, and have good toughness, which can be used for high-performance fiber materials and sports equipment materials.

Description

Technical Field

[0001] This application relates to the field of artificial protein fiber technology, specifically to a biomimetic spider silk fusion protein, artificial protein fibers made from the fusion protein, and a method for preparing the artificial protein fibers. Background Technology

[0002] Natural spider silk possesses both high strength and good toughness, making it an important biomimetic model for protein-based high-performance fibers. However, the direct acquisition of natural spider silk is limited because spiders are difficult to cultivate on a stable, large scale like silkworms. Therefore, the field has gradually developed recombinant spider silk protein and biomimetic spider silk protein fiber technologies to obtain artificial protein fibers with properties close to natural spider silk through engineering. Existing artificial protein fibers typically obtain spider silk-like proteins through genetic engineering, then formulate the resulting proteins into spinning solutions, and shape them through methods such as wet spinning. The nascent fibers after shaping require traction, stretching, or other post-treatments to further orient the protein chains, thereby improving the fiber's tensile properties. To improve the mechanical properties of protein fibers, existing technologies introduce aldehyde cross-linking treatment after fiber shaping. For example, aldehyde cross-linking agents such as glutaraldehyde or formaldehyde react with active groups such as amino groups in the protein, forming additional connections within the fiber. This treatment can improve fiber strength and structural stability to a certain extent and is therefore often used as a reinforcing method in the post-treatment of protein fibers.

[0003] However, after the aldehyde crosslinking treatment is completed, further cleaning and drying are usually required. The post-processing is relatively complicated, resulting in higher costs. Furthermore, for fiber materials that need to come into contact with the human body or be used for a long time, residual aldehyde crosslinking agents may cause irritation and pungent odor. Summary of the Invention

[0004] The purpose of this application is to solve the problem that the post-processing steps are relatively complicated when existing protein fibers are reinforced by aldehyde crosslinking, and the residual aldehyde crosslinking agent may limit the application of fiber materials. This application discloses a biomimetic spider silk fusion protein, as well as artificial protein fibers made from the fusion protein and the corresponding preparation method.

[0005] The applicant discovered that the aforementioned problem arose because aldehyde crosslinking is an additional chemical reinforcement step after fiber forming. The aldehyde crosslinking agent needs to penetrate the fiber interior and react with active groups such as amino groups in the protein to form the necessary intermolecular bonds for reinforcement. After the crosslinking treatment, unreacted crosslinking agent or small molecule residues may still remain on the fiber surface and inside. These residues are difficult to eliminate on their own through the fiber forming process alone, therefore, post-processing steps such as cleaning and drying are usually required.

[0006] The applicant further discovered that reducing reliance on aldehyde crosslinking treatment is not simply a matter of removing the aldehyde crosslinking agent from the post-processing steps. This is because, in existing technologies, the aldehyde crosslinking agent plays a crucial role in reinforcing and stabilizing the fiber structure after forming. Reducing this additional chemical bonding necessitates that the fiber's strength and toughness be provided more by the modular structure of the fusion protein itself, the wet spinning process, and the post-stretching and orientation process. Therefore, the fusion protein itself needs a structural basis more suitable for fiber formation and stretching orientation.

[0007] To achieve the above objectives, the following technical solution is adopted: In at least one embodiment, a biomimetic spider silk fusion protein is disclosed. Along the N-terminus to C-terminus direction, the biomimetic spider silk fusion protein comprises a spider silk protein N-terminal domain, a core repeat region, an elastin-like block, and a spider silk protein C-terminal domain connected in sequence. The amino acid sequence of the spider silk protein N-terminal domain is shown in SEQ ID NO:2. The core repeat region consists of m consecutive tandem RP units, where m is an integer from 12 to 24, and the amino acid sequence of the RP unit is shown in SEQ ID NO:1. The elastin-like block consists of n consecutive tandem VPGKG units, where n is an integer from 5 to 20, and the elastin-like block, as a whole, is located between the core repeat region and the spider silk protein C-terminal domain. The amino acid sequence of the spider silk protein C-terminal domain is shown in SEQ ID NO:3.

[0008] In the above design, the domains are not simply connected in series, but rather form a continuous coordination along the same protein chain, from dissolution to fiber assembly. The N-terminal domain of the spider silk protein is located at the beginning of the protein chain, which helps maintain the processability of the fusion protein during expression and purification, and also facilitates the subsequent formulation of spinning solutions. The continuous core repeat region is located in the middle region, providing the basis for spider silk-like repetitive sequences for subsequent fiber formation and providing the main mechanical support after fiber stretching and orientation. The elastin-like block is placed after the continuous core repeat region to regulate the chain segment state without breaking the core repeat region. The C-terminal domain of the spider silk protein is located at the end of the protein chain, which facilitates the continued entry of the protein chain into the assembly and fiber formation process during the shaping process.

[0009] Furthermore, the core repeat region is formed by multiple RP units connected in series. This continuous arrangement allows the spiderweb-like repeat sequence to form a relatively complete mechanical support foundation along the fiber length, preventing the core repeat region from being frequently interrupted by other functional sequences. If elastin-like sequences are dispersed and inserted between multiple RP units, although it may improve local hydrophilicity or segment flexibility, it will disrupt the continuity of the core repeat region, making it difficult for the spiderweb-like repeat sequence to fully exert its strength support function. In this application, elastin-like blocks are placed as a whole after the core repeat region, so that the core repeat region maintains a continuous structure first, and then the elastin-like blocks regulate the protein chain state, thereby taking into account both mechanical support and processing requirements.

[0010] The elastin-like block is formed by multiple VPGKG units linked together. Lysine residues enhance the hydrophilicity and charge characteristics of this region, making the fusion protein less likely to completely behave as a difficult-to-handle hydrophobic repeating protein during dissolution and spinning solution preparation. Located between the core repeat region and the C-terminal domain of spider silk protein, this block forms a transition region between the continuous core repeat region and the terminal assembly domain. On the one hand, the block does not insert into the core repeat region, thus not weakening its continuous mechanical basis; on the other hand, it can regulate the state of the chain segments following the core repeat region, making the protein chain more suitable for orientation and fiber formation during wet spinning and post-stretching processes.

[0011] Therefore, in this biomimetic spider silk fusion protein, the continuous core repeating region provides the strength foundation, the independent elastin-like blocks improve the processing conditions, and the C-terminal domain continues to participate in fiber assembly. This synergistic relationship means that the fiber properties derive more from the modular structure of the protein molecules themselves and the subsequent spinning orientation process, rather than relying on aldehyde crosslinking reinforcement after fiber formation. This provides a structural basis for achieving high breaking strength and good breaking elongation in artificial protein fibers while reducing aldehyde crosslinking treatment, and also helps improve fiber toughness; simultaneously, it reduces the post-processing burden caused by washing and drying steps and the control of crosslinking agent residues.

[0012] In the biomimetic spider silk fusion protein disclosed in at least one embodiment, preferably, m is an integer from 16 to 20, and n is an integer from 8 to 12. More preferably, m is 18, and n is 10.

[0013] In the above design, the number of RP units directly affects the length of the core repeat region and the proportion of spider-like sequences, while the number of VPGKG units affects the degree to which the elastin-like blocks regulate the protein processing state. Controlling both within the above ranges is beneficial for forming a more balanced structural basis between the protein expression processing window and the fiber mechanical support. The implementation with m = 18 and n = 10 corresponds to the representative fusion protein CSEP-2, which is the specific implementation supported by the fiber preparation and mechanical testing data obtained in this application.

[0014] In the biomimetic spider silk fusion protein disclosed in at least one embodiment, preferably, a flexible linker peptide is disposed between the elastin-like block and the C-terminal domain of the spider silk protein, and the amino acid sequence of the flexible linker peptide is GSG.

[0015] In the above design, the GSG linker peptide is located between the elastin-like block and the C-terminal domain of the spider silk protein. Because the GSG linker peptide is short and has flexible connection characteristics, it can reduce the spatial constraints caused by direct connection between adjacent modules, thus facilitating a smoother transition between the elastin-like block and the spider silk protein C-terminal domain on the same protein chain.

[0016] In the biomimetic spider silk fusion protein disclosed in at least one embodiment, preferably, the biomimetic spider silk fusion protein further includes an N-terminal additional residue located on the N-side of the N-terminal domain of the spider silk protein and a purification tag located on the C-side of the C-terminal domain of the spider silk protein. The amino acid sequence of the N-terminal additional residue is MASMTGGQQMGRGS, and the purification tag is HHHHHH. More preferably, the amino acid sequence of the biomimetic spider silk fusion protein is as shown in SEQ ID NO:4.

[0017] In the above design, the N-terminal appended residues can be derived from the expression and construction process and are located on the N-side of the N-terminal domain of the spider silk protein. The purification tag is placed on the C-side of the C-terminal domain of the spider silk protein, enabling the fusion protein to be obtained through affinity purification. The full-length sequence shown in SEQ ID NO:4 further defines the module boundaries and connectivity of the representative CSEP-2 fusion protein, which is beneficial for providing a stable protein source for subsequent artificial protein fiber preparation.

[0018] At least one embodiment discloses an artificial protein fiber made from the aforementioned biomimetic spider silk fusion protein. Preferably, the artificial protein fiber is made from a biomimetic spider silk fusion protein with m = 18 and n = 10 through wet spinning and subsequent stretching. Under test conditions of a clamping gap of 10 mm, a stretching rate of 5 mm / min, a test temperature of 25°C, and a relative humidity of 55%, the artificial protein fiber exhibits a tensile strength of ≥400 MPa, a tensile elongation at break of ≥30%, and a toughness of ≥150 MJ / m³.

[0019] In the above design, the continuous core repeating region of the fusion protein provides a spider-like sequence basis for the fiber, while the independent VPGKG-like elastin blocks help improve protein dissolution and spinning conditions. Wet spinning dehydrates the protein solution in a coagulation bath, and post-stretching further orients the resulting fibers along their length. The above structure and processing work together to enable representative synthetic protein fibers to achieve good overall tensile properties under non-aldehyde conditions.

[0020] At least one embodiment discloses a method for preparing artificial protein fibers, the method comprising: dissolving the above-mentioned biomimetic spider silk fusion protein in hexafluoroisopropanol to obtain a spinning solution; extruding the spinning solution into a methanol-water solution coagulation bath to coagulate the spinning solution into nascent fibers; collecting the nascent fibers and subjecting them to softening, stretching, and drying to obtain the artificial protein fibers. The method does not involve the addition of an aldehyde crosslinking agent.

[0021] In the above method, hexafluoroisopropanol is used to formulate the biomimetic spider silk fusion protein into a spinning solution suitable for extrusion. The spinning solution undergoes dehydration and coagulation in a methanol-water coagulation bath, thus forming nascent fibers that can be drawn and collected. A softening treatment brings the nascent fibers into a stretchable state, and subsequent stretching further adjusts the orientation of the protein chains within the fibers. Because this method does not use aldehyde crosslinking agents, it reduces additional crosslinking steps and minimizes application limitations caused by residual aldehyde crosslinking agents.

[0022] In the preparation method disclosed in at least one embodiment, preferably, the concentration of the biomimetic spider silk fusion protein in the spinning solution is 100 mg / mL to 200 mg / mL, and the volume fraction of methanol in the methanol-water coagulation bath is 85% to 95%. Preferably, the spinning solution is injected into the methanol-water coagulation bath through a needle with a specification of 28G to 32G, the injection rate of the spinning solution is 8 μL / min to 10 μL / min, and the collection rate of the nascent fibers is 0.4 m / min to 0.6 m / min. Preferably, the softening treatment is carried out using a methanol-water solution with a volume fraction of 70% to 80%, the softening treatment time is 2 min to 3 min, and the post-stretching ratio is 2 to 4 times.

[0023] In the above design, protein concentration affects the extrudable state of the spinning solution, while the volume fraction of methanol in the coagulation bath affects the dehydration and coagulation rate of the protein solution. Needle specifications, feed rate, and collection speed collectively limit the forming state of the spinning solution after entering the coagulation bath. Softening treatment conditions and post-stretching ratio further affect the stretchability of the nascent fibers and the mechanical properties of the fibers after drying. Controlling these parameters within the specified ranges is beneficial for obtaining continuous synthetic protein fibers capable of post-stretching.

[0024] More preferably, in the preparation method disclosed in at least one embodiment, the biomimetic spider silk fusion protein is a biomimetic spider silk fusion protein with m = 18 and n = 10, or a biomimetic spider silk fusion protein with an amino acid sequence as shown in SEQ ID NO:4. The concentration of the biomimetic spider silk fusion protein in the spinning solution is 130 mg / mL to 170 mg / mL; the volume fraction of methanol in the methanol-water coagulation bath is 88% to 92%; the spinning solution is injected into the methanol-water coagulation bath through a 28G to 32G needle at a propulsion rate of 8 μL / min to 10 μL / min; the collection rate of the nascent fibers is 0.45 m / min to 0.55 m / min; the softening treatment is performed using a methanol-water solution with a volume fraction of 73% to 78% for a softening time of 2 min to 3 min; and the post-stretching ratio is 2.5 to 3.5 times.

[0025] In the above design, the preferred conditions match those of a representative embodiment of the CSEP-2 fusion protein. The spinning solution concentration, coagulation bath composition, and feed rate enable the protein solution to stably enter the coagulation bath and form nascent fibers. The collection rate, softening treatment time, and post-stretch ratio together define the processing window from formation to orientation of the nascent fibers, thus facilitating the acquisition of synthetic protein fibers with a relatively balanced breaking strength, elongation at break, and toughness. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments are briefly described below. Obviously, the drawings described below are only some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the biomimetic spider silk fusion protein module structure involved in the embodiments of this application.

[0028] Figure 2 This is a schematic diagram of a wet spinning apparatus for artificial protein fibers according to an embodiment of this application.

[0029] Figure 3 This is a schematic diagram of the clamping state for a single fiber tensile test according to an embodiment of this application.

[0030] Explanation of key figure labels: 1. Micro-injection pump; 2. Syringe; 3. Needle; 4. Coagulation bath container; 5. Methanol-water solution coagulation bath; 6. Nascent fiber; 7. Rotary collecting roller; 8. Fiber collecting device; 9. Clamp. Detailed Implementation

[0031] The technical solution of this application will be described below with reference to the accompanying drawings and specific embodiments. The described embodiments are for the purpose of helping to understand this application and should not be construed as limiting the scope of protection. Unless otherwise stated, the numerical ranges used in this application include endpoint values.

[0032] like Figure 1 As shown, the biomimetic spider silk fusion protein of this application comprises, from the N-terminus to the C-terminus, the following components: an N-terminal domain of spider silk protein; a core repeat region; an elastin-like block; and a C-terminal domain of spider silk protein. The core repeat region is formed by multiple RP units connected in series. The elastin-like block is formed by multiple VPGKG units connected in series and is located as a whole block between the core repeat region and the C-terminal domain of spider silk protein. This arrangement allows the core repeat region to maintain a continuous spider silk protein repeat structure, while introducing independent elastic blocks that can improve the extensibility of the chain segment near the C-terminal domain.

[0033] In one embodiment, the amino acid sequence of the RP unit is shown in SEQ ID NO:1. The amino acid sequence of the N-terminal domain of the spider silk protein is shown in SEQ ID NO:2. The amino acid sequence of the C-terminal domain of the spider silk protein is shown in SEQ ID NO:3. The elastin-like block is composed of repeated VPGKG units, and the amino acid sequence of each VPGKG unit is shown in SEQ ID NO:5.

[0034] The number of RP units in the core repeat region is denoted as m, where m can be 12 to 24. The number of VPGKG units in the elastin-like block is denoted as n, where n can be 5 to 20. Preferably, m is 16 to 20 and n is 8 to 12. More preferably, m is 18 and n is 10. For the representative fusion protein CSEP-2 with m of 18 and n of 10, its full-length amino acid sequence is shown in SEQ ID NO:4.

[0035] In a preferred embodiment, a GSG linker peptide is disposed between the elastin-like block and the C-terminal domain of the spider silk protein. The GSG linker peptide provides a shorter, flexible linker region, resulting in a smoother spatial transition between the elastin-like block and the C-terminal domain of the spider silk protein. In a more specific embodiment, the N-terminus of the fusion protein also includes the additional residue MASMTGGQQMGRGS, and the C-terminus of the fusion protein includes an HHHHHH tag to facilitate recombinant expression and affinity purification.

[0036] The artificial protein fiber of this application can be manufactured according to... Figure 2 The wet spinning apparatus shown is prepared as follows. The apparatus includes a micro-injection pump 1; a syringe 2; a needle 3; a coagulation bath container 4; a methanol-water solution coagulation bath 5; nascent fibers 6; a rotating collecting roller 7; and fiber collection 8. The syringe 2 is fixed to the micro-injection pump 1, and the outlet of the needle 3 is located in the coagulation bath container 4. The spinning solution is injected into the methanol-water solution coagulation bath 5 through the needle 3 to form nascent fibers 6, which are then drawn and collected by the rotating collecting roller 7.

[0037] The method for preparing artificial protein fibers according to this application includes protein preparation, spinning solution preparation, wet spinning, and fiber post-treatment. This method does not involve the addition of aldehyde crosslinking agents. The aldehyde crosslinking agents include glutaraldehyde and formaldehyde, as well as other crosslinking reagents capable of crosslinking with protein amino groups through aldehyde groups.

[0038] During protein preparation, the nucleic acid sequence encoding the biomimetic spider silk fusion protein can be designed according to the codon preferences of the host cell. The coding sequence is inserted into an expression vector, and the host cell expresses the target fusion protein. The expression product is purified by cell disruption, centrifugation, and affinity chromatography. The purified protein is then dialyzed and lyophilized to obtain a protein powder suitable for spinning.

[0039] When preparing the spinning solution, the protein powder is added to hexafluoroisopropanol and allowed to dissolve completely. The concentration of the biomimetic spider silk fusion protein in the spinning solution can be between 100 mg / mL and 200 mg / mL. If the protein concentration is too low, the ability of the spinning solution to form continuous fibers in the coagulation bath decreases. If the protein concentration is too high, the viscosity of the spinning solution increases, which may affect the stability of needle extrusion and the uniformity of fiber diameter.

[0040] During wet spinning, the spinning solution is extruded through needle 3 into a methanol-water coagulation bath 5. The volume fraction of methanol in the methanol-water solution can be 85% to 95%. The needle size can be 28G to 32G. The spinning solution feed rate can be 8 μL / min to 10 μL / min. The nascent fibers 6 are drawn and collected by a rotating collecting roller 7 at a collection speed of 0.4 m / min to 0.6 m / min.

[0041] The collected nascent fibers can be softened in a 70% to 80% methanol aqueous solution for 2 to 3 minutes. The softened fibers are then post-stretched, with a stretch ratio of 2 to 4 times. Post-stretching allows the protein chains within the fibers to further orient along the fiber length, thereby improving the fiber's breaking strength and toughness. After air drying, the post-stretched fibers yield synthetic protein fibers.

[0042] like Figure 3 As shown, the mechanical properties of the fiber can be obtained through a single-fiber tensile test. Before the test, the fiber is observed using an optical microscope, and the fiber diameter is measured. A single fiber is clamped between clamps 9 arranged vertically or horizontally, with a clamping distance of 10 mm. The tensile rate is 5 mm / min, the test temperature is 25℃, and the relative humidity is 55%. The breaking strength is calculated by dividing the breaking load by the fiber cross-sectional area. The breaking elongation is calculated as the ratio of the elongation at break to the initial clamping distance. The toughness is calculated based on the area under the stress-strain curve.

[0043] Example 1 This embodiment prepares a representative biomimetic spider silk fusion protein, CSEP-2, and further prepares CSEP-2 artificial protein fibers. The modular structure of CSEP-2 is as follows: Figure 1As shown, from the N-terminus to the C-terminus, it consists of: N-terminal appended residues MASMTGGQQMGRGS; the N-terminal domain of spider silk protein; 18 consecutive RP units; 10 consecutive VPGKG units; GSG linker peptide; the C-terminal domain of spider silk protein; and the HHHHHH tag. Its modular structure can be represented as: MASMTGGQQMGRGS-NTD-(RP)18-(VPGKG)10-GSG-CTD-HHHHHH The full-length amino acid sequence of CSEP-2 is shown in SEQ ID NO:4, with a sequence length of 914 aa. The RP unit in CSEP-2 is shown in SEQ ID NO:1, the N-terminal domain of spider silk protein is shown in SEQ ID NO:2, the C-terminal domain of spider silk protein is shown in SEQ ID NO:3, and the elastin-like block composed of 10 consecutive VPGKG units is shown in SEQ ID NO:6.

[0044] A nucleic acid sequence encoding CSEP-2 was designed based on the codon preference of *E. coli*. The coding sequence was cloned into the pET-28a(+) expression vector via NdeI and XhoI sites. After confirmation by sequencing, the recombinant expression vector was transformed into *E. coli* BL21(DE3). Positive clones were inoculated and cultured, and after expansion, IPTG was added to induce expression of the target protein.

[0045] After induction, bacterial cells were collected. The cells were lysed and centrifuged, and the soluble supernatant was subjected to nickel affinity chromatography. The eluted protein was dialyzed and lyophilized to obtain a white, flocculent CSEP-2 protein powder. SDS-PAGE analysis showed a single major band at approximately 75 kDa. Approximately 50% of the target protein was present in soluble form. About 25 to 30 mg of purified protein was obtained per liter of TB medium, with a protein purity exceeding 90%.

[0046] CSEP-2 protein powder was added to hexafluoroisopropanol to prepare a spinning solution with a concentration of 150 mg / mL. After gentle stirring at room temperature for 5 h, a clear and homogeneous spinning solution was obtained.

[0047] according to Figure 2 The apparatus shown is used for wet spinning. The spinning solution is loaded into a 1 mL syringe equipped with a 30G needle. The syringe is fixed to a micro-injection pump, and the spinning solution is injected into a coagulation bath containing a 90% methanol aqueous solution at a feed rate of 8 μL / min. After entering the coagulation bath, the spinning solution undergoes dehydration and coagulation to form continuous nascent fibers.

[0048] Nascent fibers were drawn and collected using a rotating collecting roller at a linear speed of 0.5 m / min. The nascent fibers were then immersed in a 75% methanol aqueous solution for 2.5 min to soften them. The softened fibers were then removed, post-stretched at a 3-fold stretch ratio, and air-dried for 3 h to obtain CSEP-2 synthetic protein fiber.

[0049] In this embodiment, no aldehyde crosslinking agent, such as glutaraldehyde or formaldehyde, was added during protein preparation, spinning, softening treatment, and post-stretching.

[0050] use Figure 3 The clamps shown were used to test the tensile properties of CSEP-2 synthetic protein fibers. The clamp spacing was 10 mm, the tensile rate was 5 mm / min, the test temperature was 25℃, and the relative humidity was 55%. Each test group contained at least 10 samples. Before testing, the fibers were observed using an optical microscope, and the fiber diameter was measured using ImageJ.

[0051] The test results are shown in the table below.

[0052]

[0053] The results above show that CSEP-2 synthetic protein fiber, without the addition of aldehyde crosslinking agent, has a breaking strength of over 400 MPa, a breaking elongation of over 30%, and a toughness of over 150 MJ / m3.

[0054] Example 2 This embodiment adopts the same overall technical route as Embodiment 1; the difference lies in the lower length of the core repeat region and the elastin-like block, and the corresponding adjustment of the spinning conditions.

[0055] The fusion protein in this embodiment is designated CSEP-1. The N-terminus of CSEP-1 is connected to a vector-introduced residue, followed by the sequential linking of an NTD, 16 RP units, and 8 VPGKG units; subsequently, a CTD is linked via a GSG linker peptide, and a His tag is attached to the terminal end. Except for the difference in the number of RP and VPGKG unit repeats, the RP unit sequence in CSEP-1 is identical to SEQ ID NO:1, the NTD sequence is identical to SEQ ID NO:2, and the CTD sequence is identical to SEQ ID NO:3.

[0056] CSEP-1 protein powder was prepared according to the construction and expression method described in Example 1. Approximately 45% of the target protein was present in soluble form, and approximately 21.6 mg of pure protein was obtained per liter of TB culture medium after purification.

[0057] CSEP-1 protein powder was mixed with hexafluoroisopropanol to prepare a spinning solution with a concentration of 130 mg / mL. The spinning solution was extruded into an 88% (v / v) methanol aqueous solution using a 30G needle at a feed rate of 8 μL / min. The nascent fibers were collected at a linear velocity of 0.45 m / min, softened in a 73% (v / v) methanol aqueous solution for 2.5 min, then post-stretched at a stretch ratio of 2.5 times, and air-dried for 3 h. No aldehyde crosslinking agent was added in this example.

[0058] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0059]

[0060] Example 3 The main difference between this embodiment and Embodiment 1 is that the core repeat region and the length of the elastin-like block are higher, and a higher protein concentration and a higher post-stretching ratio are used.

[0061] The fusion protein in this embodiment is designated CSEP-3. The N-terminus of CSEP-3 is connected to a vector-introduced residue, followed by the sequential linking of an NTD, 20 RP units, and 12 VPGKG units; subsequently, a CTD is linked via a GSG linker peptide, and a His tag is attached to the terminal end. Except for the difference in the number of RP and VPGKG unit repeats, the RP unit sequence in CSEP-3 is identical to SEQ ID NO:1, the NTD sequence is identical to SEQ ID NO:2, and the CTD sequence is identical to SEQ ID NO:3.

[0062] CSEP-3 protein powder was prepared according to the construction and expression method described in Example 1. Approximately 42% of the target protein was present in soluble form, and approximately 18.8 mg of pure protein was obtained per liter of TB culture medium after purification.

[0063] CSEP-3 protein powder was mixed with hexafluoroisopropanol to prepare a spinning solution with a concentration of 170 mg / mL. The spinning solution was extruded into a 92% (v / v) methanol-water solution using a 30G needle at a feed rate of 8 μL / min. The nascent fibers were collected at a linear velocity of 0.55 m / min, softened in a 78% (v / v) methanol-water solution for 2.5 min, then post-stretched at a 3.5-fold draw ratio, and air-dried for 3 h. No aldehyde crosslinking agent was added in this example.

[0064] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0065]

[0066] Comparative Example 1 This comparative study investigated the effects of independent elastin-like blocks on fusion protein expression, spinning, and mechanical properties.

[0067] The fusion protein in this comparative example includes, from the N-terminus to the C-terminus, vector-introduced residues, the NTD, and 18 RP units; subsequently, the CTD is linked via a GSG linker peptide, and a His tag is attached to the end. Compared to Example 1, this comparative example removes 10 VPGKG units.

[0068] The comparative fusion protein was constructed, expressed, and purified using the same method as in Example 1. Approximately 28% of the target protein was present in soluble form, and approximately 12.4 mg of pure protein was obtained per liter of TB medium after purification. When using the same spinning conditions as in Example 1, the clarity of the spinning solution decreased, the continuity of the nascent fibers formed in the coagulation bath was poor, and the breakage rate increased during post-stretching.

[0069] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0070]

[0071] Comparative Example 2 This comparative study investigated the effect of the N-terminal domain of spider silk protein on fusion protein expression and fiber formation.

[0072] The fusion protein in this comparative example comprises, from the N-terminus to the C-terminus, vector-introduced residues, 18 RP units, and 10 VPGKG units; subsequently, a CTD is linked via a GSG linker peptide, and a His tag is attached to the end. Compared to Example 1, this comparative example omits the NTD.

[0073] The comparative fusion protein was constructed, expressed, and purified using the same method as in Example 1. Approximately 31% of the target protein was present in soluble form, and approximately 14.6 mg of purified protein was obtained per liter of TB medium. Under the same spinning conditions as in Example 1, nascent fibers could be formed, but the fibers were more prone to localized breakage during collection and post-stretching.

[0074] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0075]

[0076] Comparative Example 3 This comparative study examines the effect of setting elastin-like sequences as independent blocks.

[0077] The fusion protein in this comparative example contains the same number of RP units and VPGKG units as in Example 1, but the 10 VPGKG units are dispersed within the core repeat region, forming a local structure in which several RP units and VPGKG units alternate adjacently. This comparative example also retains the NTD, GSG linker peptide, CTD, and His tag.

[0078] The comparative fusion protein was constructed, expressed, and purified using the same method as in Example 1. Approximately 40% of the target protein was present in soluble form, and approximately 18.9 mg of pure protein was obtained per liter of TB medium after purification. Continuous fibers could be formed under the same spinning conditions as in Example 1, but the post-stretch stability was lower than that in Example 1.

[0079] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0080]

[0081] Comparative Example 4 This comparative study investigated the effect of the stretching step on the mechanical properties of synthetic protein fibers.

[0082] This comparative example uses the same CSEP-2 fusion protein as Example 1, and is constructed, expressed, purified, and wet-spun using the same methods as in Example 1. The difference is that the nascent fibers in this comparative example, after being softened with a 75% (v / v) methanol aqueous solution, are not subjected to a 3x stretch, but are instead directly dried in air for 3 hours.

[0083] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0084]

[0085] Comparative Example 5 This comparative study investigated the effect of aldehyde crosslinking treatment on the comprehensive mechanical properties of CSEP-2 fibers.

[0086] This comparative example uses the same CSEP-2 fusion protein as in Example 1, and is constructed, expressed, purified, and wet-spun using the same methods as in Example 1. The difference is that the nascent fibers are treated with glutaraldehyde aqueous solution before post-stretching, followed by 3-fold post-stretching and air drying.

[0087] Tensile tests were performed according to the test method in Example 1, and the following results were obtained.

[0088]

[0089] Examples 1 to 3 all yielded continuous artificial protein fibers, demonstrating that setting an independent VPGKG-like elastin block after the continuous RP core repeat region can maintain a comprehensive state of expression, spinnability, and post-stretchability within a certain number of repeats and process window. Among them, Example 1 showed superior performance in terms of expression yield, fiber continuity, and overall tensile properties.

[0090] In Comparative Example 1, the removal of the elastin-like block resulted in a decrease in the soluble expression rate and purified yield of the fusion protein, and a significant reduction in the strength and toughness of the resulting fibers. These results indicate that the independent VPGKG elastin-like block is beneficial for improving protein treatment status and contributes to the overall mechanical properties of the fibers.

[0091] Comparative Example 2 showed that the N-terminal domain of spider silk protein was removed, and while fiber formation was possible, post-stretch stability decreased. This result demonstrates that the NTD is not merely an optional terminal sequence, but also has a practical impact on the processing state of the fusion protein and the fiber-forming process.

[0092] Comparative Example 3, where the same number of VPGKG units were dispersed within the core repeat region, resulted in fiber performance lower than that of Example 1. This result demonstrates that placing elastin-like sequences as independent blocks between the core repeat region and the CTD is more beneficial for maintaining the mechanical support of the continuous RP core region than simply dispersing them within the core repeat region.

[0093] Comparative Example 4, without post-stretching, resulted in fibers with reduced strength and toughness. This result demonstrates that the post-stretching step plays a crucial role in improving protein chain orientation and fiber mechanical properties.

[0094] In Comparative Example 5, the addition of aldehyde crosslinking treatment improved the fiber's tensile strength, but reduced its elongation at break and toughness. Example 1, without the addition of aldehyde crosslinking agent, achieved high tensile strength while maintaining good elongation at break and toughness, making it more suitable for protein fiber applications requiring comprehensive tensile properties and a simplified process.

[0095] The above results indicate that by setting independent VPGKG-type elastin blocks between the continuous RP core repeat region and the C-terminal domain of spider silk protein, and combining wet spinning and post-stretching treatment, this application can obtain artificial protein fibers with high tensile strength and toughness without adding aldehyde crosslinking agents.

[0096] The foregoing description of the specifications and embodiments is used to explain the technical solutions of this application, but does not constitute a limitation on the scope of protection. Modifications, equivalent substitutions, or other improvements made by those skilled in the art based on the disclosure of this application without inventive effort should be included within the scope of protection of this application.

[0097] sequence list Sequence List Description This sequence listing is an amino acid sequence. SEQ ID NO:4 is recorded according to the established full-length amino acid sequence of CSEP-2, with a length of 914 aa and a C-terminal ending with HHHHHH.

[0098] The GSG linker peptide is 3 aa in length and is not numbered separately; it is stated directly in the main text of the instruction manual.

[0099]

[0100] SEQ ID NO:1 Name: RP unit. Length: 39 aa.

[0101] GGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAG SEQ ID NO:2 Name: N-terminal domain of spider silk protein. Length: 90 aa.

[0102] AQQSAAAAAAEAAQSAQQSAAAAAAEAAQSAQQSAAAAAAEAAQSAQQSAAAAAAEAAQS AQQSAAAAAAEAAQSAQQSAAAAAAEAAQS SEQ ID NO:3 Name: C-terminal domain of spider silk protein. Length: 49 aa.

[0103] GYGPGAGSGGSGGSGRGGYGAGSGSGRGGAGQGGSGGQGAAAAAAAGGY SEQ ID NO:4 Name: CSEP-2 full-length amino acid sequence. Length: 914 aa. Module structure: MASMTGGQQMGRGS - NTD - (RP)18 - (VPGKG)10 - GSG - CTD - HHHHHH MASMTGGQQMGRGSAQQSAAAAAAEAAQSAQQSAAAAAAEAAQSAQQSAAAAAAEAAQSA QQSAAAAAAEAAQSAQQSAAAAAAEAAQSAQQSAAAAAAEAAQSGGAGQGGYGGLSGSQA GSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAG AGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSG RSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGG GAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQ GYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAG QGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYG GQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGG YGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQG AGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGG LSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGA AAAAAGAGGGAGQGGYGGLSGSQAGSGRSQGYGGQGAGAAAAAAGAGGGAGQGGYGGLSG SQAGSGRSQGYGGQGAGAAAAAAGAGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGK GVPGKGVPGKGVPGKGGSGGYGPGAGSGGSGGSGRGGYGAGSGSGRGGAGQGGSGGQGAA AAAAAGGYHHHHHH SEQ ID NO:5 Name: VPGKG unit. Length: 5 aa.

[0104] VPGKG SEQ ID NO:6 Name: Elastin-like block. Length: 50 aa.

[0105] VPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG SEQ ID NO:7 Name: N-terminal appended residue. Length: 14 aa.

[0106] MASMTGGQQMGRGS SEQ ID NO:8 Name: His6 tag. Length: 6 aa.

[0107] HHHHHH

Claims

1. A biomimetic spider silk fusion protein, characterized in that, Along the N-terminus to C-terminus direction, the biomimetic spider silk fusion protein includes a spider silk protein N-terminal domain, a core repeat region, an elastin-like block, and a spider silk protein C-terminal domain connected in sequence. The amino acid sequence of the N-terminal domain of the spider silk protein is shown in SEQ ID NO:2; The core repeating region consists of m consecutive tandem RP units, where m is an integer from 12 to 24, and the amino acid sequence of the RP unit is shown in SEQ ID NO:1; The elastin-like block consists of n consecutively tandem VPGKG units, where n is an integer from 5 to 20, and the elastin-like block is located as a whole block between the core repeat region and the C-terminal domain of the spider silk protein. The amino acid sequence of the C-terminal domain of the spider silk protein is shown in SEQ ID NO:

3.

2. The biomimetic spider silk fusion protein according to claim 1, characterized in that, m is an integer from 16 to 20, and n is an integer from 8 to 12.

3. The biomimetic spider silk fusion protein according to claim 2, characterized in that, m is 18 and n is 10.

4. The biomimetic spider silk fusion protein according to any one of claims 1 to 3, characterized in that, A flexible linker peptide is disposed between the elastin-like block and the C-terminal domain of the spider silk protein, and the amino acid sequence of the flexible linker peptide is GSG.

5. The biomimetic spider silk fusion protein according to any one of claims 1 to 3, characterized in that, The biomimetic spider silk fusion protein also includes N-terminal additional residues located on the N-side of the N-terminal domain of the spider silk protein and a purification tag located on the C-side of the C-terminal domain of the spider silk protein. The amino acid sequence of the N-terminal appended residue is MASMTGGQQMGRGS, and the purification tag is HHHHHH.

6. The biomimetic spider silk fusion protein according to claim 5, characterized in that, The amino acid sequence of the biomimetic spider silk fusion protein is shown in SEQ ID NO:

4.

7. A synthetic protein fiber, characterized in that, The artificial protein fiber is made from the biomimetic spider silk fusion protein according to any one of claims 1 to 6.

8. The artificial protein fiber according to claim 7, characterized in that, The artificial protein fiber is prepared by wet spinning and post-stretching of the biomimetic spider silk fusion protein as described in claim 6. Under test conditions of a clamping distance of 10 mm, a tensile rate of 5 mm / min, a test temperature of 25℃, and a relative humidity of 55%, the synthetic protein fiber exhibits a breaking strength of over 400 MPa, a breaking elongation of over 30%, and a toughness of over 150 MJ / m3.

9. A method for preparing artificial protein fibers, characterized in that, include: The biomimetic spider silk fusion protein according to any one of claims 1 to 6 is dissolved in hexafluoroisopropanol to obtain a spinning solution; The spinning solution is extruded into a methanol-water coagulation bath to coagulate the spinning solution into nascent fibers. The nascent fibers are collected and then softened, stretched, and dried to obtain the artificial protein fiber. The preparation method does not involve the addition of an aldehyde crosslinking agent.

10. The preparation method according to claim 9, characterized in that, The concentration of the biomimetic spider silk fusion protein in the spinning solution is 100 mg / mL to 200 mg / mL, and the volume fraction of methanol in the methanol aqueous solution coagulation bath is 85% to 95%.

11. The preparation method according to claim 9 or 10, characterized in that, The spinning solution is injected into the methanol-water coagulation bath through a needle with a specification of 28G to 32G. The injection rate of the spinning solution is 8 μL / min to 10 μL / min, and the collection rate of the nascent fibers is 0.4 m / min to 0.6 m / min.

12. The preparation method according to claim 9 or 10, characterized in that, The softening treatment is carried out using a methanol aqueous solution with a volume fraction of 70% to 80%, and the softening treatment time is 2 to 3 minutes. The stretching ratio after stretching is 2 to 4 times.

13. The preparation method according to claim 9, characterized in that, The biomimetic spider silk fusion protein is the biomimetic spider silk fusion protein according to claim 6; The concentration of the biomimetic spider silk fusion protein in the spinning solution is 130 mg / mL to 170 mg / mL; The volume fraction of methanol in the methanol-water coagulation bath is 88% to 92%. The spinning solution is injected into the methanol aqueous solution coagulation bath through a 28G to 32G needle at a rate of 8 μL / min to 10 μL / min. The collection rate of the nascent fibers is 0.45 m / min to 0.55 m / min; The softening treatment is carried out using a methanol aqueous solution with a volume fraction of 73% to 78%, and the softening treatment time is 2 to 3 minutes. The stretching ratio of the post-stretch is 2.5 to 3.5 times.

Images