A recombinant spider silk protein and its synthesis method

Abstract

The present invention relates to the technical field of recombinant protein design and synthesis, and specifically discloses a recombinant spider silk protein and its synthesis method. The recombinant spider silk protein provided by the present invention undergoes optimized design such as replacement of the N-terminal and C-terminal domains of different spider species and different spider silk types, and large fragment recombination of the middle functional domain repeat sequences. A series of recombinant spider silk proteins are obtained through cell-free protein synthesis methods and prokaryotic fermentation expression. Compared with natural spider dragline silk proteins or other existing recombinant spider silk proteins, they generally have good composite mechanical properties and high-temperature resistance characteristics. At the same time, they have a relatively small molecular weight, significantly improving the total expression level and soluble expression, providing new raw materials and design and synthesis methods for the development of high-performance biomaterials.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein design and synthesis technology, specifically to a recombinant spider silk protein and its synthesis method. Background Technology

[0002] There are over 53,000 known spider species worldwide. Web-building spiders possess seven types of silk glands, and each spider species can secrete more than ten types of spider silk proteins (including subtypes). This results in a high degree of diversity in the sequence, structure, and properties of natural spider silk proteins, offering immense potential for recombinant design. Spider silk is a type of natural high-molecular-weight protein fiber secreted by spider silk glands. The traction silk, in particular, possesses excellent mechanical properties, unique physical characteristics, and rich bioactivity. Therefore, spider silk proteins have broad application prospects in high-performance biomaterials, tissue engineering, smart materials, and functional fibers.

[0003] Spiders exhibit cannibalistic behavior and produce very low silk yields, making it difficult to accumulate natural spider silk. Spider webs themselves are multi-component mixtures, making separation and purification challenging. Spider silk proteins possess numerous repetitive sequences in their intermediate functional domains, rich in higher-order structures such as α-helices, β-sheets, and β-turns, exhibiting significant amino acid bias. Their expression levels are extremely low in heterologous hosts, and they are prone to misfolding to form inclusion bodies. Natural spider silk protein fibers and fermentation-expressed inclusion bodies are extremely insoluble in conventional aqueous solutions, typically requiring organic solvents such as hexafluoroisopropanol, urea, guanidine hydrochloride, and formic acid for dissolution. These solvents are toxic, can damage protein activity, and limit their application as biomaterials in green aqueous systems.

[0004] Due to current technological limitations, the construction of recombinant spider silk protein expression plasmids requires reducing GC content and addressing issues such as mismatches in PCR amplification of repetitive sequences and difficulties in sequencing. Recombinant spider silk proteins typically require truncating the repetitive sequences of the intermediate functional domains of natural spider silk proteins, resulting in a significant reduction in performance while increasing expression levels. Alternatively, simple multi-fold repetition designs using sequences from a single spider species may be employed, or sequences from other non-spider-derived proteins may be fused. Furthermore, the development of heterologous host expression and purification processes remains limited, making it difficult to efficiently synthesize high-performance recombinant spider silk proteins.

[0005] In summary, due to the large molecular weight, complex sequence, high diversity, and poor solubility of natural spider silk proteins in aqueous solutions, most recombinant spider silk proteins exhibit low heterologous expression levels, are prone to inclusion body formation, and show significantly weakened performance. Current technologies still cannot meet the needs for diversified recombinant design of spider silk proteins, screening for improved composite performance, and achieving soluble, high-expression, and efficient synthesis. Therefore, it is necessary to develop a new recombinant spider silk protein and its synthesis method, enabling it to possess high performance and a low molecular weight. This method should facilitate cell-free protein synthesis, achieving efficient and high-fidelity amplification of recombinant spider silk protein expression plasmids, efficient transcription and translation, efficient purification, and performance verification. Furthermore, it should facilitate fermentation engineering scale-up to overcome existing technological bottlenecks and provide more possibilities for scientific research, recombinant design, development, and application of high-performance spider silk proteins. Summary of the Invention

[0006] In order to overcome the above-mentioned defects and shortcomings of existing recombinant design and synthesis methods of spider silk protein, this invention proposes a recombinant spider silk protein and its synthesis method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A recombinant spider silk protein, the amino acid sequence of which includes: an N-terminal domain of spider silk protein, two or more repeating functional domains in the middle and a C-terminal domain;

[0009] The N-terminal domain is selected from the N-terminal sequence of the large ampulla gland spider silk protein MaSp1 from the Australian funnel-web spider.

[0010] The two or more repeating functional domain sequences in the middle are selected from the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, or a recombinant sequence of at least one of the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, the MaSp1 sequence of the large ampullae gland silk protein of the Australian funnel-web spider, and the MaSp2 sequence of the large ampullae gland silk protein of the clouded spider; and the first functional domain sequence connected to the N-terminus is the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider;

[0011] The C-terminal domain is a hydrophilic domain that maintains the biological properties of spider silk proteins. The Latin names of the Australian funnel-web spider, cloud-spotted spider, Darwin's bark spider, and large-bellied orb-weaver spider are respectively: Australian funnel-web spider (Atrax robustus), cloud-spotted spider (Araneidae Cyrtophora), Darwin's bark spider (Caerostrisdarwini), and large-bellied orb-weaver spider (Araneus ventricosus).

[0012] The recombinant spider silk protein includes two or more of the following: mechanical properties, high-temperature resistance, and significantly improved solubility and heterologous expression levels of different spider traction silk proteins after recombinant design.

[0013] Optionally, 10-18 amino acids can be used as a linker to connect different spider species and different functional domain repetitive sequences, wherein the proportion of glycine in the linker is greater than 50%.

[0014] Optionally, a purification tag is fused to its N-terminus or C-terminus, the purification tag being selected from one of Histidine tag (His tag), Hemagglutinin tag (HA tag), and FLAG epitope tag (Flag tag).

[0015] Optionally, its amino acid sequence has more than 60% homology with the amino acid sequence shown in SEQ ID NO.1.

[0016] Optionally, the C-terminal domain may be selected from the C-terminal sequence of the small ampullae gland silk protein MiSp of Orb-weaver spider or the C-terminal sequence of the large ampullae gland silk protein MaSp2 of Orb-weaver spider.

[0017] Optionally, its amino acid sequence is SEQ ID NO.1. The N-terminus is selected from the N-terminal sequence of the funnel-web spider MaSp1, the middle functional domain repeat sequence is selected from the Darwin bark spider MaSp4 sequence and the funnel-web spider MaSp1 sequence, and the C-terminus is selected from the C-terminal sequence of the orb-weaver spider MiSp.

[0018] Optionally, its amino acid sequence is SEQ ID NO.2. Its N-terminus is selected from the N-terminal sequence of the funnel-web spider MaSp1, the middle functional domain repeat sequence is selected from the sequence of the Darwinian bark spider MaSp4 and the MaSp2 sequence of the clouded spider, and the C-terminal sequence is selected from the C-terminal sequence of the clouded spider MaSp2. This sequence shows 60.08% homology with the sequence of SEQ ID NO.1.

[0019] Optionally, its amino acid sequence is SEQ ID NO.3. Its N-terminus is selected from the N-terminal sequence of the funnel-web spider MaSp1, the middle functional domain repeat sequence is selected from the Darwinian bark spider MaSp4, and the C-terminus is selected from the C-terminal sequence of the orb-weaver spider MiSp. This sequence shows 83.56% homology with the sequence of SEQ ID NO.1.

[0020] The present invention also provides a DNA sequence for expressing the recombinant spider silk protein.

[0021] The present invention also provides an expression plasmid for expressing the recombinant spider silk protein, which is obtained by the following method:

[0022] After codon optimization according to host preference, the DNA sequence of the recombinant spider silk protein was artificially synthesized and constructed into an expression vector to obtain the expression plasmid of the recombinant spider silk protein.

[0023] Optionally, the expression vector is the pJL1 expression vector or the pET30a expression vector.

[0024] After codon optimization according to host preference, the DNA sequence of the recombinant spider silk protein was artificially synthesized and constructed into the pJL1 expression vector (a plasmid vector from CFPS of Jiangsu Yugong Biotechnology Co., Ltd.) for cell-free protein synthesis. It was then constructed into the pET30a expression vector (from Addgene, catalog number 85761) for fermentation expression evaluation, yielding the expression plasmid of the recombinant spider silk protein. The host selected for fermentation expression evaluation was a conventional Escherichia coli expression strain, or the phage-resistant strain BL21(DE3), or the rare codon strain BL21 condplys(DE3).

[0025] The present invention also provides a method for synthesizing the recombinant spider silk protein, specifically: using the expression plasmid of the recombinant spider silk protein, after amplification, expression and purification, the recombinant spider silk protein is obtained.

[0026] Optionally, the method for synthesizing the recombinant spider silk protein includes the following specific steps:

[0027] 1) The expression plasmid of the recombinant spider silk protein was subjected to isothermal rolling circle amplification. The isothermal rolling circle amplification reaction system included the recombinant expression plasmid, DNA polymerase, buffer, dNTPs and random primers to obtain the amplified expression plasmid of the recombinant spider silk protein. The temperature of the rolling circle amplification reaction system was 37℃, and the isothermal amplification was performed for 1-3 hours. The random primers were ordered from Universal Biotech and named 6 Random Primer. The ordered sequence was designed as NNNN*N*N, with 3' modification to phosphothioester and no 5' modification.

[0028] 2) The expression plasmid of recombinant spider silk protein, amplified by isothermal rolling circle, was used to express the protein, followed by purification using a magnetic bead kit to obtain the recombinant spider silk protein. The cell-free protein synthesis kit used was the Cell-Free Protein Synthesis Kit (CFPS) from Jiangsu Yugong Biotechnology Co., Ltd., catalog number: EG24301. The magnetic bead kit used was the BeaverBeads® IDA-Nickel magnetic bead kit from Suzhou Beaver Biomedical Engineering Co., Ltd., catalog number: No. 70501-K10.

[0029] Further optionally, in step 1), in the isothermal rolling circle amplification reaction system, the concentration of the recombinant expression plasmid is 1-5 ng / μL, the DNA polymerase is Phi29 DNA polymerase from Jiangsu Yugong Biotechnology Co., Ltd., catalog number: EG25102, and the random primers are synthesized by General Biotechnology (Anhui) Co., Ltd., with 3' end modified to thiophosphate and no 5' end modification, and their ordered sequence is represented as NNNN*N*N, where N represents any base and "*" indicates that the phosphodiester bond between adjacent nucleotides is replaced by a thiophosphate bond.

[0030] Optionally, in step 2), the cell-free protein synthesis system is supplemented with 1-3 mM of one or more of glycine, alanine, proline, and serine, along with 20-100 μg / mL of the isothermal rolling circle amplification recombinant expression plasmid. The cell-free protein synthesis temperature is 16-25℃, the time is 16-32 h, and the shaker speed is 200-400 rpm. The magnetic bead kit is used for purification at 4℃ or room temperature, with rotational incubation for 1-3 h. After elution, a recombinant spider silk protein solution is obtained, which can be ultrafiltered, concentrated, and replaced with an aqueous ionic solution. The cations in the aqueous ionic solution include H+. + Na + K + Ca 2+ One or more of the following, including anions including Cl. - PO4 3- One or more of them.

[0031] Compared with the prior art, the present invention has the following technical effects:

[0032] This invention optimizes the design by replacing the N-terminal and C-terminal domains of different spider species and different types of spider silk, and by recombining large fragments of repetitive sequences in the middle functional domain. Without adding any non-spider-derived sequence fragments except for the linker and purification tag, a composite spider traction silk with mechanical properties and high-temperature resistance is obtained. It can efficiently synthesize cells-free proteins and achieve fermentation-soluble expression of recombinant spider silk proteins.

[0033] 1) It has the characteristics of high thermal stability and low molecular weight.

[0034] The recombinant spider silk protein of this invention possesses a complete structure with N-terminal, C-terminal, and intermediate repeat sequences. It is derived from large-fragment recombinant sequences of various spider MaSp subtypes and MiSp sequences, and contains virtually no other fusion protein sequences from non-spider sources. A series of recombinant spider silk proteins with a molecular weight less than 71 kDa and high-temperature resistance were successfully constructed and screened, with the 49 kDa recombinant spider silk protein exhibiting superior high-temperature resistance. This recombinant design effectively avoids the problems of high molecular weight, difficulty in heterologous expression, and easy formation of inclusion bodies in natural spider silk proteins, as well as the defects of loss of higher-order structure and performance weakening after significant truncation. It achieves modular and diversified construction of the multifunctional domain core sequence of high-performance spider silk proteins, which is beneficial for improving the tolerance to high-temperature treatment during material preparation and reducing degradation losses during processing.

[0035] 2) High-efficiency synthesis and purification can be achieved through a cell-free system.

[0036] The recombinant spider silk protein of this invention can overcome host limitations and be synthesized in a cell-free environment. Instead of using linear DNA and mRNA, a recombinant expression plasmid is constructed to achieve efficient and high-fidelity isothermal rolling circle amplification. Taking into account the amino acid preferences of the recombinant spider silk protein, a suitable synthesis method is obtained in a cell-free open system. The terminally fused purification tag is exposed and specifically captured by magnetic beads, rather than folded into higher-order structures, which is beneficial for high-throughput synthesis screening and performance verification of the recombinant spider silk protein.

[0037] 3) The E. coli fermentation system significantly enhances soluble expression.

[0038] The recombinant spider silk protein of this invention can also be constructed into the pET30a expression vector and induced to express intracellularly in *E. coli*. Compared with natural spider puller silk protein, it significantly increases the soluble expression level in the cell lysis supernatant while reducing the expression of inclusion bodies in the cell lysis precipitate, thus avoiding the use of large amounts of organic solvents for purification and refolding. Compared with other published recombinant spider silk proteins, it significantly improves the overall expression level and soluble expression level while maintaining high-temperature resistance, which is beneficial for the fermentation scale-up, large-scale production, and green manufacturing of new biomaterials for high-performance recombinant spider silk proteins.

[0039] In summary, this invention utilizes innovative original designs, such as N-terminal and C-terminal domain substitution across spider species and spider silk types, and large-fragment recombination of repetitive sequences in the intermediate functional domain, to construct a series of recombinant expression plasmids. By optimizing the system and process, it achieves synergistic effects of efficient cell-free protein synthesis and fermentation amplification. The invention screens and verifies recombinant spider silk proteins with advantages such as high-temperature resistance, low molecular weight for easy heterologous synthesis and amplification, significantly improved overall and soluble expression, and avoidance of the effects of inclusion bodies and organic solvents. This provides new raw materials and design and synthesis methods for recombinant spider silk proteins for the development of high-performance biomaterials. Attached Figure Description

[0040] Figure 1 The figure shows the results of Western Blot analysis of the total expression levels of CSY (85 kDa), a natural spider silk protein synthesized from the spider silk of the tarantula, and 3R-CS (71 kDa), a recombinant spider silk protein synthesized from the tarantula.

[0041] Figure 2 The results of CSY expression by fermentation in Escherichia coli BL21(DE3) and detection of expression level and solubility by SDS-PAGE and Western blot are shown in the figure.

[0042] Figure 3 The expression level and solubility of 3R-CS were detected by SDS-PAGE and Western blot in Escherichia coli BL21(DE3) fermentation.

[0043] Figure 4 Western blot results of CSY (a cell-free protein synthesizer) after heating at 100℃ for different times;

[0044] Figure 5 Western blot results of 3R-CS, a cell-free protein synthesizer, after heating at 100℃ for different times;

[0045] Figure 6 Western blot results of 3R-3R (molecular weight 56kDa), a cell-free protein synthesized by heating at 100℃ for different times;

[0046] Figure 7 Figure 1 shows the results of SDS-PAGE and Western blot analysis for the expression level and solubility of 3R-3R in Escherichia coli BL21(DE3) fermentation.

[0047] Figure 8 Western blot results of 3R-2R (molecular weight 49kDa), a cell-free protein synthesized by heating at 100℃ for different times;

[0048] Figure 9 Figure 1 shows the results of SDS-PAGE and Western blot analysis of the expression level and solubility of 3R-2R in Escherichia coli BL21(DE3) fermentation.

[0049] Figure 10 Figure 1 shows the results of SDS-PAGE and Western blot analysis of the expression level and solubility of 2R (molecular weight 33 kDa) in Escherichia coli BL21(DE3) fermentation.

[0050] Figure 11 Coomassie Brilliant Blue staining results of 2R purified for fermentation expression after heating at 100℃ for different times. Detailed Implementation

[0051] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. The vectors for recombinant expression plasmids, the reagent kits used for cell-free protein synthesis and magnetic bead purification involved in the examples are all common vectors and commercially available reagents in the art.

[0052] Example 1

[0053] 1.1 Construction of recombinant spider silk protein expression plasmid

[0054] Designed according to the following recombinant spider silk protein sequence:

[0055] The amino acid sequence includes: the N-terminal sequence of the funnel-web spider MaSp1, the middle functional domain repeat sequence of the Darwin bark spider MaSp4 and the clouded spider MaSp2, and the C-terminal sequence of the clouded spider MaSp2, named 3R-CS. The amino acid sequence is shown in SEQ ID NO.2.

[0056] Between the repetitive sequences of functional domains of different spider species, a 14-amino acid linker is used to provide a flexible region for the complex structure. The sequence of the linker is shown in SEQ ID NO.9.

[0057] Its C-terminus is fused with a purification tag, which is selected from GSSHHHHHH in the His tag, as shown in sequence SEQ ID NO.12.

[0058] The natural clouded spider tractor silk protein MaSp2, with a His tag purified label GSSHHHHHH (SEQ ID NO. 12) fused to its C-terminus, is named CSY, and its amino acid sequence is shown in SEQ ID NO. 4.

[0059] After codon optimization according to E. coli preferences, the DNA sequences of 3R-CS and CSY were artificially synthesized as shown in SEQ ID NO.6 and SEQ ID NO.8, respectively. The DNA sequences were constructed into the pJL1 expression vector to obtain recombinant expression plasmids 3R-CS-pJL1 and CSY-pJL1. The DNA sequences were also constructed into the pET30a expression vector to obtain recombinant expression plasmids 3R-CS-pET30a and CSY-pET30a.

[0060] Among them, the pJL1 expression vector is a plasmid vector from CFPS of Jiangsu Yugong Biotechnology Co., Ltd., and the pET30a expression vector is from Addgene, catalog number 85761.

[0061] 1.2 Cell-free synthesis of recombinant spider silk proteins

[0062] The commercially available Phi29 DNA polymerase from Jiangsu Yugong Biotechnology Co., Ltd. was used to perform rolling circle amplification of cell-free expression plasmids 3R-CS-pJL1 and CSY-pJL1. The reaction system was 50 μL, with the concentration of recombinant expression plasmid at 5 ng / μL. Phi29 DNA polymerase, its buffer, dNTPs, and random primers were added according to the usage instructions. Amplification was performed isothermally at 37℃ for 2 h to obtain the recombinant expression plasmid after rolling circle replication. The specific components of the 50 μL rolling circle amplification system are shown in Table 1.

[0063] Table 1. Components of the Rolling Circle Amplification System

[0064]

[0065] 3R-CS-pJL1 and CSY-pJL1 were expressed using the Cell-Free Protein Synthesis Kit (CFPS) from Jiangsu Yugong Biotechnology Co., Ltd. The cell-free reaction system was 1 mL, the reaction temperature was 20℃, the reaction time was 24 h, and the shaker speed was 300 rpm.

[0066] The specific components of 1 mL of cell-free protein synthesis are shown in Table 2:

[0067] Table 2 Cell-free protein synthesis components

[0068]

[0069] 1.3 Purification of recombinant spider silk protein

[0070] Because the recombinant spider silk proteins mentioned above all contain His tags, they can be separated and purified. Purification was performed using the commercially available BeaverBeads® IDA-Nickel magnetic bead kit from Suzhou Beaver Biomedical Engineering Co., Ltd. The imidazole concentrations used in the prepared binding buffer, washing buffer, and elution buffer were 50 mM, 100 mM, and 500 mM, respectively. The specific operating steps are as follows:

[0071] 1) Magnetic bead equilibration: Add 20 μL of IDA-Nickel magnetic beads to every 50 μL of cell-free expression reaction system. Take 400 μL of resuspended magnetic beads in 1 mL of reaction solution, let it stand on a magnetic rack, discard the liquid, add 400 μL of binding buffer to wash, and fully resuspend in a vortex apparatus. Repeat the washing operation twice.

[0072] 2) Magnetic bead incubation: Resuspend the equilibrated magnetic beads in 4 mL binding buffer, add 1 mL of cell-free reaction solution, place on a vertical rotating shaker, and ensure that the liquid can be inverted within a single rotation cycle. Incubate at room temperature for 2 hours.

[0073] 3) Magnetic bead washing: After incubation, use a vortex mixer to shake thoroughly, collect the magnetic beads with a magnetic rack, discard the supernatant, add 1 mL of washing buffer to each sample and vortex, repeatedly invert and resuspend thoroughly, collect the magnetic beads with a magnetic rack, discard the supernatant, and repeat the washing process twice.

[0074] 4) Elution of protein: Add 1 mL of elution buffer to the washed magnetic beads, vortex thoroughly and let stand for 10 min, vortex once more and place on a magnetic rack. After the magnetic beads are completely adsorbed, collect the supernatant to obtain the purified recombinant spider silk protein.

[0075] To further improve the concentration and purity of recombinant spider silk protein and replace the eluent components, the supernatant was concentrated by ultrafiltration using an Amicon® Ultra ultrafiltration tube (3 kDa MWCO) from Merck, Germany. The centrifugation conditions were 10000g for 10min. An aqueous ionic solution was added, 10mM Na2HPO4, 300mM sodium chloride, and NaH2PO4 to adjust the pH to neutral, thus obtaining 3R-CS and CSY spider silk protein solutions.

[0076] 1.4 Detection of soluble expression of recombinant spider silk protein

[0077] The above 3R-CS and CSY spider silk protein solutions were electrophoresed using a 4-20% gradient pre-prepared SDS-PAGE gel. The target protein was then transferred to a 0.2 μm PVDF membrane for antibody incubation and ECL chemiluminescence imaging detection. The HRP conjugated antibody used was Immunoway's commercially available His-Tag Rabbit pAb (HRP) (catalog number: YM2079).

[0078] Western Blot results are as follows: Figure 1 As shown, the results indicate that the 3R-CS recombinant spider silk protein obtained by selecting 457 amino acids after the 480th amino acid of the natural clouded spider puller silk protein MaSp2, including the intermediate repeat sequence and the C-terminus, the N-terminus of the recombinant funnel-web spider MaSp1 (133 amino acids), and the repeat sequence of the Darwinian bark spider MaSp4 (150 amino acids), with a 14-amino acid intermediate linker, and fusing a His-tag tag (SEQ ID NO.12) at the C-terminus, significantly increases the overall expression level in a cell-free system compared to CSY.

[0079] The CSY-PET30a plasmid and the 3R-CS-PET30a plasmid were transformed into Escherichia coli BL21(DE3) for fermentation expression.

[0080] Single clones were picked and inoculated into 4 mL LB tubes containing 30 μg / mL kanamycin, and cultured in a shaker at 37°C and 200 rpm. When the OD600 reached 0.6, 0.5 mM IPTG was added to the tubes, and the tubes were cultured at 15°C for 16 h and at 37°C for 4 h.

[0081] Take 450 μL of the precipitate after centrifugation of the culture medium, resuspend it in 300 μL of lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 5 v / v% glycerol, 0.5 v / v% Triton X-100, pH 8.0), and sonicate for 1 min.

[0082] Whole bacterial sample: Take 100 μL of lysis buffer, mix it with 50 μL of 5x loading buffer, heat at 100℃ for 10 min, and centrifuge at 15000 rpm for 2 min;

[0083] Periplasmic space preparation: Take 900 μL of the precipitate after centrifugation of culture medium, resuspend it in 100 μL of lysis buffer A (50 mM Tris, 150 mM NaCl, 500 mM sucrose, pH 8.0), and incubate at 200 rpm and 15 °C for 60 min. Then add 100 μL of lysis buffer B (50 mM Tris, 150 mM NaCl, pH 8.0), and continue incubation at 200 rpm and 15 °C for 60 min. Centrifuge at 15000 rpm for 10 min, and collect the supernatant and cell pellet separately. Mix 50 μL of 5x loading buffer into the supernatant to obtain the periplasmic space protein sample. Heat at 100 °C for 10 min, and centrifuge at 15000 rpm for 2 min for later use.

[0084] Supernatant and inclusion body samples: Collect the cell pellet and resuspend it in 300 μL of lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 5 v / v% glycerol, 0.5 v / v% Triton X-100, pH 8.0), and sonicate for 1 min. Centrifuge at 15000 rpm for 10 min, and collect the supernatant and pellet separately. Mix 90 μL of 5x loading buffer into 180 μL of supernatant to obtain the supernatant sample. Resuspend the pellet in 130 μL of 5x loading buffer to obtain the inclusion body sample. Heat at 100 °C for 10 min, then centrifuge at 15000 rpm for 2 min, ready for loading.

[0085] The total expression level of spider silk protein, its expression in soluble supernatant, and its expression in precipitated inclusion bodies were detected by SDS-PAGE and Western blot.

[0086] The fermentation expression results of CSY-PET30a are as follows: Figure 2 As shown, where:

[0087] Lane M1::Protein marker

[0088] Lane M2: Western blot Protein marker

[0089] Lane BSA: 1μg (left), 2μg (right)

[0090] Lane NC: Uninduced whole cells;

[0091] Lane 1: Whole cells induced at 15℃ for 16 hours;

[0092] Lane 2: Whole cells induced at 37℃ for 4 hours;

[0093] Lane NC1: Uninduced periplasm;

[0094] Lane 3: Periplasmic cells induced at 15℃ for 16 hours;

[0095] Lane 4: Periplasm induced at 37℃ for 4 hours;

[0096] Lane NC2: Uninduced cell lysis supernatant;

[0097] Lane 5: Cell lysis supernatant after 16 h of induction at 15℃;

[0098] Lane 6: Cell lysis supernatant after induction at 37℃ for 4 hours;

[0099] Lane NC3: Uninduced cell lysis precipitate;

[0100] Lane 7: Cell lysis and precipitation induced at 15℃ for 16 h;

[0101] Lane 8: Cell lysis and precipitation induced at 37℃ for 4 hours;

[0102] The antibody used in Western blot was an anti-His antibody.

[0103] The fermentation expression results of 3R-CS-PET30a are as follows: Figure 3 As shown, where:

[0104] Lane M1: Protein marker

[0105] Lane M2: Western blot Protein marker

[0106] Lane BSA: 1μg (left), 2μg (right)

[0107] Lane NC: Uninduced whole cells;

[0108] Lane 1: Whole cells induced at 15℃ for 16 hours;

[0109] Lane 2: Whole cells induced at 37℃ for 4 hours;

[0110] Lane NC1: Uninduced cell lysis supernatant;

[0111] Lane 3: Cell lysis supernatant after 16 h of induction at 15℃;

[0112] Lane 4: Cell lysis supernatant after induction at 37℃ for 4 hours;

[0113] Lane NC2: Uninduced cell lysis precipitate;

[0114] Lane 5: Cell lysis and precipitation induced at 15℃ for 16 h;

[0115] Lane 6: Cell lysis and precipitation induced at 37℃ for 4 hours;

[0116] The antibody used in Western blot was an anti-His antibody.

[0117] The results showed that the overall expression level of 3R-CS and the proportion of soluble expression in the supernatant were significantly higher than those of CSY.

[0118] 1.5 Determination of the high-temperature resistance properties of recombinant spider silk protein

[0119] The cell-free synthesized CSY and 3R-CS spider silk protein solutions were heated at 100℃ for 30 min and 60 min, respectively, and the results are as follows: Figure 4 , Figure 5 As shown, after heating at 100℃ for 30 min and 60 min, CSY was almost completely degraded, while 3R-CS still retained 45.2% and 8.2% respectively, demonstrating that the recombined composite has certain high-temperature resistance properties.

[0120] Example 2

[0121] Designed according to the following recombinant spider silk protein sequence:

[0122] The amino acid sequence comprises: an N-terminal sequence selected from the funnel-web spider MaSp1, a repeating sequence of the central functional domain selected from the Darwinian bark spider MaSp4, and a C-terminal sequence selected from the orb-weaver spider MiSp, named 3R-3R, as shown in SEQ ID NO.3. 3R-3R shares the same N-terminal domain and 150 identical amino acid sequences in the central functional domain as 3R-CS.

[0123] Between the 3R repeat sequences in the middle, a 14-amino acid linker is used, the sequence of which is shown in SEQ ID NO.9.

[0124] Its N-terminus is fused with a purification tag, which is selected from HHHHHHGSS in the His tag, as shown in sequence SEQ ID NO.13.

[0125] After codon optimization according to E. coli preferences, the above-mentioned 3R-3R DNA sequence was artificially synthesized as shown in SEQ ID NO.7. The DNA sequence was constructed into the pJL1 expression vector to obtain the recombinant expression plasmid 3R-3R-pJL1.

[0126] Following the cell-free protein synthesis and purification method described in Example 1, a 3R-3R recombinant spider silk protein solution was obtained. After heating at 100°C for 30 min and 60 min, the results were as follows: Figure 6 As shown, 3R-3R has a smaller molecular weight than 3R-CS, but its high-temperature resistance is similar when heated at 100℃ for 30 min. When heated at 100℃ for 60 min, 24.5% is still retained, demonstrating relatively better high-temperature resistance.

[0127] Referring to the fermentation evaluation plasmid construction and fermentation expression method described in Example 1, the total expression level of spider silk protein, soluble supernatant expression, and precipitated inclusion body expression were detected using SDS-PAGE and Western blot.

[0128] The fermentation expression results of 3R-3R-PET30a are as follows: Figure 7 As shown, where:

[0129] Lane M1: Protein marker

[0130] Lane M2: Western blot Protein marker

[0131] Lane BSA: 1μg (left), 2μg (right)

[0132] Lane NC: Uninduced whole cells;

[0133] Lane 1: Whole cells induced at 15℃ for 16 hours;

[0134] Lane 2: Whole cells induced at 37℃ for 4 hours;

[0135] Lane NC1: Uninduced cell lysis supernatant;

[0136] Lane 3: Cell lysis supernatant after 16 h of induction at 15℃;

[0137] Lane 4: Cell lysis supernatant after induction at 37℃ for 4 hours;

[0138] Lane NC2: Uninduced cell lysis precipitate;

[0139] Lane 5: Cell lysis and precipitation induced at 15℃ for 16 h;

[0140] Lane 6: Cell lysis and precipitation induced at 37℃ for 4 hours;

[0141] The antibody used in Western blot was an anti-His antibody.

[0142] The results showed that the overall expression level of 3R-3R was significantly higher than that of 3R-CS. Optimizing expression conditions at lower temperatures for longer periods could significantly increase the proportion of soluble expression in the supernatant.

[0143] Example 3

[0144] Designed according to the following recombinant spider silk protein sequence:

[0145] The amino acid sequence comprises: an N-terminal sequence selected from the funnel-web spider MaSp1; a central functional domain repeat sequence selected from the Darwinian bark spider MaSp4 and the funnel-web spider MaSp1; and a C-terminal sequence selected from the orb-weaver spider MiSp, named 3R-2R. The amino acid sequence is shown in SEQ ID NO.1. Compared to 3R-CS, 3R-2R has the same N-terminal domain and 150 identical amino acid sequences in the central functional domain.

[0146] Between the functional domain repeat sequences of different spider species, a 14-amino acid linker is used, the sequence of which is shown in SEQ ID NO.9.

[0147] Its N-terminus is fused with a purification tag, which is selected from HHHHHHGSS in the His tag, as shown in sequence SEQ ID NO.13.

[0148] The published recombinant spider silk protein sequence uses the N-terminal sequence of the funnel-web spider MaSp1, the middle domain of which consists of only two repeats from the MaSp1 sequence, and the C-terminal sequence of the orb-weaver spider MiSp. The N-terminus is fused with the purified His tag HHHHHHGSS (SEQ ID NO.13), and is named 2R. Its amino acid sequence is shown in SEQ ID NO.10. Compared to 3R-2R, 2R only lacks the 150 amino acids of the Darwinian bark spider MaSp4 sequence in its middle functional domain.

[0149] After codon optimization according to E. coli preferences, the above-mentioned 3R-2R DNA sequences were artificially synthesized, as shown in SEQ ID NO. 5, and the 2R DNA sequence, as shown in SEQ ID NO. 11. The DNA sequences were constructed into the pJL1 expression vector to obtain recombinant expression plasmids 3R-2R-pJL1 and 2R-pJL1.

[0150] Following the cell-free protein synthesis and purification method described in Example 1, a 3R-2R recombinant spider silk protein solution was obtained. After heating at 100°C for 30 min and 60 min, the results were as follows: Figure 8As shown, 3R-2R has a smaller molecular weight than 3R-CS and 3R-3R. However, after heating at 100℃ for 30 min and 60 min, 3R-2R still retains 61.44% and 31.7% of its molecular weight, respectively, demonstrating relatively better high-temperature resistance.

[0151] Referring to the fermentation evaluation plasmid construction and fermentation expression method described in Example 1, the total expression level of spider silk protein, soluble supernatant expression, and precipitated inclusion body expression were detected using SDS-PAGE and Western blot.

[0152] The fermentation expression results of 3R-2R-PET30a are as follows: Figure 9 As shown, where:

[0153] Lane M1: Protein marker

[0154] Lane M2: Western blot Protein marker

[0155] Lane BSA: 1μg (left), 2μg (right)

[0156] Lane NC: Uninduced whole cells;

[0157] Lane 1: Whole cells induced at 15℃ for 16 hours;

[0158] Lane 2: Whole cells induced at 37℃ for 4 hours;

[0159] Lane NC1: Uninduced cell lysis supernatant;

[0160] Lane 3: Cell lysis supernatant after 16 h of induction at 15℃;

[0161] Lane 4: Cell lysis supernatant after induction at 37℃ for 4 hours;

[0162] Lane NC2: Uninduced cell lysis precipitate;

[0163] Lane 5: Cell lysis and precipitation induced at 15℃ for 16 h;

[0164] Lane 6: Cell lysis and precipitation induced at 37℃ for 4 hours;

[0165] The antibody used in Western blot was an anti-His antibody.

[0166] The results showed that the overall expression level and soluble expression level of 3R-2R were significantly higher than those of 3R-CS and 3R-3R.

[0167] Referring to the fermentation evaluation plasmid construction and fermentation expression method described in Example 1, the total expression level of spider silk protein, soluble supernatant expression, and precipitated inclusion body expression were detected using SDS-PAGE and Western blot.

[0168] The fermentation expression results of 2R-PET30a are as follows: Figure 10 As shown, where:

[0169] Lane M1: Protein marker

[0170] Lane M2: Western blot Protein marker

[0171] Lane BSA: 1μg (left), 2μg (right)

[0172] Lane NC: Uninduced whole cells;

[0173] Lane 1: Whole cells induced at 15℃ for 16 hours;

[0174] Lane 2: Whole cells induced at 37℃ for 4 hours;

[0175] Lane NC1: Uninduced cell lysis supernatant;

[0176] Lane 3: Cell lysis supernatant after 16 h of induction at 15℃;

[0177] Lane 4: Cell lysis supernatant after induction at 37℃ for 4 hours;

[0178] Lane NC2: Uninduced cell lysis precipitate;

[0179] Lane 5: Cell lysis and precipitation induced at 15℃ for 16 h;

[0180] Lane 6: Cell lysis and precipitation induced at 37℃ for 4 hours;

[0181] The antibody used in Western blot was an anti-His antibody.

[0182] The results showed that, compared with the classic recombinant spider silk protein 2R, 3R-2R significantly increased the overall expression level and the soluble expression level at higher temperatures and shorter times. The optimized recombinant design is beneficial for high-density fermentation scale-up.

[0183] The 2R recombinant spider silk protein solution expressed by 2R-PET30a fermentation was heated at 100℃ for 30 min and 60 min, followed by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. The results are as follows: Figure 11As shown, 2R is almost completely degraded, indicating that 2R itself does not have high temperature resistance, and the recombination design of 3R-2R combines excellent high temperature resistance properties.

[0184] In summary, the recombinant spider silk proteins 3R-CS, 3R-3R, and 3R-2R, designed and screened, possess the same N-terminal domain and 150 identical amino acid sequences in the intermediate functional domain. Recombinant studies of natural spider puller silk protein CS and the commonly used spider silk protein 2R both exhibited high-temperature resistance, maintaining a certain degree of thermal stability for extended periods in a boiling water bath. Furthermore, through a specific cell-free protein synthesis system and process parameters, efficient expression and screening of the recombinant spider silk proteins were achieved. Using *E. coli* fermentation, the recombinant spider silk proteins significantly increased the total heterologous expression level and soluble expression level. The recombinant spider silk proteins and their synthesis method of this invention achieved a series of beneficial effects, demonstrating broad potential for the development and application of high-performance biomaterials.

Claims

1. A recombinant spider silk protein, characterized in that: Its amino acid sequence includes: the N-terminal domain of spider silk protein, two or more repeating functional domains in the middle, and the C-terminal domain; The N-terminal domain is selected from the N-terminal sequence of the large ampulla gland spider silk protein MaSp1 from the Australian funnel-web spider. The two or more repeating functional domain sequences in the middle are selected from the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, or a recombinant sequence of at least one of the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, the MaSp1 sequence of the large ampullae gland silk protein of the Australian funnel-web spider, and the MaSp2 sequence of the large ampullae gland silk protein of the clouded spider; and the first functional domain sequence connected to the N-terminus is the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider; The C-terminal domain is a hydrophilic domain that can maintain the biological properties of spider silk protein; The amino acid sequence of the recombinant spider silk protein is SEQ ID NO.

1.

2. A recombinant spider silk protein, characterized in that: Its amino acid sequence includes: the N-terminal domain of spider silk protein, two or more repeating functional domains in the middle, and the C-terminal domain; The N-terminal domain is selected from the N-terminal sequence of the large ampulla gland spider silk protein MaSp1 from the Australian funnel-web spider. The two or more repeating functional domain sequences in the middle are selected from the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, or a recombinant sequence of at least one of the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, the MaSp1 sequence of the large ampullae gland silk protein of the Australian funnel-web spider, and the MaSp2 sequence of the large ampullae gland silk protein of the clouded spider; and the first functional domain sequence connected to the N-terminus is the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider; The C-terminal domain is a hydrophilic domain that can maintain the biological properties of spider silk protein; The amino acid sequence of the recombinant spider silk protein is SEQ ID NO.

2.

3. A recombinant spider silk protein, characterized in that: Its amino acid sequence includes: the N-terminal domain of spider silk protein, two or more repeating functional domains in the middle, and the C-terminal domain; The N-terminal domain is selected from the N-terminal sequence of the large ampulla gland spider silk protein MaSp1 from the Australian funnel-web spider. The two or more repeating functional domain sequences in the middle are selected from the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, or a recombinant sequence of at least one of the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider, the MaSp1 sequence of the large ampullae gland silk protein of the Australian funnel-web spider, and the MaSp2 sequence of the large ampullae gland silk protein of the clouded spider; and the first functional domain sequence connected to the N-terminus is the MaSp4 sequence of the large ampullae gland silk protein of Darwin's bark spider; The C-terminal domain is a hydrophilic domain that can maintain the biological properties of spider silk protein; The amino acid sequence of the recombinant spider silk protein is SEQ ID NO.

3.

4. DNA for expressing the recombinant spider silk protein according to any one of claims 1-3.

5. An expression plasmid for expressing the recombinant spider silk protein of any one of claims 1-3, characterized in that: After codon optimization according to host preference, the DNA sequence of the recombinant spider silk protein was artificially synthesized and constructed into an expression vector to obtain the expression plasmid of the recombinant spider silk protein.

6. The recombinant expression plasmid of spider silk protein as claimed in claim 5, wherein: The expression vector is either the pJL1 expression vector or the pET30a expression vector.

7. The method of synthesizing a recombinant spider silk protein of any one of claims 1-3, characterized in that: The recombinant spider silk protein is obtained by amplification, expression, and purification using the expression plasmid of the recombinant spider silk protein according to claim 5 or 6.

8. The method for synthesizing recombinant spider silk protein as described in claim 7, characterized in that: The specific steps include the following: 1) The expression plasmid of the recombinant spider silk protein was subjected to isothermal rolling circle amplification, wherein the isothermal rolling circle amplification reaction system included the recombinant expression plasmid, DNA polymerase, buffer, dNTPs and random primers; 2) The expression plasmid of the recombinant spider silk protein after isothermal rolling circle amplification was expressed using a cell-free protein synthesis kit, and the protein was purified using a magnetic bead kit to obtain the recombinant spider silk protein.

9. The method for synthesizing recombinant spider silk protein as described in claim 8, characterized in that: The DNA polymerase is Phi29 DNA polymerase; And / or the cell-free protein synthesis system contains 20-100 μg / mL of recombinant spider silk protein expression plasmid; And / or in the cell-free protein synthesis system, 1-3 mM of one or more of glycine, alanine, proline, and serine may be added. And / or the temperature for the cell-free protein synthesis is 16-25℃, the time is 16-32h, and the shaking speed is 200-400rpm; The purification of the magnetic bead kit is carried out at a temperature of 4°C or room temperature for 1-3 hours.

Images