Bionic spider silk protein dimer with improved self-assembly performance and application thereof

Abstract

The invention discloses a bionic spider silk protein monomer and a dimer thereof. By replacing the original C-terminal sequence of spider silk protein MaSp1s and introducing cysteine, the modified spider silk protein monomer rMaSp1s forms a dimer through a disulfide bond. The bionic spider silk protein dimer disclosed by the invention has relatively high self-assembly performance and is beneficial to application in an industrial range.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and in particular relates to a method for preparing a spider silk protein dimer with improved molecular weight and self-assembly performance. Background Art

[0002] Spider silk is a naturally occurring high-strength fiber, which can be divided into primary ampullate gland silk, secondary ampullate gland silk, etc. according to the glands where it is produced. It has the advantages of good biocompatibility, high tensile strength, good ductility, and environmental friendliness, and is one of the natural materials with excellent performance. However, spiders are cannibalistic and it is difficult to raise them on a large scale to obtain a large amount of natural spider silk protein. Heterologous synthesis is an effective way to obtain spider silk protein.

[0003] The spider's major ampulla silk protein MaSp has a very high molecular weight (250-350 kDa). Its core domain contains a large number of glycine and alanine residues and is composed of dozens or hundreds of short repeating units of 19 to 46 amino acids. Each short repeating unit contains varying numbers of repeating motifs such as PolyA (i.e. An), (GA)n, GGX and GPGGX. These repeating motifs give spider silk fibers excellent strength and maintain a certain degree of elasticity during the silk-forming process. Many studies have shown that molecular weight is one of the key determinants of the excellent mechanical properties of spider silk. However, these characteristics of natural spider silk proteins, such as high GC content of genes, highly repeated amino acid sequences in the repeating core region, high content of specific amino acids, and large molecular weight, have brought great difficulties to heterologous expression.

[0004] In recent years, Han et al. discovered a new type of short major ampullate spider silk protein (MaSp1s) in Cyrtophora moluccensis. Compared with natural MaSp, its core domain does not have the ability to form fibers with excellent mechanical properties. Its molecular weight is 40kDa lower, and it only contains a short core region (191AA), without typical An and repeat sequences, and flanked by typical conserved terminal domains (439AA in total). However, Thamm et al. found that on the basis of similar molecular weight, MaSp1s is easier to express than the fibers produced by MaSp, and the fibers formed by it have similar ductility to MaSp fibers, slightly higher stiffness and strength, and better overall toughness. Despite this, the low molecular weight of MaSp1s still limits the mechanical properties of the fibers it forms to a certain extent, and cannot meet the needs of industrialization. Studies on the relationship between the sequence, structure, molecular weight and spinning performance of spider silk proteins have shown that there is still a lot of room for improvement in the sequence design and spinning performance of recombinant spider silk proteins. With the development of genetic engineering and molecular biology, improving the self-assembly performance of spider silk proteins by optimizing the sequence-structure-function relationship of spider silk proteins and expressing recombinant spider silk proteins using efficient exogenous protein expression systems are effective ways to further improve the industrial application of recombinant spider silk proteins. Summary of the invention

[0005] The purpose of the present invention is to provide a bionic spider silk protein dimer with improved self-assembly performance and application thereof.

[0006] The invention replaces the original C-terminal sequence of the spider silk protein MaSp1s and introduces cysteine, so that the modified spider silk protein monomer forms a dimer, improves its self-assembly performance, and is conducive to its application in the industrial range.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A biomimetic spider silk protein monomer (rMaSp1s), the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0009] The present invention replaces the natural MaSp1s C-terminal sequence, introduces a cysteine ​​residue at a suitable position of the MaSp1s sequence, and then induces the cysteine ​​residues of the two monomers to form a disulfide bond, thereby forming a dimer. The protein dimer structure of the present invention is as follows Figure 1 shown.

[0010] In the present invention, the amino acid sequence of the natural spider silk protein MaSp1s is shown in SEQ ID NO:2, wherein the replaced amino acid sequence of the C-terminus is the 342nd to 439th amino acids thereof; and the nucleotide sequence is shown in SEQ ID NO:5.

[0011] The present invention further provides a DNA molecule encoding the bionic spider silk protein monomer of the present invention.

[0012] The present invention further provides an expression vector comprising the above DNA molecule. The expression vector is a plasmid, a bacteriophage, a virus or a host cell. The host cell can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma. Escherichia coli is preferred.

[0013] The present invention also provides a bionic spider silk protein dimer, which is formed by disulfide bond polymerization of the thiol groups of the cysteine ​​at the C-termini of two spider silk protein monomers.

[0014] The present invention further provides the application of the bionic spider silk protein monomer and dimer in the preparation of tissue engineering materials, biomacromolecule fixation materials, biosensors, protective materials, etc.

[0015] Advantages of the present invention:

[0016] The present invention optimizes the gene sequence based on low molecular weight short major ampullate spider silk protein (MaSp1s) and performs semi-rational design, and successfully obtains a dimer of bionic spider silk protein (rMaSp1s) with significantly improved molecular weight and self-assembly performance in vitro.

[0017] The monomer and dimer of the spider silk protein described in the present invention were stirred in a 60°C aqueous solution. After stirring for 48 hours, the morphology of the monomer rMaSp1s and the dimer was significantly different. The monomer was almost unable to form nanofibers and could only slightly aggregate into a particle morphology. Compared with the monomer, under the same incubation conditions (60°C, 48 hours), the dimer described in the present invention exhibited a mesh-like nanofiber structure with a diameter of about 2nm ( Figure 3 c). The spider silk protein with mesh fiber structure has broad application prospects in the fields of wearable electronics, optical sensors, intelligent control and energy harvesting. The research results of the present invention show that the spider silk protein dimer with improved molecular weight has stronger self-assembly performance than the spider silk protein monomer, and the material made of it has better mechanical strength and toughness, which can better meet the needs of industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 Schematic diagram of the C-terminal dimer of the biomimetic spider silk protein rMaSp1s of the present invention ( Figure 1a is the C-terminal protein of Latrodectus hesperus MaSp1 obtained through screening to replace the C-terminal of natural MaSp1s, Figure 1 b is a schematic diagram of the spider silk protein rMaSp1s dimer after C-terminal substitution).

[0020] Figure 2 This is the SDS-PAGE analysis of the biomimetic spider silk protein monomer and its dimer according to the present invention. Figure 2 a is the protein purification result of the diploid spider silk protein MaSp1s (MaSp1s-2Rep), Figure 2 b is the SDS-PAGE comparative analysis of rMaSp1s monomer purification and rMaSp1s dimer).

[0021] Figure 3 This is an atomic force microscopy comparative analysis of the solution structure of the spider silk protein monomer and dimer as well as the spider silk protein MaSp1s diploid according to the present invention. Figure 3 a is the atomic force microscopy characterization result of spider silk protein rMaSp1s monomer protein, Figure 3 b is the atomic force microscopy characterization and analysis results of the spider silk protein MaSp1s diploid (MaSp1s-2Rep). Figure 3 c shows the atomic force microscopy characterization results of the spider silk protein rMaSp1s dimer). DETAILED DESCRIPTION

[0022] The specific steps of the present invention are described below by way of examples, but are not limited to the examples.

[0023] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0024] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that this embodiment is only for illustrating the present invention and is not intended to limit the scope of the present invention in any way.

[0025] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0026] The present invention is further described below in conjunction with specific embodiments.

[0027] Example 1 Construction of spider silk protein rMaSp1s monomer

[0028] 1. Comparison and screening of C-terminal domains of spider silk proteins from different sources

[0029] Comparison of the C-termini of spider silk proteins from different sources using NCBI and PDB databases:

[0030]

[0031] The C-terminal domain of Latrodectus hesperus (Lh.CT, ABR68856.1) was selected based on the composition of the C-terminal amino acid residues of spider silk proteins and the spatial distance between the residues. The cysteine ​​residue at position 51 and the distance between the two residues were very close (4.0A), which met the criteria for forming a disulfide bond ( Figure 1 ).

[0032] 2. Construction of the expression vector of the spider silk protein monomer rMaSp1s described in the present invention

[0033] The C-terminal domain of the spider silk protein MaSp1 from Latrodectus hesperus (Accession number: ABR68856.1) and the core domain of the spider silk protein MaSp1s from Cyrtophora moluccensis (Accession number: AGQ04592.1) were fused:

[0034] Using the original sequence pET21a(+)-MaSp1s constructed on the pET21a(+) plasmid as a template, the vector and the CT gene (Lh.CT) of Latrodectus hesperus MaSp1 were first linearized by PCR using a one-step cloning primer. After the fragment was purified, the target gene fragment -Lh.CT- and -pET21a(+)-NT-MaSp1s(core)- linearized vector were connected using a one-step cloning kit. The two were cyclized under the catalysis of Exnase II to successfully construct the pET21a(+)-NT-rMaSp1s-Lh.CT (pET21a(+)-rMaSp1s) plasmid. The company sequenced to confirm that the fusion construction was successful, and then the recombinant bacteria expressing rMaSp1s were obtained by transformation.

[0035] The specific experimental steps are as follows:

[0036] (1) Vector linearization: One-step cloning primers were designed. The primers are shown in Table 1. First, inverse PCR was used to obtain a linearized vector - pET21a(+)-NT-MaSp1s(core)-. The PCR system is shown in Table 2.

[0037] (2) Insert fragment PCR amplification: The insert fragment is also obtained by PCR amplification using the designed one-step cloning primers. After the PCR is completed, the 5' and 3' ends of the insert fragment carry homologous sequences (20 bp) that correspond to the two ends of the linearized vector. After the PCR of both the vector and the fragment is completed, a small amount of the product is taken for agarose gel electrophoresis to verify the product, and then the fragment is purified using a kit and finally used for the subsequent one-step cloning.

[0038] (3) The two PCR products obtained were verified by agarose gel electrophoresis at 180V, 400mA, 10min, and the sizes of the linearized vector and the inserted fragment were 7212bp and 334bp, respectively. After verification, the dephosphorylase DpnI was added according to the system shown in Table 3, and the dephosphorylase DpnI was placed in a constant temperature metal bath at 37°C for 30min to digest and remove the old DNA template and methylated sequence.

[0039] Table 1 Primers for one-step cloning

[0040]

[0041]

[0042] Table 2 PCR system

[0043]

[0044] Table 3 Digestion system

[0045]

[0046] (4) One-step cloning: The digested linearized vector and the insert fragment are mixed according to the ratio in Table 4. Under the catalysis of Exnase II, the mixture is placed in a dry thermostatic metal bath at 37°C for 30 min to complete the recombination reaction and realize the in vitro circularization of the two linear vectors and the insert fragment.

[0047] Table 4 One-step cloning system

[0048]

[0049] (5) Transformation of competent cells: Thaw competent cells BL21 (DE3) on ice, take 10 μL of recombinant product and add it to 100 μL of competent cells, gently blow and mix, and place on ice for 30 minutes; after the standing period, heat shock in a 42°C water bath for 95 seconds, and immediately place on ice to cool for 2-3 minutes; then add 800 μL LB medium (without antibiotics), and shake the bacteria at 37°C, 180 rpm for 40 minutes; after the completion, centrifuge the bacterial solution at 12000 rpm for 1 minute, discard 900 μL of supernatant, resuspend the bacteria with the remaining medium, and gently spread it on an ampicillin-resistant plate with a sterile spreader; place the plate in an incubator at 37°C and incubate it upside down for 12-14 hours. (6) Colony PCR: Pick a monoclonal strain and perform colony PCR according to the system in Table 5.

[0050] Table 5 PCR reaction system

[0051]

[0052] The obtained PCR product was recovered by gel and verified by agarose gel electrophoresis at 180V, 400mA, 10min, and the correct size was 414bp.

[0053] In order to investigate the effect of molecular weight changes on the function of spider silk proteins, a diploid spider silk protein MaSp1s (MaSp1s-2Rep) was constructed for comparison.

[0054] Referring to the above method, the original sequence pET21a(+)-MaSp1s constructed on the pET21a(+) plasmid was used as a template. The core region gene (Core) of the vector and MaSp1s was first linearized by PCR using the one-step cloning primers, and the repeat module gene (Core) was amplified using the Gene-F and Gene-R primers in Table 6. The 2-Core length was 1212bp. At the same time, the target plasmid pET21a(+)-MaSp1s was linearized using the Gene-F and Gene-R primers in Table 6. Finally, the linearized target plasmid was connected to the PCR amplified target fragment using Exnase II of the one-step cloning kit to form a complete circular DNA molecule: pET21a-MaSp1s-2rep. The company sequenced to confirm that the construction was successful, and the recombinant bacteria expressing MaSp1s-2rep were subsequently obtained by transformation.

[0055] The specific experimental steps are as follows:

[0056] (1) Vector linearization: One-step cloning primers were designed. The primers are shown in Table 6. First, inverse PCR was used to obtain the linearized vector - pET21a(+)-MaSp1s(core)-. The PCR system is as described in Table 2.

[0057] (2) Insert fragment PCR amplification: The insert fragment is also obtained by PCR amplification using the designed one-step cloning primers. After the PCR is completed, the 5' and 3' ends of the insert fragment carry homologous sequences (20 bp) that correspond to the two ends of the linearized vector. After the PCR of both the vector and the fragment is completed, a small amount of the product is taken for agarose gel electrophoresis to verify the product, and then the fragment is purified using a kit and finally used for the subsequent one-step cloning.

[0058] (3) The two PCR products obtained were verified by agarose gel electrophoresis at 180V, 400mA, 10min, and the sizes of the linearized vector and the inserted fragment were 7503bp and 624bp, respectively. After verification, the dephosphorylase DpnI was added according to the system shown in Table 3, and the product was placed in a constant temperature metal bath at 37°C for 30min to digest and remove the old DNA template and methylated sequence.

[0059] Table 6 Primers for one-step cloning

[0060]

[0061] (4) One-step cloning: The digested linearized vector and the insert fragment are mixed in the proportions shown in Table 4. Under the catalysis of Exnase II, the mixture is placed in a dry thermostatic metal bath at 37°C for 30 min to complete the recombination reaction and realize the in vitro circularization of the two linear vectors and the insert fragment.

[0062] (5) Transformation of competent cells: Thaw competent cells BL21 (DE3) on ice, take 10 μL of recombinant product and add it to 100 μL of competent cells, gently blow and mix, and place on ice for 30 minutes; after the standing period, heat shock in a 42°C water bath for 95 seconds, and immediately place on ice to cool for 2-3 minutes; then add 800 μL LB medium (without antibiotics), and shake the bacteria at 37°C, 180 rpm for 40 minutes; after the completion, centrifuge the bacterial solution at 12000 rpm for 1 minute, discard 900 μL of supernatant, resuspend the bacteria with the remaining medium, and gently spread it on an ampicillin-resistant plate with a sterile spreader; place the plate in an incubator at 37°C and incubate it upside down for 12-14 hours. (6) Colony PCR: Pick a monoclonal strain and perform colony PCR according to the system in Table 5.

[0063] The obtained PCR product was recovered by gel and verified by agarose gel electrophoresis at 180V, 400mA, 10min, and the correct size was 1212bp.

[0064] A recombinant bacterium expressing MaSp1s-2rep was obtained. The amino acid sequence of the expressed spider silk protein MaSp1s-2rep was shown in SEQ ID NO:3; and the nucleotide sequence was shown in SEQ ID NO:6.

[0065] Example 2 Obtaining spider silk protein monomers, diploids and dimers

[0066] (1) Obtaining spider silk protein monomers and diploids

[0067] The spider silk protein monomer rMaSp1s and diploid MaSp1s-2rep recombinant bacteria constructed in Example 1 were inoculated into 50 mL of LB liquid culture medium, and ampicillin was added to a final concentration of 100 μg / mL, and cultured overnight at 180 rpm and 37°C; the seed solution cultured overnight was inoculated into fresh 300 mL of LB liquid culture medium at an inoculum size of 2%, and cultured at 180 rpm and 37°C for 1.5 h (OD600 was 0.6-0.8), and the inducer IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) was added, and expression was induced at 30°C for 24 h.

[0068] Take the fermentation broth induced by expression, centrifuge at 5000rpm for 30min, discard the supernatant, then resuspend the bacteria with 40ml of pure water, ultrasonically disrupt for 30min, and centrifuge at 12000rpm for 10min to obtain the supernatant (S). Discard the supernatant and add 10mL of pure water to suspend and mix to obtain the insoluble precipitate (IB). Perform SDS-PAGE electrophoresis detection, the concentration of concentrated gel is 4%, the concentration of separation gel is 12.5%, the sample and loading buffer are mixed in a ratio of 3:1, and the reaction is carried out in a boiling water bath for 5-10min for loading electrophoresis. Set the initial voltage of the electrophoresis instrument to 80V, increase the voltage to 180V when the sample moves to the separation gel, and end the electrophoresis when the sample moves to the bottom of the electrophoresis tank.

[0069] Since six histidines (6×His) are fused to the end of spider silk protein, the nickel chloride in the Ni column can bind to proteins containing His tags and can also bind to imidazole. Therefore, the target protein is purified using a nickel column. The protein elution is achieved by increasing the imidazole concentration, thereby obtaining a purified target protein. After the fermentation-induced expression bacterial solution is centrifuged and crushed, the obtained supernatant is filtered through a 0.22μm filter membrane to obtain a crude protein solution, and then the target protein is separated and purified using a nickel column. The steps are as follows:

[0070] a. Rinse the nickel column with pure water at a flow rate of 2 mL / min to wash away the packing protection liquid and compact the packing;

[0071] b. Rinse the nickel column with at least 5 volumes of Buffer A (20 mM Tris-HCl, 140 mM NaCl, pH 8.0) at a flow rate of 2 mL / min to balance the pH in the nickel column;

[0072] c. Use a syringe to inject the pretreated protein sample into the injection loop at a flow rate of 1.5 mL / min, and collect the penetration peak protein of the sample; d. Flush the nickel column again with at least 5-10 volumes of Buffer A (20 mM Tris-HCl, 140 mM NaCl, pH 8.0) at a flow rate of 2 mL / min to balance the pH in the nickel column until no protein is eluted;

[0073] e. Use gradient elution (5%, 10%, 15%, 30%, 60%, 100%), wash the nickel column with at least 5 volumes of Buffer B (500 mM imidazole, 20 mM Tris-HCl, 300 mM NaCl, pH 8.0) at a flow rate of 2 mL / min for each gradient, and collect the absorption peak protein of each gradient until no protein is eluted;

[0074] f. Rinse the nickel column with at least 5-10 times the volume of pure water at a flow rate of 2 mL / min until the purifier displays a no ion curve and protein absorption peak;

[0075] g. Add 20% ethanol to preserve the nickel column;

[0076] h. The collected protein solutions of different gradients were verified by SDS-PAGE.

[0077] i. The purified protein solution was dialyzed overnight to obtain a relatively pure protein.

[0078] j.SDS-PAGE electrophoresis analysis showed that Figure 2 As shown in a, the molecular weight of spider silk protein rMaSp1s is 40kDa, and the molecular weight of spider silk protein MaSp1s-2Rep is 61kDa. After induction, spider silk protein rMaSp1s monomer and MaSp1s-2Rep diploid have obvious bands at 40kDa and 61kDa, indicating that both spider silk protein monomer and diploid can be successfully induced to express and purified.

[0079] (2) Obtaining spider silk protein dimers

[0080] Using the recombinant spider silk protein monomers purified by (1) above, the redox conditions were adjusted by controlling the different buffer conditions (oxidized glutathione (GSSG) / reduced glutathione (GSH)) of the dimerization reaction according to the method in Table 7 to induce the dimerization of rMaSp1s monomers to form spider silk protein dimers. The SDS-PAGE electrophoresis results are as follows: Figure 2 As shown in b.

[0081] Table 7 Different buffer conditions for dimerization reaction

[0082]

[0083] Example 3 Verification of the self-assembly properties of spider silk protein monomers, diploids and dimers

[0084] Atomic force microscopy was used to characterize the protein monomers, diploids, and dimers after dialyzation.

[0085] The collected relatively pure spider silk protein monomer and dimer stock solutions were used to determine the protein concentration and then the molecular weight was 3 kDa.

[0086] The / Por dialysis membrane was dialyzed against a buffer solution (20 mM Tris-HCl pH 8) at 4°C overnight.

[0087] Atomic force microscopy (AFM) was used to determine the ability of recombinant spider silk protein monomers, diploids and dimers to self-assemble into fibers.

[0088] The three protein stock solutions were diluted to 0.5 mg / ml. In the AFM analysis, the 40 μL of the fibrin dilution was spotted on a ELISA kit (V1 stage, Plano GmbH, Germany) and incubated for 5 min, followed by 4 washes with 50 μL of ddH 2 O. The AFM samples were dried at room temperature for 16 h before imaging.

[0089] AFM was used to characterize its self-assembly ability and morphology. Figure 3 ), Figure 3 a shows that rMaSp1s monomers can hardly form nanofibers and can only slightly aggregate into particles, which may be due to the low molecular weight of the protein (the core region contains only 191 amino acids). Compared with monomers, under the same incubation conditions (60°C, 48h), Figure 3 As shown in Figure b, its diploid MaSp1s-2rep can form a 1-2nm protein layer with uniform particle size and local order. After 72h of assembly, MaSp1s-2rep formed aggregates with a height of 10-15nm. The rMaSp1s dimer has the best self-assembly ability, such as Figure 3 As shown in Figure 3, it can assemble a distinct mesh nanofiber structure with a diameter of about 2 nm. The mesh fibers have broad application prospects in wearable electronics, optical sensors, intelligent control and energy harvesting.

Claims

1. A bionic spider silk protein monomer, characterized in that The amino acids are shown in SEQ ID NO:

1.

2. A DNA molecule, characterized in that Encode the biomimetic spider silk protein monomer as described in claim 1.

3. An expression vector comprising the DNA molecule of claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid, phage, virus or host cell.

5. The expression vector according to claim 4, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

6. The expression vector according to claim 5, characterized in that The host cell is Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

7. A bionic spider silk protein dimer, characterized in that The bionic spider silk protein monomers described in claim 1 are polymerized by forming a disulfide bond through the thiol group of the cysteine ​​residue at the C-terminus.

8. Use of the bionic spider silk protein monomer according to claim 1 or the bionic spider silk protein dimer according to claim 7 in the preparation of tissue engineering materials, biomacromolecule fixation materials, biosensors, and protective materials.

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