Recombinant bionic spider silk protein based on computer intelligent screening and application thereof

Abstract

According to the invention, a beta-sheet microcrystalline region gene is obtained from spider silk proteins derived from Borreelia burgdorferi B31 and Streptomyces globiporus, a polyalanine sequence in a repeated region is replaced with a gene sequence MaSp1 of araneus ventricosus main ampulla gland silk as a template, and password optimization is carried out, so that a recombinant bionic spider silk protein with an amino acid sequence as shown in SEQ ID NO: 1-4 is obtained. The recombinant bionic spider silk protein disclosed by the invention not only has high beta-sheet content, but also can be subjected to efficient soluble expression in engineering bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of protein engineering, and specifically relates to a class of recombinant bionic spider silk proteins based on computer intelligent screening and applications thereof. Background Art

[0002] Spider silk is a high-performance protein fiber in nature. Its unique high tensile strength and ductility, especially its unparalleled flexibility, fatigue resistance, tensile strength similar to that of steel, breathability, moisture absorption, and biocompatibility have broad application prospects in aerospace, materials science, tissue engineering, biomedicine and other fields, and have become a research hotspot in recent years.

[0003] In the field of chip manufacturing, recombinant spider silk protein was used to prepare photoresists with high mechanical strength, breaking through the limitations of traditional materials and achieving ultra-high precision (≤100nm) 3-D electron beam lithography, which is expected to promote my country's chip manufacturing technology to take a big step forward. In the medical field, remarkable progress has been made in the use of spider silk protein to make nanofiber scaffolds for the treatment of cardiovascular diseases. Recombinant spider silk protein and polylactic acid caprolactone are blended and electrospun as nanofiber scaffolds, which have higher tensile strength and elongation than traditional nanofiber scaffolds; as a carrier for targeted drug delivery and controlled drug release, it is used for targeted delivery of stem cells and pH-responsive drug release. In the fields of military industry, environment, etc., magnetic field sensing is performed by winding spider silk on a conical single-mode optical fiber and combining streptavidin-biotin with spider silk. The proposed magnetic field sensor has the advantages of easy manufacturing and simple structure.

[0004] However, due to the characteristics of protein materials such as natural spider silk proteins, such as large molecular weight, high content of specific amino acids (about 50% Ala+Gly), highly repetitive amino acid sequences and extremely high GC content in the core region genes of spider silk proteins, their heterologous expression level is extremely low, far from meeting or expanding actual needs such as application. Even through the transformation of chassis cells in microbial systems such as Escherichia coli, the design and transformation of expression elements, the adaptation transformation of target gene expression, the regulation of protein synthesis pathways and the global regulation transformation, it is difficult to achieve a significant breakthrough in the expression of high-performance recombinant bionic spider silk proteins in prokaryotic cells. Summary of the invention

[0005] Based on the design and optimization of characteristic motifs of the microcrystalline region of spider silk protein, the present invention designs a type of microcrystalline region motif between folded sheets that can form cross-β-spine characteristics, and prepares high-performance recombinant bionic spider silk protein in Escherichia coli.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The recombinant bionic spider silk protein has the amino acid sequence shown in SEQ ID No: 1-4.

[0008] Another object of the present invention is to provide a DNA molecule encoding the above-mentioned recombinant bionic spider silk protein and an expression vector containing the DNA molecule. The expression vector is a plasmid, a bacteriophage, a virus or a host cell.

[0009] In a specific example of the present invention, the DNA molecule has a nucleotide sequence shown in SEQ ID No: 6-9.

[0010] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0011] More preferably, the host cell is Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

[0012] The present invention also provides an engineering strain capable of efficiently expressing the recombinant bionic spider silk protein of the present invention. The engineering strain uses Escherichia coli BL21 (DE3) as a host strain.

[0013] The recombinant bionic spider silk protein of the present invention can be used to prepare tissue engineering materials, biomacromolecule fixing materials, biosensors and protective materials.

[0014] Advantages of the present invention:

[0015] The present invention uses a semi-rational design based on a computer deep learning algorithm to analyze the correlation between the microcrystalline region of spider silk protein and its adjacent sequences and the self-assembly performance, screen out the recombinant bionic spider silk protein sequence and optimize the expression. Soluble recombinant bionic spider silk protein with significantly improved mechanical properties and β-folding content was successfully obtained in vitro.

[0016] The recombinant bionic spider silk protein of the present invention is significantly different from the wild type in expression and solubility. And the β-fold content in the protein can be significantly increased by temperature induction. The recombinant bionic spider silk protein of the present invention has a cross-β-spine characteristic structure and has broad application prospects in the fields of military industry, environment, etc. In combination with multi-scale simulation and experimental data analysis, the present invention uses a computer algorithm to screen the β-fold characteristic motif information of different biological sources in the PDB database, and prepares a recombinant bionic spider silk protein with a high β-fold content. It has stronger self-assembly performance than the unmodified spider silk protein, and is expected to obtain high-performance recombinant bionic spider silk protein materials with practical application value, and also provide a reference for the optimization design and production of other polymer materials with complex macroscopic conformations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1.Schematic diagram of the spatial folding process of β-sheet characteristic motifs screened by computer intelligent screening.

[0018] Figure 2 .SDS-PAGE verified the expression of recombinant biomimetic spider silk proteins KTAAWN, ITVQQ, GVLEGV, SVSVSVS, and VVVKI in Escherichia coli (S represents soluble component; IB represents inclusion body).

[0019] Figure 3 .SDS-PAGE verified the expression of wild-type spider silk protein MaSp1 in Escherichia coli.

[0020] Figure 4 .Significance analysis of recombinant bionic spider silk proteins KTAAWN, ITVQQ, GVLEGV, SVSVSVS and VVVKI and wild-type spider silk protein MaSp1 and unmodified spider silk protein.

[0021] Figure 5 .SDS-PAGE verified the purification of biomimetic spider silk proteins GVLEGV, KTAAWN and ITVQQ in Escherichia coli.

[0022] Figure 6 .Circular dichroism spectrum of recombinant bionic spider silk protein GVLEGV bionic spider silk protein.

[0023] Figure 7 .Circular dichroism spectrum of recombinant bionic spider silk protein KTAAWN bionic spider silk protein.

[0024] Figure 8 .Circular dichroism spectrum of recombinant bionic spider silk protein ITVQQ bionic spider silk protein.

[0025] Fig. 9 .Circular dichroism spectrum of wild-type spider silk protein MaSp1. DETAILED DESCRIPTION

[0026] The content of the present invention is further described in detail below in conjunction with specific embodiments, and the purpose of the embodiments is only to illustrate and describe the current best mode of the present invention. The protection scope of the present invention is not limited in any way by the embodiments described herein, and any equivalent replacement in the field implemented according to the content or principle disclosed by the present invention belongs to the protection scope of the present invention.

[0027] In the following examples, various processes and methods not described in detail are conventional methods known to those of ordinary skill in the art.

[0028] Example 1 Intelligent screening and rational design of recombinant bionic spider silk protein microcrystalline β-sheet

[0029] First, the natural spider silk protein sequence data information was obtained through web crawler technology. After data preprocessing, the obtained spider silk protein sequence and function and other unstructured data were converted into structured data and stored in the local database. Mafft and Muscle software were used to perform large-scale multiple sequence alignment and analysis on the established sequence data cluster; in view of the high specificity of the spider silk protein REP domain, a machine learning algorithm was used to improve the accuracy of feature motif extraction. 1) Obtain basic features, considering features that are not related to the length of the spider silk protein REP domain sequence, such as the mean, variance, skewness coefficient, standard deviation, etc. of all points in the spider silk protein sequence data, and the extracted statistical features were used as algorithm input; 2) Obtain time-series related features. This time, a recurrent neural network (RNN) was selected to use the output of the previous time point and the input of the current time point to generate the output of the current time point, repeat to the end of any sequence, and obtain the output at the end of the sequence as the sequence feature. The Top n data features of the spider silk protein data were integrated as a training set to determine the spider silk protein feature motif. For example, the sequence training data set of the spider silk protein REP domain (X1, Y1), (X2, Y2), ... (X n , Y n ). After data input, the optimal algorithm model was obtained through training and parameter tuning, which was expressed as a conditional probability distribution function. Feature information such as repeat unit characteristic motifs (poly(GA), polyA, GGX, GPX, QQ, GSG) and secondary structures (such as α-helix, β-sheet, β-turn, random coil, etc.) were extracted, and the types and usage frequencies of amino acid residues at various positions in the sequence were summarized and analyzed. The sequence analysis structure was evaluated by algorithm using k-fold cross validation and independent testing, and the secondary structure was optimized using explicit solvent equilibrium. The influence of different characteristic motifs on the formation of secondary structure was determined by analyzing its secondary structure and dihedral angles. Through strategies such as multiple sequence analysis, highly homologous templates were found and determined from the PDB structure database, and the overall structure was determined using software such as Modeller 9.15. The highest-scoring model was energy optimized using molecular dynamics simulation, and the loop structure was optimized using ab initio calculation to obtain a reliable three-dimensional structural model of spider silk protein. Visualization software such as VMD and DS were used to compare and analyze the structure of spider silk proteins to characterize the mapping relationship between the sequence characteristics and structure of the spider silk protein REP domain and the functional property values ​​of its spider silk fibers, forming an interface spider silk protein characteristic motif-structure-function network map (as shown in Table 1).

[0030] Table 1 Screening of soluble β-sheet sequences of recombinant biomimetic spider silk proteins

[0031]

[0032]

[0033] The β-folding sequence in the microcrystalline region shown in Table 1 was selected, and the gene sequence MaSp1 of the main ampulla of the spider was used as a template to replace the polyalanine sequence in the repeat region and perform code optimization to obtain the recombinant bionic spider silk proteins GVLEGV, KTAAWN, ITVQQ, SVSVSVS and VVVKI, the amino acid sequences of which are shown in SEQ ID No: 1-5, and the corresponding gene sequences are shown in SEQ ID No: 6-10. The schematic diagram of the spatial folding process of the β-folding characteristic motif of each corresponding recombinant bionic spider silk protein is shown in Figure 1 .

[0034] Case study 2: Construction of an engineering strain for efficient expression of recombinant bionic spider silk protein

[0035] Combining the theories and information provided by traditional biotechnology and synthetic biology, Escherichia coli was used as the host bacteria, and the pSE380 expression system was used to efficiently express spider silk protein. The engineered strain for efficiently expressing spider silk protein in the present invention was mainly constructed by the following method:

[0036] The five gene fragments shown in SEQ ID No: 6-10 in Example 1 and the gene of MaSp1 were cut and inserted into a vector using restriction endonucleases NdeI and HindIII. The vector was plasmid pSE380. The company sequenced to confirm that the fusion construction was successful. The recombinant bacteria expressing the gene were subsequently transformed:

[0037] Transform the recombinant vector into the recipient cells E.coli BL21 (DE3): thaw the competent cells BL21 (DE3) on ice, take 10 μL of the recombinant product and add it to 100 μL of the competent cells, gently blow to mix, and let it stand on ice for 30 minutes; after standing, heat shock in a 42°C water bath for 90 seconds, and immediately cool it on ice for 5 minutes; then add 800 μL LB medium (without antibiotics), and shake the bacteria at 37°C, 180 rpm for 60 minutes; after the end, centrifuge the bacterial solution at 12000 rpm for 1 minute, discard 900 μL of the supernatant, resuspend the bacteria with the remaining culture medium, and gently spread it on the ampicillin-resistant plate with a sterile spreading stick; place the plate in a 37°C incubator and incubate it upside down for 12-14 hours.

[0038] Colony PCR: Pick the monoclonal strain on the plate and perform colony PCR according to the system in Table 2. The primers are shown in Table 3.

[0039] Table 2 PCR reaction system

[0040]

[0041] The obtained PCR products were recovered by gel and verified by agarose gel electrophoresis at 180V, 400mA, 10min, and the correct size was about 1000bp.

[0042] Table 3 Colony PCR primers

[0043]

[0044] The verified recipient cells were inoculated into LB medium containing ampicillin resistance, and IPTG was added to the medium containing the recipient cells to induce the expression of recombinant spider silk proteins in the recipient cells (named as recombinant spider silk proteins GVLEGV, KTAAWN, ITVQQ, SVSVSVS and VVVKI).

[0045] The specific steps are as follows:

[0046] (1) Plasmids containing the characteristic motifs of GVLEGV, KTAAWN, ITVQQ, SVSVSVS or VVVKI and the gene containing MaSp1 were respectively transferred into the culture medium containing Escherichia coli BL21 (DE3), and PTG was added at a final concentration of 0.1 mmol / L. The Escherichia coli BL21 (DE3) was induced to produce spider silk protein MaSp1 and recombinant spider silk protein at room temperature of 30°C for 24 hours.

[0047] (2) After induction, the E. coli BL21 (DE3) bacterial culture was collected, and the E. coli was disrupted using a cell disruptor, and the supernatant was collected.

[0048] SDS-PAGE electrophoresis analysis of recombinant biomimetic spider silk proteins KTAAWN, ITVQQ, GVLEGV, SVSVSVS, and VVVKI expressed in Escherichia coli Figure 2 As shown in Figure 2, SDS-PAGE electrophoresis analysis of spider silk protein MaSp1 expressed in Escherichia coli is shown in Figure 2. Figure 3 After using grayscale scanning to quantify protein concentration, the results are as follows Figure 4 The results showed that the expression level of recombinant spider silk protein VVVKI was lower than that of the other four proteins, and was significantly higher than that of the unmodified spider silk proteins. The recombinant spider silk proteins GVLEGV, KTAAWN and ITVQQ were expressed intracellularly as soluble proteins, while the recombinant spider silk protein SVSVSVS existed in the form of inclusion bodies and needed to be renatured and purified in subsequent steps. The recombinant spider silk proteins GVLEGV, KTAAWN, ITVQQ and SVSVSVS were selected for subsequent research.

[0049] (3) Purify the supernatant using an AKTA protein purifier. 2+After the agarose column is completely bound, it is eluted using an elution buffer (500 mmol / L imidazole, 20 mM Tris, 300 mM NaCl, adjusted to pH 8.0 using HCL) to obtain a recombinant spider silk protein solution.

[0050] The fused spider silk protein solution obtained above was frozen at -80°C and dried to obtain protein powder. In order to completely freeze-dry, the freeze-drying time was required to be not less than 48 hours.

[0051] Example 3 Scale-up production of the recombinant biomimetic spider silk protein of the present invention

[0052] The proteins obtained in Example 1 and Comparative Example 1 and their purity were detected by SDS-PAGE electrophoresis. The specific operation steps are as follows:

[0053] (1) 600 ml of Escherichia coli containing the recombinant plasmids GVLEGV, KTAAWN and ITVQQ were cultured and induced with 0.1 mM IPTG at 30°C for 36 h (ITVQQ induced for 12 h). The cells were collected after centrifugation at 8000 rpm for 15 min at 4°C. Then, 60 ml of pure water was added to resuspend the cells and the cells were crushed using a cell crusher to obtain crushed cells.

[0054] (2) Centrifuge the broken bacteria in the previous step at 4°C, 12,000 rpm for 15 min and collect the supernatant.

[0055] (3) The supernatant containing the target protein was purified by affinity chromatography on a Ni-NTA column. The protein solution was loaded into a nickel column, and the column was washed with a washing buffer (20 mM Tris, 140 mM NaCl, adjusted to pH 8.0 with HCL). The protein on the column was eluted with an appropriate amount of elution buffer (500 mmol / L imidazole, 20 mM Tris, 300 mM NaCl, adjusted to pH 8.0 with HCL), and dialyzed overnight with pure water at 4°C to obtain the purified recombinant protein.

[0056] (4) Draw the BCA standard curve according to the instructions of the Novozyme BCA kit. Mix solution A and solution B in the kit at a ratio of 50:1 to make a working solution. Take 20 μL of the sample and place it in a 96-well microplate. Perform three parallel experiments for each sample. Add 200 μL of the working solution and incubate at 37°C for half an hour. Use an enzyme reader to measure the OD 562 .

[0057] (5) After complete dialysis, the recombinant protein solution was frozen at -80°C overnight and then placed in a freeze dryer for 48 hours or more to prepare lyophilized powders of GVLEGV, KTAAWN and ITVQQ.

[0058] (6) Take 30 μL of the supernatant after centrifugation and add 10 μL of 5× SDS-loading solution. Boil the sample at 100°C for 8 min. Take 10 μL of the sample and perform SDS-PAGE electrophoresis, Coomassie brilliant blue staining and decolorization.

[0059] The recombinant bionic spider silk proteins GVLEGV, KTAAWN and ITVQQ were purified in E. coli and the SDS-PAGE verification results were as follows Figure 5 As shown, the results showed that GVLEGV, KTAAWN and ITVQQ proteins were successfully obtained.

[0060] Example 4 Analysis of the secondary structure of recombinant bionic spider silk protein

[0061] The dialyzed solution was characterized using circular dichroism. The specific steps are as follows:

[0062] (1) The protein concentration of the purified recombinant bionic spider silk protein stock solution prepared in Example 3 was measured and then dialyzed with pure water at 4° C. overnight.

[0063] (2) The secondary structure composition of the recombinant biomimetic spider silk proteins GVLEGV, KTAAWN and ITVQQ was analyzed by circular dichroism (CD). Circular dichroism tests were performed using a circular dichroism spectrometer. The protein concentrates were dissolved in pure water and the final concentration of the protein solution was maintained at 0.3-0.5 mg / mL. The diluted protein solution can be used for circular dichroism tests.

[0064] The circular dichroism spectrum of the recombinant biomimetic spider silk protein GVLEGV is shown in Figure 6 As shown in the figure, under room temperature conditions, the recombinant bionic spider silk protein GVLEGV has an obvious negative absorption peak at 208nm, corresponding to the characteristic peak of the α-helix structure. The key characteristic peaks in the spectrum were determined by baseline correction and feature recognition, and the spectral data were quantitatively analyzed using the CONTINLL algorithm. The results showed that the content of α-helix structure in the protein was 22.6%, the content of β-folding structure was 23.4%, the content of random curling was 37.4%, and the content of other secondary structures (such as turns, etc.) was low. It is inferred that the main secondary structures of the protein are α-helix and β-folding. In addition, after induction at 65℃ for 48h, the content of α-helix structure of the protein at room temperature decreased to 12.8%, while the content of β-folding structure increased to 33.7%.

[0065] The circular dichroism spectrum of the recombinant biomimetic spider silk protein KTAAWN is shown in Figure 7As shown in the figure, quantitative analysis based on the CONTINLL algorithm shows that the random curling of the recombinant biomimetic spider silk protein KTAAWN at 25°C is its main structure, accounting for more than half of the total protein secondary structure content. After being kept at 45°C for 5 minutes and machine scanning for 5 minutes, the α-helix and random curling contents of the protein decreased to 5.2% and 54.0%, respectively, while the β-folding content increased to 27.9%. After being kept at 85°C for 5 minutes and machine scanning for 5 minutes, the β-folding content of the protein reached a peak of 33.4%.

[0066] The circular dichroism spectrum of recombinant biomimetic spider silk protein ITVQQ is shown in Figure 8 As shown in the figure, quantitative analysis based on the CONTINLL algorithm shows that the recombinant bionic spider silk protein ITVQQ has α-helix as the main structure at 25°C, accompanied by β-folding and random coiling. After being kept at 45°C for 5 minutes and then machine scanned for 5 minutes, the α-helix and random coiling content of the protein decreased slightly, while the β-folding content increased. After being kept at 65°C for 5 minutes and then machine scanned for 5 minutes, the β-folding content of the protein increased sharply to 81.3%.

[0067] The circular dichroism spectrum of the unmodified spider silk protein MaSp1 at room temperature is shown in the figure below. Fig. 9 As shown, in solution the main conformation is α-helix.

[0068] The above results show that at room temperature, the unmodified spider silk protein MaSp1 is mainly composed of α-helix, accounting for 97.7%, and no β-fold content is detected. The modified recombinant bionic spider silk proteins KTAAWN, GVLEGV and ITVQQ have a low α-helix content and have formed a β-fold secondary structure (7.5%, 22.6% and 38.0% respectively). After induction at 45, 65 and 85°C, the secondary structure content of all proteins changed significantly. After induction at 65°, the β-fold content of the unmodified spider silk protein was the highest, reaching 41.5%, while the β-fold content of the recombinant bionic spider silk proteins GVLEGV and KTAAWN was similar when induced at 65°C and 85°C, increasing to 33.7 and 33.4% respectively, while the β-fold content of ITVQQ was the highest after induction at 65°C, reaching nearly twice that of the unmodified spider silk protein, with a value of 81.3%. High β-fold content is a key factor for the high strength of spider silk fibers. This indicates that the recombinant bionic spider silk protein of the present invention has a higher β-folding content than the spider silk protein MaSp1, and has cross-β-spine characteristic folding sheets.

Claims

1. Recombinant bionic spider silk protein, characterized in that: It has the amino acid sequence shown in SEQ ID No: 1-4.

2. A DNA molecule, characterized in that Encodes the recombinant biomimetic spider silk protein 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 8, characterized in that The host cell is Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

7. Use of the recombinant bionic spider silk protein as claimed in claim 1 in the preparation of tissue engineering materials, biomacromolecule fixation materials, biosensors, and protective materials.

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