Easily-purified spider silk protein modified body as well as modification method and purification method thereof

Abstract

The invention provides a spider silk protein modified body easy to purify as well as a modification method and a purification method thereof, a plurality of amino acid residues in flexible regions on two sides of a spider silk protein microcrystalline region are replaced with acidic amino acids or alkaline amino acids, so that the proportion of charged amino acids in the flexible regions is greater than or equal to 7%, and the spider silk protein modified body is obtained; the spider silk protein modified body is purified in the form of an inclusion body through a temperature change process of heating and cooling. The modified body can be simply purified with high recovery rate through simple heating and cooling processes, the total recovery rate is up to 98% or above, the purity is 90%-99% or above, and meanwhile, the formed artificial spider thread is endowed with higher tensile strength by strengthening the interaction of beta-sheet on the two sides of a microcrystal region, especially between different chains. A foundation is further laid for large-scale efficient production and low-cost separation and purification of the high-performance spider silk protein, and the application possibility of the spider silk protein in various fields is greatly enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to an easily purified spider silk protein modified body, a modification method thereof and a purification method. Background Art

[0002] Spider silk is a naturally occurring, high-strength fiber, classified as primary ampullate silk and secondary ampullate silk, depending on the gland in which it is produced. Its advantages include good biocompatibility, high tensile strength, good ductility, and environmental friendliness, making it one of the most high-performance natural materials. Therefore, spider silk holds broad application prospects in fields such as biomedicine and materials science.

[0003] One type stands out for its exceptional mechanical properties: MaSp, also known as dragline. It possesses the strongest overall performance, with a lower tensile strength (1 to 2 GPa) than Kevlar but a higher ductility (50-60% strain at failure). This exceptional performance is due to its layered structure, where the molecular chains of the microcrystalline and flexible regions are well aligned along the long axis of the fiber, forming fibrils. It has a typical A n and GGX (X can be tyrosine, leucine, etc.) domains, where A n It is a unit that usually contains n = 6-12 alanine amino acids, forming antiparallel β-sheets and their nanocrystals, while GGX forms α-helices and random coils in the flexible region. It is generally believed that the highly oriented crystalline region plays a decisive role in the strength and toughness of spider silk, while the flexible region determines the elasticity and ductility of spider silk ( Adv. Mater ,2009, 21, 487-492).

[0004] Since spiders are cannibalistic, it is difficult to obtain large quantities of natural spider silk proteins through large-scale breeding. Heterologous synthesis is an effective way to obtain spider silk proteins. The development of biomimetic spider silk proteins is more conducive to large-scale production and application than natural proteins. The biomimetic spider silk proteins designed based on the natural spider silk protein sequence have solved the problem of heterologous expression to a certain extent. However, in large-scale production, there are still problems such as easy inclusion of impurities and precipitation. It is difficult to achieve high recovery rate purification with relevant reagents, which still cannot meet the needs of industrialization. ( Adv. Sci , 2022, 9, 2103965) There is still much room for improvement in our current understanding of the relationship between the sequence, structure and various properties of spider silk proteins, as well as the design of biomimetic spider silk proteins.

[0005] In addition, spider silk protein is a hard protein with poor water solubility. When expressed in E. coli, it tends to wrap around foreign proteins and precipitate as the yield accumulates, which inevitably leads to separation and purification problems ( PNAS, 2010, 107, 14059-14063). However, existing technologies for solving the problem of separation and purification of spider silk proteins usually weaken the original properties of spider silk proteins. Summary of the Invention

[0006] The first purpose of the present invention is to provide a method for modifying spider silk protein that is easy to purify, thereby solving the problem of purifying spider silk protein without weakening or even strengthening the self-assembly ability of spider silk protein or enhancing its tensile strength.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for modifying spider silk protein that is easily purified, characterized by replacing several amino acid residues in the flexible region on both sides of the spider silk protein microcrystalline region with acidic or basic amino acids so that the proportion of charged amino acids in the flexible region is ≥7%, thereby obtaining a modified spider silk protein; The spider silk protein modification body is purified in the form of inclusion bodies through a temperature-changing process of heating and cooling.

[0008] In some embodiments of the present invention, after amino acid substitution in the flexible region, the molar ratio of acidic amino acids to basic amino acids is 3:1 to 1:3, preferably 1:1. Purification using the temperature-switching method can be achieved even when the ratio of basic amino acids to acidic amino acids after substitution is within a wide range (e.g., 1:7). However, a large difference in the number of acidic and basic amino acids can lead to a decrease in the tensile strength of the protein. Therefore, in the present invention, the ratio of acidic to basic amino acids is preferably 1:1 to 1:3 (which can be 1:3 or 3:1), and more preferably 1:1. At a 1:1 ratio, the modified spider silk protein exhibits optimal tensile strength.

[0009] In some embodiments of the present invention, the number of amino acid residues replaced is 4-40. Replacing fewer amino acids results in little improvement, making it impossible to purify the protein using temperature-variable purification. Replacing too many amino acids significantly impacts the properties of the original spider silk protein. More preferably, 12-28 amino acid residues are replaced.

[0010] In some embodiments of the present invention, the replacement of amino acid residues avoids the GGY motif in the flexible region to reduce the impact on the mechanical properties of the original spider silk protein.

[0011] In some embodiments of the present invention, the replaced amino acid residue is located in the flexible region close to the microcrystalline region on both sides of the microcrystalline region. The closeness means: the distance from the microcrystalline region is ≤ 4 amino acids.

[0012] The replacement of amino acid residues in the present invention can be the replacement of amino acid residues between adjacent flexible regions (for example, the flexible regions on both sides of a microcrystalline region), or the replacement of acidic and basic amino acid residues in several flexible regions (for example, there is no replacement in the flexible region between two adjacent microcrystalline regions, while amino acid replacement is performed on the left side of the first microcrystalline region and on the right side of the second microcrystalline region. This replacement is an amino acid replacement that separates a section of the flexible region). When amino acid replacement is performed in the flexible region, amino acid replacement can be performed in the middle section of the flexible region, or in the region of the flexible region close to the microcrystalline region. Preferably, amino acid replacement is performed in the region close to the microcrystalline region. The purpose of replacing acidic and basic amino acids at different positions is to form salt bonds in the flexible region in a targeted manner, to further stabilize the formation of its β-sheet microcrystalline region, and to improve the ability of protein self-assembly.

[0013] In some embodiments of the present invention, the amino acid residue replacements are primarily glycine (G). Primarily glycine (G) replacements refer to the preference for glycine when replacing amino acid residues, while also avoiding the GGY motif (and preferably locating replacements near the microcrystalline region). To adhere to this principle and ensure a sufficient amino acid supply, other amino acids, such as arginine (R), tryptophan (T), serine (S), and glutamate (Q), may also be selected for replacement. Glycine-based replacements can further reduce the GC content of spidroin protein genes, overall improving the self-assembly ability of spidroin proteins while significantly enhancing their polarity and water solubility.

[0014] In some embodiments of the present invention, the spider silk protein or spider silk protein modified body is polyploidized. When the expression level of the modified body cannot meet the requirement for inclusion body formation, the original protein and / or modified body can be polyploidized to meet the expression level requirement, for example, to obtain 6, 12, 24, 48 rep polyploid bodies of the modified body.

[0015] A second objective of the present invention is to provide a modified spider silk protein obtained using the aforementioned modification method, wherein the modified spider silk protein exhibits an expression level of ≥15% (w / w) per unit volume. In the present invention, the modified spider silk protein must be purified as inclusion bodies. At lower expression levels, the protein primarily exists in a soluble form, which is insufficient for purification through temperature-induced phase transition. Only inclusion bodies can achieve high-recovery temperature-variable purification. The modified protein obtained by the modification method of the present invention, which involves substitution of acidic and basic amino acids in the flexible region, can achieve a certain increase in expression level relative to the original protein. When the modified protein exhibits an expression level of ≥15% (w / w) per unit volume, the protein primarily exists in the form of inclusion bodies, allowing for high-recovery purification through temperature-variable purification.

[0016] In some embodiments of the present invention, the spidroin protein is a major ampullate gland spidroin protein (MaSp) or an artificial spidroin protein. Artificial spidroin proteins can include, for example, chimeric spidroin proteins and amyloid motif chimeric spidroin proteins. Natural major ampullate gland spidroin proteins (such as MaSp1) have poor heterologous expression and are difficult to form inclusion bodies at high expression levels. Artificially modified spidroin proteins generally have better expression. Therefore, considering the performance of spidroin proteins, artificial spidroin proteins are more preferred. More preferably, the original spidroin protein with the highest possible expression level is selected to ensure that more protein in the modified form forms inclusion bodies, which can achieve better results during temperature-variable purification.

[0017] Preferably, the (original) spider silk protein and the spider silk protein modified body have the following structural composition: N-terminal domain NT + repeat region rep domain + C-terminal domain CT; Optionally, the N-terminus is derived from the natural spider silk protein MaSp1 ( Euprosthenops australis ) and the C-terminal amino acid sequence is derived from the natural spider silk protein MiSp ( Araneus ventricosus The amino acid sequence of the N-terminal domain NT is shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal domain CT is shown in SEQ ID NO: 2.

[0018] In some embodiments of the present invention, provided are spidroin protein polyploids with a repeated core region, such as MaSp1-6rep (amino acid sequence as shown in SEQ ID NO: 3, i.e., the structural form of NT-6rep-CT); or spidroin protein polyploids with an amyloid motif as the original protein, such as Amy-6rep (amino acid sequence as shown in SEQ ID NO: 9, Amy-6rep is a modified form of the chimeric spidroin protein NT-2rep-CT, wherein the polyalanine motif AAAAAAA is replaced by the amyloid motif FGAILSS in the microcrystalline region, and the rep is doubled. The microcrystalline region may also be replaced by other amyloid motifs); or recombinant spidroin protein with IS-modified microcrystalline region (amino acid sequence as shown in SEQ ID NO: 17, wherein the polyalanine motif GAAAAAAA (G) is replaced by the isoleucine-serine motif IASASAAA (I) in the microcrystalline region, constituting the IS-modified recombinant spidroin protein IS-6rep).

[0019] The present invention provides different modified forms of the above-mentioned MaSp1-6rep, Amy-6rep, and IS-6rep as the original proteins, including modified forms of MaSp1-6rep MaSp1-ab-1 / 2 / 3 / 4 / 5 (the amino acid sequences of the rep domains in the repeat regions are shown in SEQ ID NOs: 4-8, respectively), modified forms of Amy-6rep Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-6rep (the amino acid sequences of the rep domains in the repeat regions are shown in SEQ ID NOs: 10-16, respectively), and modified forms of IS-6rep Ie-1 / 2-6rep (the amino acid sequences of the rep domains in the repeat regions are shown in SEQ ID NOs: 18-19, respectively). The doubling of the original proteins here is mainly for the purpose of increasing expression and facilitating the doubling of the rep domains of the modified forms. In addition, the modified forms can also be further subjected to rep doubling.

[0020] This type of modification significantly improves the water solubility of the protein, easing purification efforts and improving the tensile strength of the resulting material. The modified form can also be polyploid, ensuring optimal expression and spinning performance.

[0021] A third object of the present invention is to provide a method for purifying the aforementioned modified body. Specifically, a precipitate containing inclusion bodies of the modified spider silk protein is mixed with pure water, first heated to a first temperature, then cooled to a second temperature for incubation, and centrifuged to obtain a second precipitate, i.e., to purify the protein. Alternatively, the second precipitate is subjected to a heating-cooling incubation process that is repeated several times, and the purified protein is obtained after centrifugation.

[0022] The first temperature and the second temperature may vary based on the difference of the original spider silk protein. Usually, the first temperature is in the range of 45-70°C, and the second temperature is in the range of 15-25°C.

[0023] The present invention uses a temperature-variable method that combines heating (e.g., 50°C) with slow cooling (e.g., 20°C) to purify recombinant spider silk protein inclusion bodies at the level of inclusion bodies. The principle is that heating causes a phase transition in the recombinant spider silk protein: the cross-links between the inclusion bodies become loose, allowing the release of impure E. coli proteins trapped within them. A temperature of 50°C also ensures that the impure proteins are not completely inactivated and form aggregates in the precipitate. Slowly cooling the temperature to 20°C maintains the stability of the protein structure and allows the inclusion bodies to reaggregate, ultimately yielding pure spider silk protein inclusion bodies free of impure proteins.

[0024] After spider silk protein is modified by the modification method of the present invention, the obtained modified body can be directly subjected to phase change by simple temperature change, that is, bionic spider silk protein with a purity of 90-99% can be efficiently obtained, with a recovery rate of more than 98% and simple operation.

[0025] The fourth object of the present invention is to provide the use of the above-mentioned spider silk protein modified body in the preparation of tissue engineering materials, biomacromolecule fixation materials, biosensors, and protective materials.

[0026] Through analysis and design, the present invention discovered that by introducing acidic and basic amino acids into the flexible region of spider silk protein to form salt bonds, a stronger molecular force can be formed to stabilize the β-sheet structure of spider silk protein and enhance the self-assembly properties of the silk protein. The introduction of acidic and basic amino acids also enhances the polarity of spider silk protein, significantly reducing the degree of tangling during protein formation at higher temperatures, resulting in a phase transition. Based on variable temperature purification and simple centrifugation, a high-purity, high-recovery spider silk protein modified form was successfully obtained. Furthermore, when the acid-base ratio in the flexible region is close, the modified form can have superior mechanical properties compared to the original protein. This protein purification method is conducive to its widespread application within the industry.

[0027] The present invention further provides an expression vector or an integrative vector comprising a gene encoding the aforementioned modified organism. The expression vector or integrative vector is a plasmid, a bacteriophage, a virus, or a host cell. The host cell can be a prokaryotic or eukaryotic cell, such as Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma. Escherichia coli is preferred.

[0028] The present invention has the following beneficial effects: (1) The present invention successfully obtains a class of typical main ampullate spidroin proteins and spidroin protein flexible region modified forms that can be easily purified by semi-rational design of the flexible region of spidroin proteins (including typical main ampullate spidroin proteins and artificial spidroin proteins) and by changing the protein polarity through substitution of acidic and basic amino acid residues. This class of modified forms does not require the use of a histidine tag for purification, but can be directly purified by a simple two-step temperature-variable treatment to efficiently obtain biomimetic spidroin proteins with a purity between 90% and 99%, which is simple to operate and has a high yield.

[0029] (2) The purification method provided by the present invention does not require the addition of denaturants such as urea or dialysis. The crushed precipitate can be directly freeze-dried after a simple heating and centrifugation treatment. It has the advantages of simple process, high separation efficiency, time saving, cost reduction, etc., and is easy to scale up industrially.

[0030] (3) When the acid-base ratio of the flexible region is close after replacing amino acids, the material preparation process of the modified body is simple and the tensile strength is significantly improved, which lays the foundation for the widespread application of biomimetic spider silk protein in the industrial field or in the field of functional materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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.

[0032] Figure 1 Schematic diagram of the modification of the polyploid protein MaSp1-6rep of the spider silk protein MaSp1 in the examples.

[0033] Figure 2 This is the protein expression of MaSp1-6rep and its transformed form.

[0034] Figure 3 Figure a shows the expression of 6-48Rep polyploids of the artificial spider silk protein amyloid motif spider silk protein, taking Amy-ab-5 as an example, according to the present invention; and Figure b shows the SDS-PAGE electrophoresis analysis of the artificial spider silk protein amyloid motif spider silk protein 12Rep control group Amy-12rep and its flexible region acid-base amino acid modified form.

[0035] Figure 4 The present invention provides an SDS-PAGE analysis of the artificial spider silk protein amyloid motif spider silk protein and its flexible region modified amyloid motif spider silk protein with equal ratio of acidic and basic amino acids (1:1) purified by the method described in the present invention; wherein a is the inclusion body lotion purification result of the amyloid motif spider silk protein (Amy-12rep) of the control group, b is the protein purification result of the amyloid motif chimeric spider silk protein flexible region modified Amy-ab-5-12rep, c is the protein purification result of the amyloid motif chimeric spider silk protein flexible region modified Amy-ab-2-12rep, and d is the protein purification result of the amyloid motif chimeric spider silk protein flexible region modified Amy-ab-3-12rep.

[0036] Figure 5 The results of temperature-variable purification of the artificial spider silk protein amyloid motif spider silk protein Amy-ab-7-12rep modified with different ratios of acidic and basic amino acids (3:1) in the flexible region according to the method of the present invention.

[0037] Figure 6 The present invention relates to SEM images of the fiber surface and cross-section of the artificial amyloid motif chimeric spider silk protein flexible region modified body.

[0038] Figure 7 This is a summary diagram of the tensile strength of artificial amyloid motif chimeric spider silk protein flexible region modified fibers involved in the present invention.

[0039] Figure 8 The results of temperature-variable purification of the modified forms Ie-1-6rep and Ie-2-6rep obtained using the method of the present invention with IS-6rep as the original spider silk protein. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific implementation methods.

[0041] The examples further illustrate the technical solution of the present invention by taking the modification of the flexible region of MaSp1 protein and the modification of the flexible region of the artificial spider silk protein amyloid motif spider silk protein Amy-6rep as examples.

[0042] Example 1 This example uses the common MaSp1 protein as an example to illustrate the modification method of the easily purified spider silk protein of the present invention.

[0043] Because native MaSp1 protein has a weak ability to express heterologously, resulting in low expression levels and an inability to form inclusion bodies, this example illustrates the present invention using the polyploid MaSp1-6rep (amino acid sequence shown in SEQ ID NO: 3), which contains six repeats of the MaSp1 core region. The spider silk protein MaSp1 originally contains a small number of charged amino acids in its flexible region, but these are all basic amino acids (arginine R). The proportion of charged amino acids is also very low, making it difficult to purify using temperature-controlled purification.

[0044] 12-28 acidic and basic amino acid residues were replaced in the flexible regions on both sides of the microcrystalline region of the typical main ampulla spider silk protein to obtain the modified forms MaSp1-ab-1, MaSp1-ab-2, MaSp1-ab-3, MaSp1-ab-4, and MaSp1-ab-5. The specific modification methods are shown in Table 1 below: Table 1 Transformation methods of MaSp1-6rep transformants

[0045] The replaced amino acid residues in the table, taking 8G / (2K, 6E) as an example, mean that 8 "G" (glycine, neutral) are replaced, 2 are replaced by "K" (lysine, basic), and 6 are replaced by "E" (glutamic acid, acidic).

[0046] Sequence comparison of MaSp1-6rep and the modified MaSp1-ab-1 / 2 / 3 / 4 / 5 Figure 1 As shown, the amino acid sequences of the repeat region rep domain of MaSp1-ab-1 / 2 / 3 / 4 / 5 are shown in SEQ ID NOs: 4-8, respectively. To facilitate a more intuitive understanding of the modification method, the slash " / " before and after respectively represents the original amino acid residue and the replaced amino acid residue. For example, "G / K" indicates that the original amino acid is "G" and the replaced amino acid is "K". "SG / DE" indicates that two consecutive amino acid residues are replaced, that is, "SG" is replaced by "DE". When replacing amino acids, glycine is preferably selected as the replacement.

[0047] The modified strains MaSp1-ab-1, MaSp1-ab-2, MaSp1-ab-3, MaSp1-ab-4, and MaSp1-ab-5 obtained above were transformed into Escherichia coli BL21 (DE3) to obtain their expression strains. Each strain was inoculated into 50 mL of LB liquid medium, and ampicillin was added to a final concentration of 100 μg / mL. The strains were cultured overnight at 180 rpm and 37°C. The overnight cultured seed solution was inoculated into 300 mL of fresh LB liquid medium at a 2% inoculum size. The strains were cultured at 180 rpm and 37°C for 1.5 h (OD600 was 0.6-0.8). Lactose was then added for induction (final concentration 20 g / L), and expression was induced at 35°C for 48 h.

[0048] Take the fermentation liquid for induced expression, centrifuge at 8000rpm for 15 minutes, discard the supernatant, then resuspend the bacteria in 60mL of pure water, ultrasonically disrupt for 30 minutes, and centrifuge at 12000 rpm for 10 minutes to obtain the supernatant (S). Discard the supernatant and add 60mL of pure water to the broken precipitate to suspend and mix to obtain the insoluble precipitate (IB). Perform SDS PAGE electrophoresis detection, the concentration of the concentrated gel is 4%, the concentration of the separation gel is 12.5%, the sample and the loading buffer are mixed in a ratio of 3:1, and the reaction is carried out in a boiling water bath for 5-10 minutes for loading electrophoresis. Set the initial voltage of the electrophoresis instrument to 80V. When the sample moves to the separation gel, increase the voltage to 180V until the sample moves to the bottom of the electrophoresis tank and end the electrophoresis. The expression is as follows Figure 2 As shown in the figure, it can be seen that after the flexible region acid-base modification method described in the present invention was used to modify MaSp1-6rep, the expression level of the main ampulla spidroin protein in the flexible region modified group was significantly increased compared to the control group (MaSp1-6rep), with the expression level of spidroin protein per unit volume accounting for 8%-12% (w / w). However, at this expression level, the protein mainly exists in a soluble form, which is insufficient for purification through temperature-induced phase transition.

[0049] Example 2 This example uses a typical artificial spider silk protein amyloid motif spider silk protein Amy-6rep as an example to illustrate the modification method. The amino acid sequence of Amy-6rep is shown in SEQ ID NO:9.

[0050] This experiment used the six-repeat amyloid motif spider silk protein sequence Amy-6rep embedded in the plasmid vector pSE380 as a template (Lin, B. et. al. Efficient Biosynthetic Fabrication of Spidroins with High Spinning Performance, Adv. Sci., 11, pages 2400128 (2024)). While maintaining a nearly uniform content of acidic and basic amino acids in the flexible region (acidic (base) / basic (acidic) amino acids ≈ 1:1.3, preferably 1:1), genetic modification was used to mutate some amino acids in the flexible region of the spider silk protein (mainly glycine, with a small amount of arginine, tryptophan, serine, and glutamate) into acidic or basic amino acids. This resulted in a class of modified proteins with enhanced flexible region binding force, as shown below: Table 2 Transformation methods of Amy-6rep transformants

[0051] The amino acid sequences of the rep domains of the Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-6rep repeat regions are shown in SEQ ID NOs: 10-16, respectively.

[0052] The amino acid sequence of the 6-rep repeat region was doubled using homozygous enzyme ligation technology (for details, please refer to the previous application CN119930779A), and the corresponding Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-12rep, Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-24rep and Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-48rep were obtained.

[0053] In this example, the amino acid sequence (mainly glycine) in the flexible region of spider silk protein is replaced with a series of acidic and alkaline charged amino acids, thereby changing the polarity of the original silk protein sequence and obtaining an amyloid protein modification that can be efficiently purified directly through a simple temperature change.

[0054] Example 3 Obtaining artificial amyloid motif spider silk protein and its flexible region modified body The recombinant expression plasmid of Amy-12rep (an amyloid motif chimeric spider silk protein without a Ni-NTA-purified 6*His tag at the N-terminus) was constructed using pSE380 as the vector: Using Amy-6rep, a spider silk protein sequence with a published six-repeat amyloid motif core region, as a template (Lin, B. et. al. Efficient Biosynthetic Fabrication of Spidroins with HighSpinning Performance, Adv. Sci., 11, pages 2400128 (2024)), 6rep was inserted into the SpeI site of the pSE380 vector using isoenzymes (SpeI, NheI) to obtain the pSE380 plasmid expression vector of Amy-12rep, which was then transformed into Escherichia coli BL21(DE3) to obtain the Amy-12rep expression strain. Amy-12rep and several flexible regions described in Example 1, modified strains of the amyloid motif chimeric spider silk protein flexible region modified by introducing acidic and alkaline amino acids in equal proportions, were inoculated into 50 mL of LB liquid medium, and ampicillin was added to a final concentration of 100 μg / mL, and cultured overnight at 180 rpm and 37°C; the overnight cultured seed liquid was inoculated into fresh 300 mL of LB liquid medium at a 2% inoculum size, and cultured at a constant temperature of 180 rpm and 37°C for 1.5 h (OD600 was 0.6-0.8), and lactose was added for induction (final concentration 20 g / L), and expression was induced at 35°C for 48 h.

[0055] Take the induced expression fermentation broth and centrifuge it at 8000 rpm for 15 minutes. Discard the supernatant, then resuspend the cells in 60 mL of purified water and disrupt them by ultrasonication for 30 minutes. After disruption, centrifuge at 12000 rpm for 10 minutes to obtain the supernatant (S). Discard the supernatant and the precipitate, resuspend and mix in 60 mL of purified water to obtain the insoluble precipitate (IB). Perform SDS-PAGE electrophoresis with a 4% stacking gel and a 12.5% ​​separating gel. Mix the sample and loading buffer in a 3:1 ratio, incubate in a boiling water bath for 5-10 minutes, and then load the sample for electrophoresis. Set the initial voltage of the electrophoresis instrument to 80 V. As the sample moves to the separating gel, increase the voltage to 180 V. Continue electrophoresis until the sample reaches the bottom of the electrophoresis tank. The expression of Amy-ab-2 / 3 / 4 / 5 / 6-12rep and Amy-ab-5-6 / 12 / 24 / 48rep with acid-base amino acid modifications in equal ratios (1:1) in the flexible region, and Amy-ab-7-12rep with acid-base amino acid modifications in different ratios (3:1) in the flexible region were the same as described above.

[0056] The results are as follows Figure 3As shown in Figure a, the molecular weights of Amy-ab-5 polyploids 6 to 48 rep are 43.3, 58.1, 90.3, and 178.9 kDa, respectively. There are obvious bands at the corresponding protein molecular weights. Ultimately, under the premise of ensuring its expression level, the corresponding 12 rep was selected in subsequent experiments. Figure 3 The molecular weight of the amyloid-like motif spider silk protein (Amy-12rep) in the control group (unmodified flexible region) shown in (b) is 58.3 kDa. The molecular weights of the several modified flexible region amyloid-like motif spider silk proteins (Amy-ab-2-12rep, Amy-ab-3-12rep, Amy-ab-4-12rep, Amy-ab-5-12rep, and Amy-ab-6-12rep) described in Example 1, in which the flexible region was modified with equal ratios of acidic and basic amino acids, are 60.2, 58.6, 59.2, 59.1, and 58.5 kDa, respectively. Another modified flexible region amyloid-like motif spider silk protein, Amy-ab-7-12rep, in which the flexible region was modified with acidic and basic amino acids in varying ratios (3:1), has a molecular weight of 57.8 kDa. After induction, both the control group and the modified flexible region group exhibited distinct bands at the corresponding protein molecular weights, consistent with the designed results. This indicates that both proteins were successfully induced for expression.

[0057] Example 4 An efficient and simple method for purifying artificial amyloid motif chimeric spider silk protein flexible region modified body In Example 1, by semi-rational design of the flexible region of the amyloid motif spider silk protein sequence Amy-6rep, introducing acidic and alkaline charged amino acids and improving its sequence composition, a class of amyloid motif chimeric spider silk protein flexible region modified forms were successfully obtained, which can be efficiently and easily purified. This example provides a purification method using variable temperature treatment for these modified forms of spider silk protein. The specific steps are as follows: Taking the amyloid motif chimeric spider silk protein flexible region modified Amy-ab-5-12rep as an example, as described in Example 2, 50 mL of induced expression fermentation broth was taken and centrifuged at 8000 rpm for 15 minutes. The supernatant was discarded, and the cells were resuspended in 50 mL of purified water and disrupted by ultrasonication for 30 minutes. After disruption, the cells were centrifuged at 12000 rpm for 10 minutes, and the supernatant (S) was discarded. The disrupted precipitate was added to 25 mL of purified water and suspended and mixed to obtain a precipitate containing spider silk protein inclusion bodies (IB). Protein samples before and after purification were analyzed by SDS-PAGE electrophoresis as shown in the following figure. Figure 4 shown.

[0058] Two-step temperature change treatment of metal bath: 1. Mix precipitate IB with an equal volume of pure water in a 50 mL centrifuge tube. Adjust the temperature to 50°C and slowly mix and heat for 20 minutes (50°C, 100 rpm, 20 minutes). Then lower the temperature to 20°C and incubate for 30 minutes. After the reaction is complete, centrifuge again and remove the supernatant to obtain precipitate I (IB-I) containing spider silk protein inclusion bodies.

[0059] 2. The precipitate containing spidroin protein inclusion bodies (IB-I) obtained in the previous step was subjected to a similar procedure: slowly heating at 60°C for 10-20 minutes, then lowering the temperature to 20°C and incubating for 30 minutes. After the reaction, the precipitate was centrifuged again and the supernatant removed to obtain precipitate II containing spidroin protein inclusion bodies (IB-II). (The remaining modified forms were treated similarly; the heat treatment time could be extended as appropriate depending on the protein content.) Finally, the collected precipitates containing different spidroin protein inclusion bodies were verified by SDS-PAGE. The results were as follows: Figure 4 shown.

[0060] 3. Purity and recovery The total amount of protein and the proportion of each component were scanned in grayscale using Quantity One version 4.62 software (Bio-Rad, USA) and ImageJ, and the average value was calculated three times.

[0061] Control group: Amy-12rep protein: The purity after one urea washing and purification is about 33%, and the protein loss is serious, such as Figure 4 As shown in a.

[0062] Flexible zone equal ratio (1:1) acid-base amino acid modification group: Amy-ab-5-12rep protein: The purity after purification is about 97%, and the recovery rate is as high as over 99%. Figure 4 As shown in b.

[0063] Amy-ab-2-12rep protein: The purity after purification is about 93%, and the recovery rate is as high as over 95%. Figure 4 As shown in c.

[0064] Amy-ab-3-12rep protein: The purity after purification is about 95%, and the recovery rate is as high as over 98%. Figure 4 As shown in d; Modification groups of acid-base amino acids with different ratios (3:1) in the flexible region: Amy-ab-6-12rep protein: After temperature-variable purification, the purity is about 97%, and the recovery rate is as high as over 98%. Figure 5 As shown in .

[0065] Combined with the comparative analysis of the low heterologous expression level of natural MaSp1 protein (Example 1), spider silk protein expressed in inclusion bodies is only suitable for the temperature-variable purification method described in the present invention when the accumulation of spider silk protein expression reaches a specific threshold (content per unit volume ≥15% (w / w)).

[0066] That is, the applicable conditions of the temperature-variable purification method are: when the proportion of recombinant spider silk protein in the unit volume of Escherichia coli is above 15% (w / w), the target protein can be expressed in the form of inclusion bodies; preferably, the temperature-variable purification effect is best when the content is greater than 20% w / w.

[0067] Example 5 Preparation and SEM analysis of artificial amyloid motif chimeric spider silk protein fibers A series of 12rep polyploids of amyloid motif chimeric spider silk protein flexible region modified bodies (Amy-ab-1 / 2 / 3 / 4 / 5 / 6 / 7-6rep, hereinafter referred to as ab-1 / 2 / 3 / 4 / 5 / 6 / 7) were selected, and the unmodified amyloid motif chimeric spider silk protein (Amy-12rep) was referred to as ab-0. Chimeric biomimetic spider silk protein materials were prepared from them respectively, and their lyophilized powder was dissolved in HFIP at a concentration of 20% w / v to prepare a spinning solution. Then, a microfluidic wet spinning machine (Janus New-Materials Co., Ltd) was used to pump it into an ethanol coagulation bath at a flow rate of 3 mL / h using a 24G injection needle, and the solution was collected at a uniform speed using a reel. Finally, the fibers were dried and peeled off at room temperature to obtain dry group fibers, and replaced in pure water for 20 minutes to obtain a series of spider silk protein flexible region modified body fibers with different tensile strength.

[0068] Use clean tweezers to cut a small amount of fiber sample (about 3-5 mm in length), and avoid touching it directly with your hands to prevent contamination. If there is grease or contaminants on the fiber surface, ultrasonically clean it with anhydrous ethanol and deionized water for 5 minutes in sequence, and dry it at room temperature for later use. Then take a small amount of fiber sample and fix it on the sample stage with conductive glue. Non-conductive samples need to be sprayed with gold. Use SEM (such as 5-10kV acceleration voltage) to observe the surface morphology, take images at different magnifications, and analyze fiber diameter, defects and other characteristics. Some results are shown below. Figure 6 As shown, all fibers formed fine fibers with a diameter of approximately 50 μm. The flexible region modified ab-1 fibers were the best, with a smooth, dense surface and uniform structure, without obvious pores or exposed microfibrils. This indicates that the introduction of acid-base amino acid residue pairs into the core flexible module resulted in the formation of different macroscopic fiber morphologies of artificial chimeric spider silk proteins. Compared with the original control group, the flexible region modified fibers showed a denser morphology and better molecular chain orientation.

[0069] Example 6 Characterization of the Mechanical Properties of Artificial Amyloid Motif Chimeric Spider Silk Protein Flexible Region Transformed Fibers The above fibers were used to prepare water-soaked and vacuum-dried groups, respectively, to obtain a series of spider silk protein flexible region modified fibers with different tensile strengths. At 25°C and 50% humidity, a single-arm tensile tester was used to test the tensile strength of several fiber materials under different conditions. The tensile strength of the modified fibers of the flexible region of the spider silk protein with different amyloid motifs was determined by measuring the ratio of the force applied to the cross-sectional area of ​​the material. The results are shown in Figure 2. Figure 7 shown.

[0070] It can be seen that the introduction of charged acid-base amino acid residue pairs in different numbers, combinations, and distances within the core flexible module allows the artificial chimeric spider silk protein to form fiber materials with different tensile strengths on a macro scale, significantly improving the mechanical properties of the material (the mechanical properties of the optimal wet fiber group increased by approximately 20 times, and the mechanical properties of the optimal dry fiber group increased by approximately 10 times). This has broadened new ideas for the functional design of recombinant spider silk protein sequences.

[0071] Example 7 In this example, the recombinant spider silk protein (IS-6rep) modified with the microcrystalline region IS was used as the original protein for flexible region modification. Acquisition of IS-6 rep gene: Synthesize spider silk proteins using the repeat region of the typical major ampullate gland spider silk protein MaSp1 as a template (Artificial Spider Silk Based Programable Woven Textile for Efficient Wound Management. Advanced Functional Materials , 2022, 32, 2107707.) The original Rep residue was modified from GAAAAAAA (G) to IASASAAA (I). The specific amino acid sequence is shown in SEQ ID NO:17. The IS-6rep gene, constructed from the cloning vector pUC57, was synthesized by GE Healthcare (Chicago, IL). The IS-6rep gene was inserted into the pSE380 expression vector or an integrative vector using a one-step cloning kit to generate the recombinant expression plasmid IS-e-0-6rep, which lacks the N-terminal 6*His tag and is suitable for Ni-NTA purification. It is hereby referred to as Ie-0-6rep.

[0072] Using Ie-0-6rep embedded in the plasmid vector pSE380 as a template, site-directed mutagenesis was used to introduce charged acidic and basic amino acid residues in varying molar ratios, combinations, and spacings within the flexible module of the spider silk protein core region, resulting in the modified Ie-1-6rep and Ie-2-6rep variants. The acidic / basic amino acid molar ratios introduced into the Ie-1 / 2-6rep flexible regions were 1 / 1 and 7 / 9, respectively. The amino acid sequences of the rep domains in these repeat regions are shown in SEQ ID NOs:18-19, respectively. Polyploids of these variants all contained identical substitutions with doubled rep numbers. These polyploids were prepared using existing homozygous enzyme ligation technology (for details, see prior application CN119930779A).

[0073] The expression of the transformed Ie-1 / 2-12rep was performed using the methods shown in Examples 3 and 4, respectively. The results of purification using the above temperature-variable purification method are shown in FIG. Figure 8 As shown, the purity and recovery are as follows: Ie-1-6rep protein: The purity after purification is about 91%, and the recovery rate is as high as over 96%; Ie-2-6rep protein: The purity after purification is approximately 95%, and the recovery rate is as high as over 95%.

[0074] The present invention realizes the simple and efficient purification of artificial spider silk protein and enhances the tensile strength of the prepared spider silk material based on the replacement of acidic and basic amino acid residues in the flexible region. By introducing acidic and basic charged amino acid pairs, its sequence composition is improved and the interaction force between peptide chains is enhanced, thereby obtaining a class of spider silk protein flexible region modified bodies with significantly enhanced tensile strength. The introduction of such acidic and basic amino acids changes the polarity of the protein, and the efficient and simple purification of artificial spider silk protein can be achieved by temperature change. They can directly capture recombinant spider silk protein from broken precipitates, and only two steps of temperature change treatment are required to obtain recombinant spider silk protein with a purity greater than 95%. The operation is simple and the yield is high, which is conducive to its industrial production. This lays the foundation for the sequence design and wide application of biomimetic spider silk protein in the industrial scope or in the field of functional materials.

Claims

1. A method for modifying spider silk protein that is easily purified, characterized in that: Replacing several amino acid residues in the flexible regions on both sides of the spider silk protein microcrystal region with acidic or basic amino acids so that the proportion of charged amino acids in the flexible regions is ≥7%, thereby obtaining a modified spider silk protein; The spider silk protein modification body is purified in the form of inclusion bodies through a temperature-changing process of heating and cooling.

2. The transformation method according to claim 1, characterized in that: After amino acid substitution in the flexible region, the molar ratio of acidic amino acids to basic amino acids is 3:1 to 1:3, preferably 1:

1.

3. The transformation method according to claim 1, characterized in that: The number of substituted amino acid residues ranges from 4 to 40.

4. The transformation method according to claim 1, characterized in that: The amino acid residues are substituted to avoid the GGY motif in the flexible region.

5. The transformation method according to claim 1, characterized in that: The replaced amino acid residues are located in flexible regions close to the microcrystalline region on both sides of the microcrystalline region; the closeness means: the distance from the microcrystalline region is ≤4 amino acids.

6. The transformation method according to claim 1, characterized in that: The replacement of the amino acid residues is mainly replacement of glycine.

7. The modified spider silk protein obtained by the modification method according to any one of claims 1 to 6, wherein the expression level per unit volume of the modified spider silk protein is ≥15% (w / w).

8. The spider silk protein modification according to claim 7, characterized in that: The spider silk protein is a main ampullate gland spider silk protein or an artificial spider silk protein.

9. The method for purifying the modified spider silk protein according to claim 7, characterized in that: The precipitate containing the modified spider silk protein inclusion body is mixed with pure water, first heated to a first temperature, then cooled to a second temperature for incubation, and centrifuged to obtain a second precipitate, i.e., the purified protein; Alternatively, the second precipitate is subjected to a heating-cooling incubation process that is repeated several times, and the purified protein is obtained after centrifugation.

10. Use of the modified spider silk protein according to claim 6 in the preparation of tissue engineering materials, biomacromolecule fixation materials, biosensors, and protective materials.

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