Method for regulating hydrophilicity and hydrophobicity of chimeric spider silk protein by adopting amyloid protein motif

Abstract

The invention relates to a method for regulating hydrophilicity and hydrophobicity of chimeric spider silk protein by adopting an amyloid protein motif. The spider silk protein comprises a core repeat region REP structural domain derived from main kettle gland silk protein; a polyalanine motif of a microcrystalline region in an REP structural domain is replaced by an amyloid protein motif with different total average hydrophilicity values, and the total average hydrophilicity value of the amyloid protein motif is between-3.133 and 0.957. The amyloid protein motif chimeric spider silk protein replaced by amyloid polypeptides with different total hydrophilicity average values forms a material with corresponding hydrophilicity and hydrophobicity macroscopically, so that protein design aiming at the hydrophilicity and hydrophobicity requirements of the material is facilitated, and spider silk protein materials with different functions are obtained; and a foundation is further laid for the industrial production of the recombinant spider silk protein and the wide application of the recombinant spider silk protein in the field of functional materials.

Description

Technical Field

[0001] The invention belongs to the technical field of functional gene modification, and in particular relates to a method for regulating the hydrophilicity and hydrophobicity of chimeric spider silk proteins by using amyloid protein motifs. Background Art

[0002] Spider silk is the natural fiber with the strongest known composite properties. Its tensile strength is more than five times that of steel of the same quality. It also has extremely high toughness, excellent biocompatibility, biodegradability and shape memory properties. Therefore, spider silk has great application potential in many fields such as biomedicine, tissue engineering and materials engineering.

[0003] Major Ampullate Spider Silk (MASS) is the dragline silk of spiders. It spreads radially on the spider web to withstand the tension of the spider web itself and the impact of prey. It has the highest tensile strength among animal fibers and is the main type of glandular silk that researchers focus on. MASS is mainly composed of Major Ampullate Spidroin (MaSp), which is rich in alanine and glycine, with the content of the two amino acids reaching 50%. The MaSp repeat core region has two characteristic motifs, namely the microcrystalline region of oligoalanine (A)n (n=4−15) motif and the flexible region (also called intercalated region) of glycine-rich GGX / GPGXX / GPGPX (X=Y, L or Q) motif. The oligoalanine (A)n motif mainly forms antiparallel β-folds in glandular silk, which is the molecular basis of the β-fold microcrystalline domain of glandular silk (referred to as microcrystalline region) and is also the key factor for the high tensile strength of spider silk. The repeating unit of MaSp is composed of a microcrystalline region and a flexible region. Since the number of these regions is usually as high as hundreds, the molecular weight of natural MaSp is generally as high as 200−350 kDa. Due to the high molecular weight of the major ampullate gland silk protein, the gene encoding the major ampullate gland silk protein has a high GC content, a highly repetitive amino acid sequence in the core region, and a high content of specific amino acids. These characteristics bring great difficulties to heterologous expression. Recombinant spider silk proteins designed based on the characteristic motifs of the major ampullate gland silk protein are still difficult to replicate the mechanical properties of natural spider silk, and the heterologous expression yield is difficult to meet the needs of industrialization. Studies on the relationship between the sequence, structure and spinning properties of spider silk proteins have shown that there is still a lot of room for improvement in the heterologous expression yield and spinning strength of recombinant spider silk proteins.

[0004] Existing studies have shown that the density of the microcrystalline region of the major ampullate gland silk protein is positively correlated with its self-assembly and filament strength ( Adv. Mater, 30: 1870250), therefore, we propose that the increase in the density of the microcrystalline regions of the major ampullate gland silk protein may increase the probability of assembly between the microcrystalline regions, thereby promoting the formation of β-folded microcrystalline structures, so as to increase the self-assembly performance of the protein to obtain a strategy for recombinant spider silk protein with improved spinning strength. In addition, inspired by the β-folded structure in spider silk proteins, the study discovered a class of amyloid motifs with a high β-folded structural tendency. Amyloid motifs can usually form highly ordered antiparallel β-folded sheets, and adjacent β sheets further form spatial zipper structures through electrostatic interactions, π-π stacking or hydrophobic effects, which gives these amyloid nanofibers extraordinary mechanical properties ( Nature 435, 773–778 (2005). Similarly, two or more β-pleated sheets in the microcrystalline region of natural spider silk proteins also form a tight complementary interface, thereby promoting the stability of the microcrystalline region and enhancing the performance of glandular silk. Zhang et al. from MIT (ACS Nano 2021, 15, 7, 11843–11853) introduced amyloid peptide motifs to replace the polyalanine motifs in the microcrystalline region of spider silk protein. The strength of the synthesized 64-fold and 128-fold chimeric spider silk proteins spun fibers reached about 0.98 GPa and the toughness reached about 161 MPa, which are higher than the recombinant spider silk proteins with the same molecular weight. The strength (2.8−3.4 times) and toughness (1.5−2.6 times) are higher, indicating that the interaction between the folded sheets of the cross-β-spine characteristics is strong, and it is easy to form a β-microcrystalline structure with stronger tensile strength. This study obtained for the first time a bionic spider silk with performance comparable to that of natural spider silk, but there is still a problem of low yield of amyloid protein motif chimeric bionic spider silk protein. Our laboratory (Adv. Sci. 2024, 11,2400128) used amyloid peptides to replace the polyalanine motif in the microcrystalline region of spider silk protein. The obtained small molecular weight amyloid protein motif chimeric biomimetic spider silk protein effectively solved the problem of low yield of amyloid protein motif chimeric biomimetic spider silk protein, and obtained nano spider silk protein fibers with good spinning performance through electrospinning. This research result greatly promoted the sequence design and application of biomimetic spider silk protein, but only discussed the confirmation of spider silk protein microcrystalline region replacement for efficient expression and protein assembly performance. Summary of the invention

[0005] The first object of the present invention is to provide a method for regulating the hydrophilicity and hydrophobicity of chimeric spider silk proteins by using amyloid protein motifs.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for regulating the hydrophilicity and hydrophobicity of a chimeric spider silk protein using an amyloid protein motif, wherein the spider silk protein comprises a core repeat region REP domain derived from a major ampullate gland silk protein; The polyalanine motif in the microcrystalline region of the REP domain is replaced by an amyloid protein motif with different overall average hydrophilicity, and the overall average hydrophilicity of the amyloid protein motif is between -3.133 and 0.957.

[0007] As a preferred embodiment, the amyloid protein motif is selected from: Any one of PDB ID: 5MGQ, ​​PDB ID: 8ENQ, PDB ID: 7LC9, PDB ID: 6G8D _A, PDB ID: 8ENR, PDB ID: 5K2F_A.

[0008] As a preferred embodiment, the method further comprises doubling the amino acid sequence of the repeat region REP of the spider silk protein after replacing the polyalanine motif, and the doubling number is between 3rep-48rep. Doubling the modified repeat segment within the range of 3rep-48rep is conducive to the improvement of protein self-assembly and spinning performance.

[0009] Furthermore, the doubling number of the amino acid sequence of the repeat region REP is 12 rep. The preferred 12 rep balances the self-assembly and spinning properties of the protein and has the best comprehensive performance.

[0010] As a preferred embodiment, the method further comprises shortening the polyalanine motif in the microcrystalline region into an oligoalanine (A)n motif with n≤7, encrypting the oligoalanine (A)n motif, and then replacing all oligoalanine motifs with the amyloid protein motif.

[0011] The oligoalanine (A)n motif encryption refers to: increasing the number of oligoalanine (A)n motifs when the sequence length is relatively fixed. Shortening the length of the oligoalanine (A)n motif and encrypting it will help improve the self-assembly performance and spinning performance of the protein. It should be noted that due to the different lengths and densities of the oligoalanine (A)n motifs in the core REP domain of different spider silk proteins, some spider silk proteins with shorter oligoalanine (A)n motifs and higher densities do not need to be shortened or encrypted.

[0012] As a preferred embodiment, the spider silk protein further comprises an N-terminal domain NT and a C-terminal domain CT, which form a chimeric spider silk protein with the repeat region REP domain; Wherein, 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 )'s C-terminal amino acid sequence.

[0013] The second object of the present invention is to provide a spider silk protein modification constructed by the above method.

[0014] Preferably, the modified body has the following structural composition: N-terminal domain NT + repeat region REP domain + C-terminal domain CT; Wherein, the amino acid sequence of the N-terminal domain NT is as shown in SEQ ID NO:2, the amino acid sequence of the C-terminal domain CT is as shown in SEQ ID NO:4, and the repeat region REP domain is an Amy-3rep sequence obtained by replacing the polyalanine motif AAAAAAA in the amino acid sequence shown in SEQ ID NO:3 with an amyloid protein motif, or a polyploid (Amy-3rep)n sequence of the Amy-3rep sequence; n represents the number of repetitions of the Amy-3rep sequence; Preferably, n is 1 to 16. More preferably, n=4.

[0015] Furthermore, the nucleotide sequence of the N-terminal domain NT is as shown in SEQ ID NO:8, the nucleotide sequence of the C-terminal domain CT is as shown in SEQ ID NO:10, and the repeat region REP domain is an Amy-3rep sequence obtained by replacing the nucleotide sequence corresponding to the polyalanine motif AAAAAAA in the nucleotide sequence shown in SEQ ID NO:9 with the nucleotide sequence corresponding to the amyloid protein motif, or a polyploid (Amy-3rep)n sequence of the Amy-3rep sequence; n represents the number of repetitions of the Amy-3rep sequence; The third object of the present invention is to provide an expression vector comprising the above spider silk protein modification. The expression vector can be a plasmid, a bacteriophage or a virus.

[0016] The fourth object of the present invention is to provide an engineered strain comprising the above spider silk protein modification. The host cell of the engineered strain can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma. Preferably, Escherichia coli.

[0017] The fifth object of the present invention is to provide the use of the above spider silk protein modification in the preparation of spider silk materials with different hydrophilicity.

[0018] Furthermore, the spider silk material prepared by the modified body can be used in the fields of medicine, tissue engineering and materials engineering, such as for the development and manufacture of new bionic functional materials such as biomembranes, artificial skin, degradable skeletons, bionic blood vessels, nanofiber scaffolds for the treatment of cardiovascular diseases, knee cartilage tissue, small-caliber artificial vascular scaffolds, medical sutures, new biomaterials for anti-infection and promoting healing, fixation materials for biomacromolecules, biosensors, etc., which use spider silk fibers as basic materials.

[0019] The present invention replaces the microcrystalline region of the chimeric spider silk protein based on a series of amyloid protein motifs with different average total hydrophilicity, designs and obtains a series of high-yield amyloid protein motif chimeric biomimetic spider silk proteins with material functionalization, and finally forms amyloid protein motif chimeric biomimetic spider silk protein materials with corresponding hydrophilicity and hydrophobicity, realizes the development of materials requiring different hydrophilicity, and further lays the foundation for the industrial production of recombinant spider silk protein and its wide application in the field of functional materials.

[0020] In addition, in a preferred embodiment, the present invention shortens the length of the polyalanine motif to reduce the alanine content and the GC content of the corresponding gene, improves the density of the spider silk protein microcrystalline region, and further improves the heterologous expression amount and spinning performance of the spider silk protein. On this basis, the modified repeat segment is doubled within the range of 3rep-48rep and combined with Euprosthenops australis The N-terminal segment of MaSp1 and the C-terminal segment of MiSp1 from Araneus ventricosus are combined into a chimeric spider silk protein, which is conducive to the self-assembly of the protein and the improvement of spinning performance. The cross-β-spine spatial zipper structure unique to the amyloid protein motif can stabilize the β-folding structure in the amyloid protein motif chimeric bionic spider silk protein, increase the self-assembly performance of the protein, and thus help increase the mechanical strength of spider silk on a macro scale, and finally obtain a series of protein materials with good performance and different hydrophilicity and hydrophobicity.

[0021] In a preferred embodiment, the present invention is directed to Euprosthenops australis The typical repeating segment MaSp-2rep of the major ampullate gland silk protein (MaSp1) from the source was rationally designed and the structural characteristics were improved to obtain an amyloid protein motif chimeric bionic spider silk protein modified body with significantly improved heterologous expression, with a maximum yield of about 300±50 purified protein mg / stem cell g, which promoted the industrial production of amyloid protein motif chimeric bionic spider silk protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 SDS-PAGE electrophoresis and grayscale scanning analysis of the target bands of the modified amyloid protein motif chimeric bionic spider silk proteins (Amy4-3rep, Amy4-6rep, Amy4-12rep, Amy4-24rep, Amy4-48rep).

[0024] Figure 2 SDS-PAGE electrophoresis analysis of modified amyloid protein motif chimeric biomimetic spider silk proteins (MaSp-8rep, Cs-12rep, Amy4-12rep, Amy5-12rep, Amy6-12rep, Amy7-12rep, Amy8-12rep, Amy9-12rep).

[0025] Figure 3 Contact angle analysis and measurement results (upright and inverted) of modified amyloid protein motif chimeric biomimetic spider silk proteins (Amy4-12rep, Amy5-12rep, Amy7-12rep, Amy9-12rep). DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, or can refer to the applicant's previous article (Adv. Sci.2024, 11, 2400128).

[0027] Example 1 Based on Euprosthenops australis Acquisition of a gene that increases the density of a typical repeat segment of the major ampullate gland silk protein (MaSp1) from The recombinant expression plasmid of MaSp-NT-2Rep-CT (NT-2Rep-CT chimeric spider silk protein without Ni-NTA purified 6*His tag at the N-terminus) was constructed using pSE380 as a vector: Using a low molecular weight chimeric spider silk protein NT-2Rep-CT as a template (Biomimicspinning of artificial spider silk from a chimeric minispidroin, Nature Chemical Biology, 13, pages 262–264 (2017)), NT-2Rep-CT was inserted into the pSE380 vector between the NcoI and HindIII sites using restriction endonucleases to obtain the pSE380 plasmid expression vector of the chimeric MaSp-NT-2Rep-CT (SEQ ID: NO.1). The structure of MaSp-NT-2Rep-CT is derived from the natural spider silk protein MaSp1( Euprosthenops australis ), the N-terminal NT domain of the spider silk protein (SEQ ID NO: 2), the two Gly and polyalanine A(n)-rich repeat region REP domains (2Rep, amino acid sequence: SEQ ID NO: 3) from the nursery web-weaving spider (E. australis), and the natural spider silk protein MiSp ( Araneus ventricosus ) is composed of the C-terminal CT domain (amino acid sequence SEQ ID NO: 4).

[0028] Using MaSp-NT-2Rep-CT chimeric on the plasmid vector pSE380 as a template, the number of key amino acid motifs of polyalanine in the microcrystalline region of the chimeric spider silk protein gene NT-2rep-CT was reduced to 7 by gene synthesis, and the number of polyalanine blocks was increased to 3 while keeping the sequence length relatively consistent. The amyloid protein motif chimeric biomimetic spider silk protein gene MaSp-NT-Cs(Crystal)-3rep-CT with increased density in the microcrystalline region was obtained. Its structure consists of the above-mentioned NT domain + the repeat region domain (3Rep, amino acid sequence: SEQ ID NO:5) after replacing the polyalanine motif and encrypting it + the above-mentioned CT domain (hereinafter referred to as Cs-3rep). It was embedded between the NcoI and HindIII sites of the pSE380 vector by the same method to obtain the pSE380 plasmid expression vector of the chimeric Cs-3rep.

[0029] Example 2 Obtaining modified amyloid peptide gene In this example, Cs-3rep embedded in the plasmid vector pSE380 was used as a template, and a series of amyloid peptide sequences with a total average hydrophilicity between -3.133 and 0.957 were selected from the amyloid peptide database (services.mbi.ucla.edu / zipperdb), namely Amy4 (ILSSTNV, PDB ID: 5MGQ), Amy5 (NSALALQ, PDB ID: 8ENQ), Amy6 (TAVANKT, PDB ID: 7LC9), Amy7 (ELNIYQY, PDB ID: 6G8D _A), Amy8 (SSVNNTQ, PDB ID: 8ENR), and Amy9 (NNQQNY, PDB ID: 5K2F_A).

[0030] The key amino acid sequence polyalanine sequence in the microcrystalline region of spider silk protein was replaced with the above-mentioned amyloid polypeptide sequence by gene synthesis, and the pSE380 expression vectors of amyloid protein motif chimeric spider silk proteins MaSp-NT-Amy4-3rep-CT, MaSp-NT-Amy5-3rep-CT, MaSp-NT-Amy6-3rep-CT, MaSp-NT-Amy7-3rep-CT, MaSp-NT-Amy8-3rep-CT and MaSp-NT-Amy9-3rep-CT were obtained, hereinafter referred to as Amy4-3rep, Amy5-3rep, Amy6-3rep, Amy7-3rep Amy8-3rep and Amy9-3rep, respectively. As an example, the structural form of Amy4-3rep is that AAAAAAA in the amino acid sequence of Cs-3rep is replaced by ILSSTNV, and its amino acid sequence is shown in SEQ ID NO:6, while Amy4-12rep is a polyploid (Amy4-3rep) 4 of Amy4-3rep, and its amino acid sequence is shown in SEQ ID NO:7. The remaining amyloid protein motif replacement structures and polyploids are all in the same replacement and doubling form.

[0031] Example 3 Construction of polyploid amyloid protein chimeric biomimetic spider silk protein This example takes the modified amyloid protein motif chimeric biomimetic spider silk protein gene Amy4-3rep as an example to illustrate the method for constructing a biomimetic spider silk protein polyploid.

[0032] First, the pSE380 plasmid of the chimeric Amy4-3rep was subjected to single enzyme digestion (the single enzyme digestion system was in accordance with the system shown in Table 1), and agarose gel electrophoresis was performed at 180 V, 400 mA, and 10 min for verification. After the inspection was correct, the band was excised and recovered, and the DNA concentration and molecular weight of the recovered fragments were detected to obtain the single-stranded vector of the spider silk protein Amy4-3rep with NheI or SpeI. At the same time, the amyloid protein motif chimeric biomimetic spider silk protein Amy4-3rep plasmid was double-digested with the same tail enzymes NheI and SpeI (the double enzyme digestion system was in accordance with the system shown in Table 2), and then verified by agarose gel electrophoresis at 180 V, 400 mA, and 10 min.

[0033] After checking, the bands were cut and recovered, and the DNA concentration and molecular weight of the recovered fragments were tested to verify the molecular weight, and the single-stranded repeat region fragment of the amyloid protein motif chimeric biomimetic spider silk protein Amy4-3rep with NheI and SpeI at the head and tail respectively was obtained.

[0034] Finally, according to the system shown in Table 3, the obtained spider silk protein Amy4-3rep repeat region gene fragment was connected with the spider silk protein Amy4-3rep single-stranded gene vector with NheI or SpeI, and the connection was carried out overnight at 16°C to obtain the polyploid amyloid protein motif chimeric spider silk protein Amy4-6rep gene expression vector connection liquid. The above process was repeated to obtain the polyploid amyloid protein motif chimeric biomimetic spider silk protein Amy4-12rep and Amy4-24rep gene expression vectors in sequence. Referring to this method, the polyploid gene expression vectors of all amyloid protein motif chimeric biomimetic spider silk proteins in this experiment were constructed.

[0035] Table 1 Single enzyme digestion reaction system

[0036] Table 2 Double enzyme digestion reaction system

[0037] Table 3 Ligation reaction system

[0038] Example 4 Expression of polyploid amyloid protein chimeric biomimetic spider silk protein Taking the modified amyloid protein motif chimeric biomimetic spider silk protein Amy4-3rep and its polyploid as an example, the competent cells NEB-10β stored in a refrigerator at -80 ℃ were placed in an ice box for thawing for 10 min. In the clean bench, the amyloid protein motif chimeric biomimetic spider silk protein expression vector Amy4-3rep / 6rep / 12rep / 24rep constructed in Example 3 was injected into the competent cells, and the preparation tube was immediately ice-bathed for 30 min, then water-bathed in a 42 ℃ water bath for 45 s, and then ice-bathed for 5 min. In the clean bench, 800uL LB culture medium (no resistance) was added to each preparation tube, mixed appropriately, and the above preparation tube was placed in a paper box and cultured in a shaker (temperature: 37 ℃, speed: 100 rpm) for 40 min. After reaching the incubation time, the preparation tube was centrifuged at 12000rpm for 1min. In the clean bench, some of the supernatant in the preparation tube after centrifugation was removed, and the remaining was mixed and transferred to the prepared amp plate (concentration of 0.1%), spread evenly with an applicator, and cultured in a 42℃ incubator for 12-14 hours. The transformed recombinant bacteria were screened by colony PCR and sequenced by Universal BioSequencing, and finally the amyloid protein motif chimeric bionic spider silk protein expression host containing the correctly connected expression vector was screened.

[0039] The strain containing the chimeric spider silk protein expression vector was inoculated into 50 mL of LB liquid medium, and ampicillin was added to make the final concentration of 100 μg / mL, and cultured overnight at 180 rpm and 37 ℃; the seed solution of overnight culture was inoculated into fresh 300 mL of LB liquid medium at an inoculum size of 2%, and cultured at 180 rpm and 37 ℃ for 2 h (OD600 was 0.4-0.6), and the inducer IPTG (isopropyl β D-thiogalactoside) (final concentration 0.3 mmol / L) was added, and the expression was induced at 16 ℃, 25 ℃, 30 ℃, and 35 ℃ for 24 h.

[0040] Take the fermentation broth of induced expression, centrifuge at 5000 rpm for 30 min, discard the supernatant, and then resuspend the bacteria with 40 mL of pure water, then ultrasonically disrupt for 30 min, and then centrifuge at 12000 rpm for 10 min to obtain the supernatant (S), discard the supernatant, add 10 mL of pure water to the supernatant, suspend and mix to obtain the insoluble precipitate (IB), freeze-dry at -81 ° C using CapableL3-65 freeze dryer to obtain freeze-dried powder. 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 sample is loaded in a boiling water bath for 5 min for electrophoresis. The initial voltage of the electrophoresis instrument is set to 120 V, and the voltage is increased to 230 V when the sample moves to the separation gel, and the electrophoresis is terminated when the sample moves to the bottom of the electrophoresis tank.

[0041] The results are as follows Figure 1 As shown, the molecular weight of the amyloid protein motif chimeric biomimetic spider silk protein Amy4-3rep is 33.2kDa, and the molecular weights of its polyploid Amy4-6rep / 12rep / 24rep are 40.4 kDa, 54.6kDa and 82.3kDa, respectively. The amyloid protein motif chimeric biomimetic spider silk protein obtained after induction has obvious bands at the corresponding protein molecular weights, which is consistent with the design. This shows that the amyloid protein motif chimeric biomimetic spider silk protein and its polyploids were successfully induced and expressed (such as Figure 2 As shown). Among them, the expression level of Amy4-24rep decreased significantly (the grayscale scanning result of the target band was 22.3%), which was not conducive to the efficient expression of the amyloid motif chimeric bionic spider silk protein, while the expression level of 12rep did not decrease significantly (the grayscale scanning result of the target band was 75.1%). This phenomenon also exists in the expression of other amyloid motif chimeric bionic spider silk proteins with different total hydrophilicity average values. In addition, studies have shown that the number of microcrystalline regions in the amyloid motif chimeric bionic spider silk protein is proportional to the silk-forming performance. Therefore, 12rep is preferred as a compromise between expression level and silk-forming performance.

[0042] Example 5 In order to prove that the amyloid artificial spider silk protein of the present application has the potential for large-scale production, this example uses a bioreactor to perform high-density fermentation on all the amyloid protein motif chimeric biomimetic spider silk proteins in this experiment. The specific operation is as follows: The pSE-380 vector expressing the amyloid protein motif chimeric biomimetic spider silk protein was introduced into the NEB-10 strain (from New England Biolabs Co., Ltd.) to construct the NEB-10 genetically engineered strain expressing the amyloid protein motif chimeric biomimetic spider silk protein, and the NEB-10 genetically engineered strain was fermented by a fed-batch fermentation process to produce the amyloid protein motif chimeric biomimetic spider silk protein.

[0043] The fermentation process includes: The bacterial cells were cultured in 200 mL of Terrific Broth (TB) medium with 50 mL of ampicillin until the OD 600 ≈5.

[0044] Subsequently, the cells were collected by centrifugation and resuspended in 2L of a customized medium consisting of a nutrient mix including 60 g / L glycerol (major carbon source), 20 g / L tryptophan, 24 g / L yeast extract, 10 g / L glucose, and mineral salts (including 0.5 g / L magnesium sulfate heptahydrate, 3.4 g / L potassium monobasic phosphate, 3.6 g / L disodium phosphate, 2.7 g / L ammonium chloride, 0.7 g / L sodium sulfate, 100.8 mg / L ferric citrate, 2.5 mg / L cobalt chloride hexahydrate, 15 mg / L manganese chloride tetrahydrate, 1.5 mg / L copper chloride dihydrate, 3 mg / L boric acid, 2.1 mg / L sodium molybdate dihydrate, 33.8 mg / L zinc acetate hydrate, and 14.1 mg / L EDTA) to support microbial growth and metabolism. The medium was supplemented with 50 mg / L ampicillin to maintain plasmid selection pressure, and 0.01% (v / v) antifoaming agent 204 was added to prevent foaming during fermentation. The resuspended cell suspension was then transferred to a 5-liter Bioflo120 fed-batch bioreactor (manufactured by Eppendorf, Hamburg, Germany) for large-scale cultivation. Throughout the fermentation, the temperature was maintained at 37°C, and the pH was strictly controlled in the range of 6.5 to 7.5, with 3 M phosphoric acid or 25% ammonia water automatically added for pH adjustment as needed. Water evaporation during fermentation was compensated by regular addition of autoclaved water. To ensure optimal aerobic conditions, the dissolved oxygen (DO) level was initially set to 70% saturation, and the DO level was maintained above 30% throughout the fermentation by adjusting the agitation speed (from 200 to 800 rpm) and the airflow rate.

[0045] After 24 hours of growth, 0.5 mM IPTG was added to induce cell expression. Induction was performed at 30-35°C to optimize protein yield. Immediately after the addition of IPTG, a customized feed medium (400 g / L glycerol, 20 g / L tryptophan, 24 g / L yeast extract, 40 g / L magnesium sulfate heptahydrate, 3.4 g / L potassium phosphate monohydrate, 3.6 g / L sodium phosphate disodium, 2.7 g / L ammonium chloride, 0.7 g / L sodium sulfate, 40 mg / L ferric citrate, 4 mg / L cobalt chloride hexahydrate, 23.5 mg / L manganese chloride tetrahydrate, 2.3 mg / L copper chloride dihydrate, 4.7 mg / L boric acid, 4 mg / L sodium molybdate dihydrate, 16 mg / L zinc acetate, 13 mg / LEDTA, 50 mg / L ampicillin) was introduced into the bioreactor at a controlled rate of 0.2 mL / min. This medium was used to provide continuous nutritional support during the expression phase. After 12 hours of induction, the fermentation process was terminated and the bacterial cells were harvested by centrifugation. The obtained cells were rich in overexpressed amyloid motif chimeric biomimetic spider silk proteins. In the experiment of this embodiment, the maximum yield of a series of amyloid motif chimeric biomimetic spider silk proteins was obtained by the weight of the biomimetic spider protein overexpressed in each gram of Escherichia coli stem cells (Table 4). In the table, 8rep (MaSp-NT-8rep-CT) is a polyploid constructed based on 2rep (MaSp-NT-2rep-CT), and the structural form of its amino acid sequence is NT-(2rep)n-CT, where n=4, and Cs-12rep is a 12rep polyploid of Cs-3rep, and the structural form of its amino acid sequence is NT-(3rep)n-CT, where n=4.

[0046] Table 4 Analysis of hydrophilicity and yield of wild-type chimeric spider silk protein and amyloid protein motif chimeric biomimetic spider silk protein

[0047] Example 6 Preparation of modified amyloid protein motif chimeric biomimetic spider silk protein material and characterization of its hydrophilicity and hydrophobicity A series of 12rep polyploids of modified amyloid motif chimeric biomimetic spider silk proteins were selected for the preparation of amyloid motif chimeric biomimetic spider silk protein materials, and their freeze-dried powder was dissolved in HFIP at a concentration of 20% w / v to prepare a film-forming solution. A cathode PDMS template was obtained by replicating the template, and then the film-forming solution was poured onto the surface of the PDMS template, dried at room temperature and peeled off to obtain a series of amyloid motif chimeric biomimetic spider silk protein films with different hydrophilicity. In a constant temperature box, at 25°C and 50% humidity, 20 μL of 25°C distilled water was evenly dripped onto the surface of a series of amyloid protein motif chimeric biomimetic spider silk protein films using a standard titration pipette. The water droplets on the surfaces of different amyloid protein motif chimeric biomimetic spider silk proteins formed different contact angles with the film surface. Within 5 seconds after the droplet was added, the morphology of the water droplets was photographed at the same position, and the contact angle between the water droplets and the film was analyzed using the angle measurement software Anglemeter. The hydrophilicity and hydrophobicity of the different amyloid protein motif chimeric biomimetic spider silk protein films were determined by the size of the contact angle. The results are shown in FIG. Figure 3 As shown in Table 5: Table 5 Correlation analysis between the hydrophilicity of amyloid protein motif and the surface contact angle of amyloid protein motif chimeric biomimetic spider silk protein film

[0048] It can be seen that the amyloid protein motif chimeric spider silk proteins replaced by amyloid peptides with different total average hydrophilicity form materials with corresponding hydrophilicity and hydrophobicity on a macro scale, which promotes the design and application of spider silk protein sequences for different functional materials.

Claims

1. A method for regulating the hydrophilicity and hydrophobicity of chimeric spider silk protein using amyloid protein motifs, characterized in that: The spider silk protein comprises a core repeat region REP domain derived from the major ampullate gland silk protein; The polyalanine motif in the microcrystalline region of the REP domain is replaced by an amyloid protein motif with different overall average hydrophilicity, and the overall average hydrophilicity of the amyloid protein motif is between -3.133 and 0.

957.

2. The method according to claim 1, characterized in that The amyloid motif is selected from: Any one of PDB ID: 5MGQ, ​​PDB ID: 8ENQ, PDB ID: 7LC9, PDB ID: 6G8D _A, PDB ID: 8ENR, PDB ID: 5K2F_A.

3. The method according to claim 1, characterized in that The method also includes doubling the amino acid sequence of the repeat region REP of the spider silk protein after replacing the polyalanine motif, wherein the doubling number is between 3 rep and 48 rep.

4. The method according to claim 3, characterized in that It also includes that the doubling number of the repeat region REP amino acid sequence is 12 rep.

5. The method according to claim 1, characterized in that The method further comprises shortening the polyalanine motif in the microcrystalline region to an oligoalanine (A)n motif with n≤7, encrypting the oligoalanine (A)n motif, and then replacing all the oligoalanine motifs with the amyloid protein motif.

6. The method according to claim 1, characterized in that The spider silk protein further comprises an N-terminal domain NT and a C-terminal domain CT, which form a chimeric spider silk protein with the repeat region REP domain; Wherein, 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 )'s C-terminal amino acid sequence.

7. A modified spider silk protein constructed by the method according to any one of claims 1 to 6.

8. An expression vector containing the spider silk protein modification product according to claim 7.

9. An engineered strain containing the spider silk protein modification according to claim 7.

10. Use of the modified spider silk protein according to claim 7 in preparing spider silk materials with different hydrophilicity and hydrophobicity.

Images