CN114805847A - Purification based on spider silk-cationic polypeptide fusion protein and preparation method of underwater adhesion hydrogel - Google Patents

Abstract

A method for purifying a spider silk-cationic polypeptide fusion protein and preparing a hydrogel for underwater adhesion, the recombinant spider silk protein gene and the cationic polypeptide gene are fused and then connected to an expression vector pET28a4 to construct a recombinant expression vector, and then the recombinant expression vector is constructed. The recombinant expression vector is introduced into the expression host cell, and the high-purity target protein is obtained after fermentative expression, separation and purification; and then the solution of the high-purity target protein is cross-linked with the tannic acid solution to obtain the underwater adhesion hydrogel. The present invention uses the negatively charged membrane protein OmpF of the host itself as a purification medium, and can obtain a large amount of high-purity fusion proteins, and is physically cross-linked with tannic acid to prepare an underwater adhesive hydrogel with excellent performance, which can be purified in batches and reduce costs. .

Description

Purification of spider silk-cationic polypeptide fusion protein and preparation of underwater adhesive hydrogel

technical field

The invention relates to a technology in the field of bioengineering, in particular to a method for purifying a spider silk-cationic polypeptide fusion protein and preparing an underwater adhesive hydrogel.

Background technique

In the field of materials science, the preparation of hydrogel materials with high underwater adhesion has always been a hot and difficult research topic. Natural polymer hydrogels such as protein, chitosan, and cellulose have attracted much attention due to their better biocompatibility and tunable degradation than chemical polymer hydrogels. The current mainstream method for preparing natural polymer hydrogels with underwater adhesion properties is to modify protein materials by means of chemical grafting groups or physical blending ions to create protein hydrogels with enhanced properties. Often, its application range is limited due to insufficient stability and insufficient biocompatibility of non-natural modifying groups. The present invention utilizes genetic engineering and fermentation engineering technology to directly produce fusion proteins modified by natural polypeptides without additional chemical or physical modification of hydrogels, avoids cytotoxic non-natural components and solves the problem of fusion of positively charged and hydrophobic polypeptides The problem that the recombinant spider silk protein is difficult to purify by nickel affinity chromatography greatly facilitates industrial production and use. The recombinant protein hydrogel prepared in this way can realize common materials such as aluminum, plastic and glass, as well as the surface of tissues and organs. Fast and stable adhesion, especially instant adhesion in underwater environment, has broad application prospects and value.

SUMMARY OF THE INVENTION

Aiming at the problems that the existing adhesive hydrogels based on natural macromolecules are relatively complex in preparation, unstable in performance and difficult to purify or have low purity of recombinant spider silk proteins fused with cationic peptide segments, the present invention proposes a spider silk-based Purification of cationic polypeptide fusion protein and preparation method of underwater adhesive hydrogel, using the host's own negatively charged membrane protein OmpF as the purification medium, a large amount of high-purity fusion protein can be obtained, and the performance is obtained by physical cross-linking with tannic acid. Excellent underwater adhering hydrogel for batch purification and cost reduction.

The present invention is achieved through the following technical solutions:

The invention relates to a preparation method of an underwater adhesive hydrogel based on a spider silk-cationic polypeptide fusion protein. The recombinant spider silk protein gene and the cationic polypeptide gene are fused and then connected to an expression vector pET28a4 to construct a recombinant expression vector, and then the recombinant expression vector is constructed. The recombinant expression vector is introduced into the expression host cell, and the high-purity target protein is obtained after fermentative expression, separation and purification; and then the solution of the high-purity target protein is cross-linked with the tannic acid solution to obtain the underwater adhesion hydrogel.

Described recombinant spider silk protein gene refers to: golden silk spider (Trichonephila clavipes) traction silk protein MaSpI core repeating sequence repeats 4, 8, 16, 32 and 64 times protein, wherein: the amino acid sequence of MaSpI core repeating sequence is as follows: shown in SEQ ID No. 1.

The construction refers to double digestion of the synthesized cationic polypeptide gene by restriction endonucleases BamHI HF and NcoI HF, and then ligated to the expression vector pET28a4 by T4 DNA ligase to obtain the expression vector of recombinant spider silk protein.

Described expression vector pET28a4 refers to: modify and add BamHI restriction enzyme site in the multi-cloning site of commercially available pET28a(+) expression vector, it adopts but is not limited to "Escherichia coli Formation and functionalization of membraneless compartment. (Wei SP, Qian ZG, Hu CF, Pan F, Chen MT, Lee SY, XiaXX. "Formation and functionalization of membraneless compartments in Escherichia coli." Nat Chem Biol, 2020, 16(10):1143-1148.) documented technical implementation.

Described cationic polypeptides include but are not limited to LL37, protamine polypeptides, polylysine, polyarginine, polyhistidine, lysine-rich, arginine-rich, histidine-rich, etc. of positively charged polypeptides.

The amino acid sequence of described LL37 is shown in SEQ ID No.2, namely:

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (from database: PBD), its codon-optimized nucleotide sequence is shown in SEQ ID No. 3, namely: 5'-CTGCTGGGCGATTTCTTCCGCAAAAGCAAAGAAAAAATCGGCAAAGAATTCAAACGCATCGTTCAGCGC ATCAAAGATTTCCTGCGCAATCTGGTTCCGCGCACCGAAAGC-3'.

The amino acid sequence of the protamine polypeptide is shown in SEQ ID No.4, namely:

RSQSRSRYYRQRQRSRRRRRRS (from database: NCBI), its codon-optimized nucleotide sequence is shown in SEQ ID No. 5, namely: 5'-CGTAGCCAGAGCCGTAGCCGTTACTACCGTCAGCGTCAGCGTAGCCGTCGTCGTCGCCGTCGTAGC-3'.

The expression hosts were E. coli BL21 (DE3) and E. coli BL21 (DE3) plysS, and the used protein expression inducer was isopropyl thiogalactoside (IPTG).

The separation and purification specifically includes: fully resuspending the Escherichia coli wet cells expressing the fusion protein in Buffer A at a mass-to-volume ratio of 1:10, and then breaking the walls through a high-pressure homogenizer at a pressure of 800-1000 bar to release intracellular substances . Adjust the pH of the broken bacteria solution after breaking the wall to Z, that is, the isoelectric point of the binding complex of the recombinant spidroin protein fused with polypeptide Y and the negatively charged membrane protein OmpF released by the broken bacteria, stir with a magnetic stirrer for 30 min, and then discard by centrifugation. The supernatant was collected and resuspended in Buffer A, and the washing was repeated 3 times. After centrifugation, the precipitate was fully resuspended in Buffer B. The supernatant was obtained by centrifugation. Distilled water, centrifuged to take the supernatant, and finally, if the protein dissolved in completely pure water is required, the supernatant can be filtered with a hydrophobic column to take the effluent.

For the separation and purification, the positively charged block of recombinant arachnid protein is combined with the negatively charged OmpF membrane protein, and the pH is adjusted to the isoelectric point Z of the conjugate to make it precipitate crudely pure, and after rinsing, it is dissolved in Buffer B. , using the strong positive charge of PEI in Buffer B to bind the negatively charged hybrid protein OmpF to make it precipitate, while the recombinant spider silk protein still exists in the supernatant to achieve further purification.

Said isoelectric point Z=isoelectric point of polypeptide Y×its content ratio in the complex+OmpF isoelectric point 4.5×its proportion in the complex.

The Buffer A is an aqueous solution containing 10 mM Tris and 50 mM NaCl, and its pH is the same as the isoelectric point Z.

The Buffer B is an aqueous solution (pH 4.5) containing 50 mM Tris, 500 mM NaCl, 8 M urea, PEI _{10 kd} (2% m/v), and Triton X-100 (1.5% v/v).

The ammonium sulfate precipitation specifically refers to: adding ammonium sulfate powder to the supernatant to 40% saturation, at this time the protein solution will produce aggregated precipitate, and centrifuge at 12000rpm (×21235g) for 30min at 25°C to get the precipitate.

The hydrophobic column packing is Phenyl Beeds 6FF, and the purpose of passing through the hydrophobic column is to remove residual TritonX-100.

The high-purity target protein solution refers to: dissolving the lyophilized protein in a 90 mM LiBr solution to prepare a 100 mg/ml protein solution.

The cross-linking reaction refers to: gradually drop the high-purity target protein solution into the tannic acid solution at a volume ratio of 1:1, and stir with a stirring bar after the dropwise addition, so as to form a solution that adheres to the stirring bar. Hydrogels.

The invention relates to an underwater adhesive hydrogel prepared by the above method, which has a macroscopic shape of a soft and plastic light brown solid, a microscopic shape of a rich pore structure, and an underwater adhesion strength of 1-100 kPa.

technical effect

Compared with the prior art, the present invention uses the negatively charged OmpF protein of the expression host itself as the purification medium to avoid the limitation of the loading volume of the column chromatography purification method, the expensive consumables, the complicated and slow flow process, and the difficulty in amplification. The entire purification process time is shortened to one tenth of that of affinity chromatography purification, the cost of consumables is extremely low, the operation is convenient, and it can be scaled up in industrial batches; The gel exhibits higher adhesion performance under the conditions of preparation and use, and has no non-natural toxic components in other adhesion hydrogels, and has fast and stable adhesion to the surface of tissues and organs and common materials such as plastics, metals, etc. Attachment.

Description of drawings

Fig. 1 is the SDS-PAGE figure of spider silk-cationic polypeptide fusion protein in the embodiment;

In the figure: #1 is LL37-MaSpI16 protein, #2 is protamine-MaSpI16 protein;

Fig. 2 is a schematic diagram of the purification process of spider silk-cationic polypeptide fusion protein in the present invention;

Fig. 3 is the SDS-PAGE figure of the purification process of the LL37-MaSpI16 protein of the embodiment;

In the figure: #P ₀ shows that LL37-MaSpI16 protein is combined with OmpF protein and precipitates to achieve crude purity, #P ₁ shows that OmpF protein is competed by PEI _10kDa for binding and precipitation, releasing LL37-MaSpI16 protein shown in #S ₂ .

Fig. 4 is the actual photo of embodiment LL37-MaSpI16 TA hydrogel, TA refers to tannic acid;

Figure 5 is a scanning electron microscope photograph of Example LL37-MaSpI16 TA hydrogel, protamine-MaSpI16 TA hydrogel and control MaSpI16 TA hydrogel, the protein concentration used for preparing the hydrogel is 100mg/ml, and the TA concentration is 200mg /ml, the volume ratio of the two is 1:1;

Figure 6 is the rheological experimental data of Example LL37-MaSpI16 TA hydrogel, protamine-MaSpI16 TA hydrogel and control MaSpI16 TA hydrogel, the protein concentration used for preparing the hydrogel is 100 mg/ml, and the TA concentration is 200mg/ml, the volume ratio of the two is 1:1;

In the figure: G’ is the elastic modulus, G” is the loss modulus;

Figure 7 is a schematic diagram of the test data of underwater adhesion performance of Example LL37-MaSpI16 TA hydrogel, protamine-MaSpI16 TA hydrogel and control MaSpI16 TA hydrogel, the protein concentration used for preparing the hydrogel is 100mg/ml, TA The concentration is 100～300mg/ml, and the volume ratio of the two is 1:1;

Figure 8 is a schematic diagram of the adhesion application of spider silk-cationic polypeptide fusion protein.

Detailed ways

This embodiment includes the following steps:

Step 1) construct a target protein expression vector, and import the expression vector into Escherichia coli E.coli BL21 (DE3) to realize the method for expression production, specifically including:

1.1) After double digestion of the synthesized cationic polypeptide gene by the restriction endonucleases BamHI HF and NcoIHF, the expression vector and protamine of the LL37-MaSpI16 fusion protein were constructed by connecting to the expression vector pET28a4-MaSpI16 by T4 DNA ligase. - Expression vector for MaSpI16 fusion protein.

1.2) For the LL37-MaSpI16 fusion protein, its expression production process is: after the expression vector pET28a4-LL37-MaSpI16 is transformed into the expression host cell, the expression host cell is placed in 4mL containing kanamycin (50mg/L). After culturing at 37°C for 6-8h in LB test tube medium, inoculate 4-6 small shake flasks containing kanamycin (50mg/L) in 50mL R/2 medium for about 8h at 37°C, All were transferred into a 2L R/2 medium fermenter containing kanamycin, cultured at 37°C to an OD600 of about 40, added 2ml of IPTG solution with a concentration of 1M, induced expression at 16°C for 12-16h and harvested.

For the protamine-MaSpI16 fusion protein, the expression production process is as follows: after the expression vector pET28a4-protamine-MaSpI16 is transformed into the expression host cell, the expression host cell is inoculated into 4mL LB containing kanamycin (50mg/L). Tubes, incubated overnight at 37°C, 220 rpm. With 1% of the inoculum, the above bacterial liquid was transferred to 100 mL of LB liquid medium containing kanamycin (50 mg/L), and cultured at 37°C and 220 rpm for 3 to 4 hours, until the OD600 of the bacterial liquid reached 3 to 4. , all the above bacterial liquids were added to 800ml TB liquid medium containing kanamycin (50mg/L), and cultured at 37°C and 220rpm. When the OD600 reached 6-8, the expression was induced, and 0.9 mL of 1M IPTG was added for induction. The induction conditions were 16 °C, 220 rpm, and 20 h.

The cloned host E.coli DH5α, the expression host E.coli BL21 (DE3); the expression plasmid pET28a4; BamHI&NcoI restriction endonucleases, T4 DNA ligase; kanamycin, chloramphenicol; LB medium , R/2 medium.

The component content of the LB medium (per liter) is: 10 g/L tryptone, 5 g/L yeast powder and 10 g/L sodium chloride, autoclaved at 121° C. for 20 min.

The component content of the TB medium (per liter) is: 13.4g/L tryptone, 26.7g/L yeast extract, 5.6g/L glycerol to configure component A, and after stirring and mixing, each 2L shakes Fill the bottle with 720 ml of component A, and sterilize by autoclaving at 121° C. for 20 min. Component B phosphate buffer: 23.1g/L KH2PO4, 164.3g/L K2HPO4·3H2O, autoclaved at 121°C for 20min. Add 80 mL of Part B to every 720 mL of Part A before use.

The component content of the described R/2 medium (per liter) is: 2g/L (NH4)2SO4, 6.75g/L KH2PO4, 0.85g/L citric acid, Trace metal 5mL/L, the medium is hydrogenated The pH was adjusted to 6.8 with potassium oxide, and sterilized by autoclaving at 121 °C for 20 min.

Step 2) Separation and purification after harvesting the fermented cells: fully resuspend the Escherichia coli wet cells expressing the fusion protein LL37-MaSpI in Buffer A at a mass-volume ratio of 1:10 to adjust the pH to Z, and then pass through a high-pressure homogenizer The high pressure of 800 ~ 1000bar breaks the wall to release intracellular substances. The pH of the broken bacteria solution after breaking the wall was adjusted to Z, and then the supernatant was discarded by centrifugation, and the precipitate was placed on a stirring table and stirred, so that it was fully resuspended in Buffer A, and then centrifuged at 12000rpm (×21235g) for 20min at 25°C to take the precipitate. The step plays the role of washing and is repeated 3 times in total. The precipitate after washing 3 times was fully stirred and resuspended in Buffer B. The supernatant was collected by centrifugation, and ammonium sulfate powder was slowly added until the saturation was 40% to salt out the protein. After standing at room temperature for 5-10 min, the pellet was kept at 12000 rpm (× 25°C). 21235g) centrifuge for 20 min to get the precipitate, then suspend the precipitate in distilled water, place it on a stirring table and stir well, and then centrifuge to get the supernatant. Finally, if the protein dissolved in completely pure water is required, the supernatant can be filtered with a hydrophobic column to obtain the effluent. The purified protein is lyophilized according to storage requirements. The purification process is shown in Figure 2.

In this example, for the LL37-MaSpI16 fusion protein, Z=6.9, and its calculation method is LL37 isoelectric point 10.61 × the content of the fusion protein in the complex 40% + negatively charged protein OmpF isoelectric point 4.5 × this The content of negatively charged protein in the complex is 60%; for the protamine-MaSpI16 fusion protein, Z=6.1, which is calculated as the isoelectric point of protamine 12.48×the content of the fusion protein in the complex 20%+the negatively charged protein OmpF, etc. The electric point is 4.5×the content of the negatively charged protein in the complex is 80%.

The Buffer A is an aqueous solution (pH=Z) containing 10 mM Tris and 50 mM NaCl.

The Buffer B contained 50 mM Tris, 500 mM NaCl, 8 M urea, 2% m/v PEI _10kd , 1.5% v/v Triton X-100 in water (pH 4.5).

The Buffer A adjusts the pH to Z in order to make the binding complex of the target protein and the negatively charged heteroprotein OmpF precipitate at the isoelectric point to achieve crude purity, 50mM Tris in Buffer B is for buffering pH changes, 500mM NaCl is for The competition weakens the electrostatic interaction between the target protein and the negatively charged heteroprotein. 8M urea is a strong destroyer of hydrogen bonds and hydrophobic interactions, so that the precipitate can be fully dissolved. 2% m/v PEI _10kd is used to bind the negatively charged heteroprotein to settle it down , 1.5% v/v TritonX-100 is the accelerant for the target protein, and pH 4.5 is to further precipitate all impurity proteins.

Step 3) Preparation of underwater adhesive hydrogel after obtaining high-purity freeze-dried protein: gradually add the freeze-dried protein to the 90 mM LiBr solution in small amounts and several times, and continue adding after the freeze-dried powder to be added is fully dissolved each time. Until the preparation is 100mg/ml protein solution. Gradually add a certain volume of the protein solution dropwise to an equal volume of 200mg/ml tannin solution, and stir while adding. Adding 5 ml of tannic acid solution can produce spherical hydrogels with a diameter of about 1 cm, as shown in Figure 4.

The solvent for dissolving the freeze-dried protein powder is LiBr solution, and the concentration is 90 mM.

The concentration of the protein solution was 100 mg/ml.

The concentration of the tannic acid solution is 200 mg/ml.

The volume ratio of the protein solution and the tannic acid solution is 1:1.

After specific practical experiments, under the conditions of using the above-mentioned expression host cells and according to the above-mentioned separation and purification operation parameters, the purification process can be quickly completed within 2 hours, and the time of the entire purification process can be shortened to one tenth of the purification by affinity chromatography. The purified protein The purity is as high as more than 90%, as shown in Figure 1.

After specific practical experiments, the obtained LL37-MaSpI16 fusion protein hydrogel was obtained under the condition that the protein concentration was 100 mg/ml, the tannic acid solution concentration was 100-300 mg/ml, and the volume ratio of protein solution and tannic acid solution was 1:1. The underwater adhesion strength is about 20～24kPa, which is 30%～100% higher than that of MaSpI16 under the same preparation and use conditions of the hydrogel; the underwater adhesion strength of the protamine-MaSpI16 fusion protein hydrogel is about 18～100%. 23kPa, compared with MaSpI16, the adhesion strength is increased by 18% to 50% under the same conditions of hydrogel preparation and use, and there is no non-natural toxic components in other adhesion hydrogels, which is harmful to the surface of tissues and organs and common materials such as plastics. , metals, etc. all have fast and stable adhesion, as shown in Figure 8.

The above-mentioned specific implementation can be partially adjusted by those skilled in the art in different ways without departing from the principle and purpose of the present invention. The protection scope of the present invention is subject to the claims and is not limited by the above-mentioned specific implementation. Each implementation within the scope is bound by the present invention.

sequence listing

<110> Shanghai Jiaotong University

<120> Purification based on spider silk-cationic polypeptide fusion protein and preparation method of underwater adhesive hydrogel

<130> fxc850e

<141> 2022-03-09

<160> 5

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> MaSpI core repeat (Trichonephila clavipes)

<400> 1

Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala

1 5 10 15

Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly

20 25 30

<210> 2

<211> 37

<212> PRT

<213> Cationic polypeptide LL37 (Artificial Sequence)

<400> 2

Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu

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Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val

20 25 30

Pro Arg Thr Glu Ser

35

<210> 3

<211> 111

<212> DNA

<213> LL37 codon-optimized sequence (Artificial Sequence)

<400> 3

ctgctgggcg atttcttccg caaaagcaaa gaaaaaatcg gcaaagaatt caaacgcatc 60

gttcagcgca tcaaagattt cctgcgcaat ctggttccgc gcaccgaaag c 111

<210> 4

<211> 22

<212> PRT

<213> protamine polypeptide (Artificial Sequence)

<400> 4

Arg Ser Gln Ser Arg Ser Arg Tyr Tyr Arg Gln Arg Gln Arg Ser Arg

1 5 10 15

Arg Arg Arg Arg Arg Ser

20

<210> 5

<211> 66

<212> DNA

<213> protamine codon-optimized sequence (Artificial Sequence)

<400> 5

cgtagccaga gccgtagccg ttactaccgt cagcgtcagc gtagccgtcg tcgtcgccgt 60

cgtagc 66

Claims (9)

1. A preparation method of underwater adhesion hydrogel based on spider silk-cationic polypeptide fusion protein is characterized in that gene fusion of recombinant spider silk protein gene and cationic polypeptide is connected to an expression vector pET28a4 to construct a recombinant expression vector, then the recombinant expression vector is introduced into an expression host cell, and high-purity protein is obtained after fermentation expression, separation and purification; then, carrying out cross-linking reaction on the high-purity mesh protein solution and a tannic acid solution to obtain underwater adhesive hydrogel;

the recombinant spider silk protein gene refers to: a protein of the silkworms (trichoneephila clavipes) dragline protein MaSpI core repeat repeated 4, 8, 16, 32 and 64 times, wherein: the amino acid sequence of the MaSpI core repetitive sequence is shown as SEQ ID No. 1;

the separation and purification, the combination of the positive electricity block of the recombinant spider silk protein and the negative electricity OmpF membrane protein, the pH value is adjusted to the isoelectric point Z of the combination to ensure that the combination is precipitated and coarse, the combination is dissolved in Buffer B after being rinsed, the strong positive charge of PEI in the Buffer B is combined with the negative electricity ompF protein to ensure that the combination is precipitated, and the recombinant spider silk protein still exists in the supernatant to realize further purification.

2. The method according to claim 1, wherein the constructing comprises: the synthesized cationic polypeptide gene was digested by restriction endonucleases BamHI HF and NcoIHF, and ligated to expression vector pET28a4 by T4 DNA ligase to obtain an expression vector of recombinant spidroin.

3. The method of claim 1 or 2, wherein the cationic polypeptide comprises: LL37, protamine polypeptides, polylysine, polyarginine, polyhistidine, lysine-rich, arginine-rich, histidine-rich, and like positively charged polypeptides.

4. The method according to claim 3, wherein the amino acid sequence of LL37 is shown in SEQ ID No.2, i.e.: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, the nucleotide sequence of which after codon optimization is shown in SEQ ID No.3, namely: 5'-CTGCTGGGCGATTTCTTCCGCAAAAGCAAAGAAAAAATCGGCAAAGAATTCAAACGCATCGTTCAGCGCATCAAAGATTTCCTGCGCAATCTGGTTCCGCGCACCGAAAGC-3', respectively;

the amino acid sequence of the protamine polypeptide is shown as SEQ ID No.4, namely: RSQSRSRYYRQRQRSRRRRRRS (from database: NCBI), having a codon-optimized nucleotide sequence as shown in SEQ ID No.5, namely: 5'-CGTAGCCAGAGCCGTAGCCGTTACTACCGTCAGCGTCAGCGTAGCCGTCGTCGTCGCCGTCGTAGC-3' are provided.

5. The method according to claim 1, wherein the expression hosts are E.coli BL21(DE3) and E.coli BL21(DE3) plysS, and the protein expression inducer is isopropyl thiogalactoside (IPTG).

6. The method according to claim 1, wherein the separation and purification comprises: e.coli wet cells expressing the fusion protein were cultured in a manner of 1: fully suspending the mixture in a Buffer A at a mass-volume ratio of 10, and breaking the walls by using a high-pressure homogenizer at a high pressure of 800-1000 bar to release intracellular substances; broken fungus liquid after will breaking the wall adjusts pH to Z, the isoelectric point of the negative electricity membrane protein OmpF that has fused polypeptide Y's recombination spider silk protein and broken fungus release combines the complex promptly, use magnetic stirrers stirring 30min, centrifugation is abandoned the supernatant and is got the deposit and resuspend in Buffer A again afterwards, abundant resuspension washing is repeated 3 times, will deposit abundant resuspending in Buffer B centrifugation after the centrifugation and get the supernatant, carry out the ammonium sulfate sediment with the supernatant, resuspend this deposit in distilled water again, the supernatant is got in the centrifugation, the protein that dissolves if needs complete pure water can select for use this supernatant to filter the outflow if at last.

7. The method according to claim 6, wherein the isoelectric point Z is the isoelectric point of the polypeptide Y x the content thereof in the complex + OmpF isoelectric point 4.5 x the content thereof in the complex;

the Buffer A is an aqueous solution containing 10mM Tris and 50mM NaCl, and the pH value of the Buffer A is the same as the isoelectric point value Z;

the Buffer B contains 50mM Tris, 500mM NaCl, 8M urea and PEI _10kd (2% m/v) Triton X-100 (1.5% v/v) in water (pH 4.5);

the ammonium sulfate precipitation specifically comprises the following steps: ammonium sulfate powder was added to the supernatant to 40% saturation, at which time the protein solution would aggregate and precipitate, and the precipitate was removed by centrifugation at 12000rpm (X21235 g) for 30min at 25 ℃.

8. The method of claim 1, wherein the solution of the high purity target protein is: dissolving the freeze-dried protein in a 90mM LiBr solution to prepare a 100mg/ml protein solution;

the crosslinking reaction is as follows: according to the following steps: gradually dropwise adding the solution of the high-purity protein into the tannic acid solution in a volume ratio of 1, and stirring by using a stirring rod after dropwise adding is finished to form the hydrogel adhered to the stirring rod.

9. An underwater adhesive hydrogel prepared by the method according to any one of claims 1 to 8, wherein the hydrogel is a soft and moldable light brown solid with abundant pore structures and the underwater adhesive strength is 1 to 100 kpa.

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