A method for producing protein HFBI

Abstract

The present invention discloses a method for producing protein HFBI. The method comprises the following steps: infecting insect cells with a recombinant baculovirus containing an insect cell expression vector expressing a fusion protein, and collecting the supernatant; the fusion protein is composed of a GP64 signal peptide and a protein HFBI; the amino acid sequence of the GP64 signal peptide is shown in SEQ ID NO:2 from the 1st to the 20th position from the N-terminus; then the supernatant is precipitated with ammonium sulfate to obtain a large amount of protein HFBI, and finally the protein is further purified by heating using the thermal stability. The purity of the protein HFBI obtained by the method provided by the present invention is more than 95%, the concentration is 6 mg / ml, and it has biological activity. It can be seen that the present invention can significantly increase the yield of protein HFBI, and the production process is simple, which can be fully applied to actual production. The present invention has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing protein HFBI. Background Art

[0002] Fungal hydrophobins are a class of small molecular weight proteins (7-15kDa) secreted by higher filamentous fungi. They can self-assemble on the surface of hyphae and spores to form amphiphilic protein films, playing a key role in different developmental stages of fungi. Fungal hydrophobins can self-assemble at the water-air interface to form amphiphilic protein films of nanometer thickness, with the hydrophilic side of the protein film facing the water layer and the hydrophobic side facing the air interface. As an amphiphilic protein, fungal hydrophobins exhibit obvious hydrophobic and hydrophilic regions on their surface, making fungal hydrophobins one of the most surface-active proteins. Good biocompatibility, unique self-assembly properties and high film-forming stability make fungal hydrophobins widely used in research in the fields of food emulsification, drug delivery, biomaterial modification and biosensors.

[0003] Fungal hydrophobins can be divided into two categories, type I and type II, based on their physical properties and hydrophilicity distribution. Type I fungal hydrophobins are highly insoluble and can only be dissolved in a few organic solvents such as formic acid and trifluoroacetic acid. Type II fungal hydrophobins can be dissolved in many solvents, such as 1-2% (m / v) SDS aqueous solution and 60% (v / v) ethanol aqueous solution. The biologically active type II fungal hydrophobin solution can form a hydrophobin film at the air-liquid interface.

[0004] Type II fungal hydrophobic proteins can be obtained by original bacteria culture, or expressed by Escherichia coli and yeast. Although original bacteria culture can obtain biologically active type II fungal hydrophobic proteins, the yield is very low. Although the type II fungal hydrophobic proteins expressed by Escherichia coli are large in quantity, they are not well folded and cannot self-assemble into films. Yeast can secrete and express biologically active type II fungal hydrophobic proteins, but methanol must be used as an inducing agent during expression, so this method limits the scale of industrial production. Methods for purifying type II fungal hydrophobic proteins include SDS extraction, two-phase system ATPS separation, and various chromatography methods, but each method has certain limitations. For example, SDS extraction and two-phase system ATPS separation require the use of a large amount of inorganic or organic reagents, and the waste liquid generated after separation needs additional treatment, and the final type II fungal hydrophobic protein solution contains a large amount of salts and pigments, which also need to be removed by subsequent processes; the scale of crude protein samples that can be processed by chromatography in practical applications is too small, and the fillers used in various chromatography are expensive, so it is not suitable for large-scale use. Up to now, there is no method for expressing and purifying type II fungal hydrophobic proteins that can be applied to large-scale industrial production. The main problems are the lack of methods for efficiently expressing type II fungal hydrophobic proteins and the complexity and high cost of existing purification methods.

[0005] The protein HFBI is a type II fungal hydrophobin and the only protein currently used to make amphiphilic membranes (FanH, Wang B, ZhangY, ZhuY, Song B, Xu H, Zhai Y, Qiao M, Sun F: Acryo-electronmicroscopy support film formedby 2D crystals ofhydrophobin HFBI. Nat Commun 2021, 12: 7257.). Summary of the invention

[0006] The object of the present invention is to produce the protein HFBI.

[0007] The present invention first protects the use of a fusion protein in producing protein HFBI. The fusion protein consists of a GP64 signal peptide and a protein HFBI. The amino acid sequence of the GP64 signal peptide may be as shown in SEQ ID NO:2 from positions 1 to 20 from the N-terminus. The amino acid sequence of the protein HFBI may be as shown in SEQ ID NO:2 from positions 21 to 95 from the N-terminus. The amino acid sequence of the fusion protein may be as shown in SEQ ID NO:2.

[0008] The use of a nucleic acid molecule encoding any of the above-mentioned fusion proteins in the production of protein HFBI also falls within the protection scope of the present invention.

[0009] The nucleotide sequence of the nucleic acid molecule encoding any of the above fusion proteins may be as shown in SEQ ID NO: 1 from position 7 to position 294 from the 5' end. The nucleotide sequence shown in SEQ ID NO: 1 from position 7 to position 66 from the 5' end encodes the GP64 signal peptide, and the nucleotide sequence shown in SEQ ID NO: 1 from position 67 to position 294 from the 5' end encodes the protein HFBI.

[0010] The use of a recombinant vector containing a nucleic acid molecule encoding any of the above fusion proteins in producing protein HFBI also falls within the scope of protection of the present invention. The recombinant vector expresses any of the above fusion proteins; the original vector constituting the recombinant vector may be an insect cell expression vector.

[0011] The insect cell expression vector may be specifically a pFastBac-1 vector. The recombinant plasmid pFastbac-HFBI may be a recombinant plasmid obtained by replacing the small DNA fragment between the restriction endonucleases BamHI and HindIII of the pFastBac-1 vector with the DNA molecule shown at positions 7 to 294 from the 5' end of SEQ ID NO:1. Recombinant plasmid

[0012] pFastbac-HFBI expresses a fusion protein whose amino acid sequence is shown in SEQ ID NO:2.

[0013] The use of a recombinant baculovirus containing any of the above-mentioned recombinant vectors in the production of protein HFBI also falls within the protection scope of the present invention.

[0014] The recombinant baculovirus can be obtained by transforming any of the above-mentioned recombinant vectors containing a nucleic acid molecule encoding any of the above-mentioned fusion proteins into DH10 Bac competent cells, and then screening with antibiotics (specific antibiotics are gentamicin, tetracycline and kanamycin) and blue-white screening.

[0015] The present invention also protects a method for producing protein HFBI, comprising the steps of (1): infecting insect cells with any of the above-mentioned recombinant baculoviruses, and collecting supernatant 1; the supernatant 1 contains protein HFBI;

[0016] The N-terminal 20 amino acids of the fusion protein are cleavable GP64 signal peptides. When the fusion protein is secreted into the culture medium through the insect cell secretion pathway, the GP64 signal peptide will be cleaved, and therefore, what is secreted into the culture medium is the protein HFBI.

[0017] In the step (1), the insect cells may be sf9 cells or High 5 cells.

[0018] In the step (1), the culture parameters for infecting insect cells with any of the above-mentioned recombinant baculoviruses may be: 25-30°C (e.g., 25-28°C, 28-30°C, 25°C, 28°C or 30°C), 100-200rpm (e.g., 100-150rpm, 150-200rpm, 100rpm, 150rpm or 200rpm), and culture for 80-100 hours (e.g., 80-96 hours, 96-100 hours, 80 hours, 96 hours or 100 hours).

[0019] The above method may also include step (2): adding ammonium sulfate to the supernatant 1 collected in step (1), mixing, and obtaining a precipitation system; then stirring, centrifuging, and separating from top to bottom into three layers: a liquid layer, a protein aggregation layer, and a precipitation layer; collecting the protein aggregation layer and dissolving it with water to obtain a protein dissolved solution.

[0020] It should be noted that, conventionally, the ammonium sulfate method is used to precipitate proteins to collect the precipitation layer, while the present application innovatively collects the protein aggregation layer, and only the protein aggregation layer contains a large amount of protein HFBI.

[0021] In the step (2), the concentration of ammonium sulfate in the precipitation system may be 1-3M (eg, 1-2M, 2-3M, 1M, 2M or 3M).

[0022] In the step (2), the stirring time may be 1-3 h (e.g., 1-2 h, 2-3 h, 1 h, 2 h or 3 h).

[0023] The above method may further include step (3): taking the protein solution obtained in step (2), treating it at 85°C-95°C (such as 85°C-90°C, 90°C-95°C, 85°C, 90°C or 95°C) for 20min-40min (such as 20min-30min, 30min-40min, 20min, 30min or 40min), then centrifuging to collect supernatant 2; supernatant 2 contains protein HFBI.

[0024] Experiments have shown that the purity of the protein HFBI produced by the method established by the present invention is more than 95%, the concentration is 6 mg / ml, and it has biological activity. It can be seen that the present invention can significantly increase the yield of protein HFBI (in one embodiment of the present invention, about 60 mg of protein HFBI was purified from 500 ml of insect cell culture fluid (specifically 500 ml of High 5 cell fluid)), and the production process is simple and can be fully applied to actual production. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SDS-PAGE result of the supernatant collected in step 4 of Example 1.

[0026] Figure 2 The biological activity identification results of the protein HFBI expressed and purified in Example 1 are shown in FIG.

[0027] Figure 3 The SDS-PAGE result is for comparative example.

[0028] Figure 4 The biological activity identification results of the proteins expressed and purified in the comparative example. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] Example 1. Expression and purification of protein HFBI

[0032] Protein HFBI (described in the following literature: T., Aro, N., Kalkkinen, N., Alatalo, E., and M. (1996) Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI, Eur. J. Biochem. 235, 248-255.) is composed of 75 amino acids and has a molecular weight of 7.5 kDa.

[0033] After a lot of experiments, the inventors of this application established the expression and purification method of protein HFBI, which is as follows:

[0034] 1. Construction of recombinant plasmid pFastbac-HFBI

[0035] 1. After a large number of experiments, the inventors of the present application designed and commissioned Shengyuan Kemeng Company to synthesize the DNA molecule shown in SEQ ID NO: 1. From the 5' end, positions 1 to 6 of SEQ ID NO: 1 are the recognition sequence of the restriction endonuclease BamHI, positions 7 to 66 encode the GP64 signal peptide, positions 67 to 294 encode the protein HFBI, and positions 295 to 300 are the recognition sequence of the restriction endonuclease HindIII.

[0036] SEQ ID NO: 1:

[0037] GGATCCATGGTCTCTGCCATCGTGTTGTACGTGCTCTTGGCTGCTGCTGCACACT

[0038] CAGCTTTCGCCTCTAACGGTAATGGAAACGTCTGTCCACCTGGCCTGTTCAGTAA

[0039] TCCACAGTGCTGTGCTACTCAGGTGCTGGGTCTCATCGGCCTCGACTGCAAAGT

[0040] GCCTTCTCAGAACGTTTACGATGGTACCGACTTCAGAAACGTCTGCGCAAAGAC

[0041] AGGTGCACAACCTCTCTGTTGTGTCGCTCCAGTGGCTGGCCAGGCACTGCTTTG

[0042] CCAAACTGCTGTCGGCGCTTAAAAGCTT

[0043] 2. Use restriction endonucleases BamHI and HindIII to digest the DNA molecule shown in SEQ ID NO: 1 and recover the DNA fragments.

[0044] 3. The pFastBac-1 vector (product of ThermoFisher, catalog number 10360014) was digested with restriction endonucleases BamHI and HindIII to recover the vector backbone.

[0045] 4. Connect the DNA fragment recovered in step 2 and the vector backbone recovered in step 3 to obtain the recombinant plasmid pFastbac-HFBI.

[0046] The recombinant plasmid pFastbac-HFBI was sequenced. The sequencing results showed that the recombinant plasmid pFastbac-HFBI was obtained by replacing the small DNA fragment between the restriction endonucleases BamHI and HindIII of the pFastBac-1 vector with the DNA molecule shown in positions 7 to 294 from the 5' end of SEQ ID NO:1.

[0047] The recombinant plasmid pFastbac-HFBI expresses a protein with an amino acid sequence as shown in SEQ ID NO: 2

[0048] GP64-HFBI. In SEQ ID NO: 2, from the N-terminus, positions 1 to 20 are the GP64 signal peptide (1-20 amino acids at the N-terminus of the baculovirus envelope protein GP64), and positions 21 to 95 are the protein HFBI.

[0049] SEQ ID NO:2:

[0050] MVSAIVLYVLLAAAAHSAFASGNGNVCPPGLFSNPQCCATQVLGLIGLDCKVPSQ

[0051] NVYDGTDFRNVCAKTGAQPLCCVAPVAGQALLCQTAVGA

[0052] 2. Expression of HFBI Protein

[0053] 1. The recombinant plasmid pFastbac-HFBI was transformed into DH10 Bac competent cells (product of ThermoFisher, catalog number 10361012), and then selected by antibiotics (specific antibiotics are gentamicin, tetracycline and kanamycin) and blue-white screening (for specific operation steps, refer to the Bac to Bac operation manual of Invitrogen) to obtain several baculoviruses.

[0054] 2. Extract the plasmids of several baculoviruses obtained in step 1 respectively and use them as templates, and use the primer pair consisting of primer M13Forward: 5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO: 3) and primer M13 Reverse: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 4) to perform PCR amplification to obtain PCR amplification products; then make the following judgment: if the PCR amplification product obtained from a baculovirus contains a band of about 2600 bp and does not have a band of about 300 bp, the baculovirus is a recombinant baculovirus (see the Bac to bac operation manual for specific explanations).

[0055] 3. Dilute sf9 cells with a survival rate greater than 98% and in the logarithmic growth phase with Grace's Insect Cell Culture Medium, Unsupplemented (Invitrogen) to obtain a cell density of 5.0×10 5 / ml of sf9 cell fluid.

[0056] 4. Mix solution A and solution B and leave at room temperature for 25 min to obtain a mixture.

[0057] Solution A consisted of 100 μl Grace's Insect Cell Culture Medium, Unsupplemented and 2.5 μg recombinant baculovirus.

[0058] Solution B consisted of 100 μl Grace's Insect Cell Culture Medium, Unsupplemented and 8 μl cellfectin II (Invitrogen).

[0059] 5. Take a plate (35 mm in size), spread 2 ml of the sf9 cell solution obtained in step 3, and culture at 28°C for 4 hours; then add the mixture obtained in step 4 dropwise to the plate, culture at 28°C for 4 hours, discard the culture medium, and then add fresh 2 ml of SFM-900II culture medium (product of ThermoFisher, catalog number 10902088), and culture at 28°C for 5 days.

[0060] After 5 days of static culture, sf9 cells were observed to swell and show obvious signs of viral infection.

[0061] 6. After completing step 5, collect the supernatant in the plate, record it as virus solution P1, and store it at 4°C away from light.

[0062] 7. After completing step 6, inoculate 200 μl of virus solution P1 into 180 ml of sf9 cell solution (cell density is 2.0×10 6 / ml) in a triangular shake flask (specification: 2L), culture at 28°C and 150rpm for 96 hours; then centrifuge at 4°C and 2000g for 5 minutes, collect the supernatant, record it as virus solution P2, and store it at 4°C in the dark.

[0063] 8. After completing step 7, inoculate 20 ml of virus solution P2 into 500 ml of High 5 cell solution (cell density is 2.0×10 6 / ml) in a 2L shake flask at 28°C and 150 rpm for 96 hours; then centrifuge at 4°C and 8000 rpm for 10 minutes to collect the supernatant.

[0064] The supernatant collected in step 8 contains the protein HFBI.

[0065] It should be noted that the N-terminal 20 amino acids of the protein GP64-HFBI are cleavable GP64 signal peptides. When the protein GP64-HFBI is secreted into the culture medium through the insect cell secretion pathway, the GP64 signal peptide has been cleaved, and therefore, what is secreted into the culture medium is the protein HFBI.

[0066] 3. Purification of HFBI Protein

[0067] 1. Take the supernatant collected in step 8 of step 2 and concentrate it 10 times using an ultrafiltration membrane system with a cutoff value of 30 kDa (Sartorius VivaFlow200) to obtain a concentrated supernatant.

[0068] 2. After completing step 1, add ammonium sulfate powder (for the purpose of precipitation) to the concentrated supernatant to obtain a precipitation system; in the precipitation system, the concentration of ammonium sulfate is 2M.

[0069] 3. After completing step 2, take the precipitation system, stir at room temperature for 2 hours, and then centrifuge at 4°C and 14000rpm for 30 minutes, and divide it into three layers from top to bottom, namely, a liquid layer, a HFBI protein aggregation layer at the gas-liquid interface, and a bottom precipitation layer. Discard the liquid layer and the bottom precipitation layer, then collect the HFBI protein aggregation layer, dissolve it with an appropriate amount of water, and obtain a protein solution.

[0070] 4. After completing step 3, place the protein solution in a 90°C water bath for 30 minutes, then centrifuge at 4°C and 14,000 rpm for 10 minutes, and collect the supernatant (about 10 ml).

[0071] The purpose of this step is to remove residual impurities.

[0072] The supernatant collected in step 4 was subjected to SDS-PAGE. The results are shown in Figure 1 (Lane 1-3 are all supernatants collected in step 4.) The results show that after ammonium sulfate precipitation and heating treatment, the purity of the protein HFBI in the supernatant collected in step 4 is above 95%.

[0073] The concentration of HFBI protein in the supernatant collected in step 4 was measured using a BCA kit (product of ThermoFisher, catalog number 23227). The results showed that the concentration of HFBI protein in the supernatant collected in step 4 was 6 mg / ml; that is, about 60 mg of HFBI protein was purified from 500 ml of insect cell culture fluid (specifically, 500 ml of High 5 cell fluid).

[0074] Example 2: Identification of biological activity of the protein HFBI expressed and purified in Example 1

[0075] A biologically active type II hydrophobin solution which can form a hydrophobin film at the air-liquid interface.

[0076] Based on this principle, the biological activity of the protein HFBI expressed and purified in Example 1 was identified. The specific steps are as follows:

[0077] 1. Place culture dish 1 (with a diameter of 90 mm) upside down in culture dish 2 (with a diameter of 150 mm), then attach a paraffin sealing film to the surface of culture dish 1, and add a certain amount of water around culture dish 1 to supplement the humidity.

[0078] 2. Dilute the supernatant collected in step 3 of Example 1 with water to obtain a protein HFBI solution with a concentration of 100 μg / ml. Drop the protein HFBI solution and water (as a control) on the paraffin sealing film, 35 μL per drop.

[0079] 3. After completing step 2, cover the culture dish 3 (with a diameter of 150 mm) with its lid and allow to equilibrate for 15 minutes.

[0080] 4. After completing step 3, open the lid and observe after 15 minutes.

[0081] Results Figure 2 (HFBI is a protein HFBI solution, and the arrow is a HFBI membrane (i.e., a hydrophobic protein membrane formed by the protein HFBI solution). The results showed that a flat top appeared on the surface of the droplet after 15 minutes, i.e., a HFBI membrane was formed. It can be seen that the protein HFBI expressed and purified by the method established in Example 1 has biological activity.

[0082] Effect of replacing GP64 signal peptide with bee venom signal peptide on expression and purification of protein HFBI in comparative example and example 1

[0083] Signal peptides are short peptide chains that guide newly synthesized proteins to transfer to the secretory pathway. Signal peptides are used to guide exogenous proteins to be secreted into specific compartments of cells, thereby improving solubility and avoiding difficulties caused by inclusion body renaturation. Studies have shown that after a variety of exogenous genes are connected to signal peptides, they are secreted and expressed in prokaryotic expression systems such as Escherichia coli, L-form bacteria, Bacillus and Lactobacillus; signal peptides are also widely used in eukaryotic expression systems such as Pichia pastoris and insect baculovirus expression systems. There are many types of secretory expression signal peptides. In order to determine whether any secretory expression signal peptide can be used to produce the active protein HFBI, the applicant conducted the following experiments:

[0084] 1. Construction of recombinant plasmid HFBI

[0085] According to the method of step 1 in Example 1, the nucleic acid molecule encoding the GP64 signal peptide (shown in positions 7 to 66 from the 5' end of SEQ ID NO: 1) was replaced with the nucleic acid molecule encoding the melittin signal peptide, and the other steps remained unchanged to obtain the recombinant plasmid HFBI.

[0086] The amino acid sequence of the melittin signal peptide is shown in SEQ ID NO:5.

[0087] Melittin signal peptide belongs to the secretory expression signal peptide.

[0088] 2. Protein Expression

[0089] According to the method of step 2 in Example 1, the recombinant plasmid pFastbac-HFBI was replaced by the recombinant plasmid HFBI, and the other steps remained unchanged, and the supernatant was collected.

[0090] 3. Protein Purification

[0091] According to the method of step 3 in Example 1, the supernatant collected in step 8 of step 2 of Example 1 is replaced by the supernatant collected in step 2, and the other steps remain unchanged.

[0092] SDS-PAGE results are shown in Figure 3 Theoretically, the protein HFBI without the signal peptide is natural HFBI, with a molecular weight of 7.5 kDa, while the fusion protein without the signal peptide has a molecular weight of 10 kDa (see Figure 3 Thus, the cleavage efficiency of the melittin signal peptide is not high, resulting in most of the purified proteins HFBI N-terminus still carrying a signal peptide, that is, the molecular weight of the protein purified using the melittin signal peptide is high.

[0093] IV. Identification of biological activity of the purified protein in step 3

[0094] According to the method of Example 2, the protein HFBI was replaced by the protein obtained in step 3, and the other steps remained unchanged.

[0095] The protein film formation results obtained in step 3 are shown in Figure 4 The results showed that no flat top appeared on the surface of the droplet after 15 minutes, that is, the HFBI film could not be generated. It can be seen that the GP64 signal peptide in the method established in Example 1 was replaced with the bee venom signal peptide, although both are secretory expression signal peptides, the use of the bee venom signal peptide cannot express and purify the biologically active protein HFBI.

[0096] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of fusion protein in producing biologically active protein HFBI; The fusion protein consists of the GP64 signal peptide and the protein HFBI; The amino acid sequence of the GP64 signal peptide is shown in SEQ ID NO: 2 from position 1 to position 20 from the N-terminus; The amino acid sequence of the protein HFBI is shown in SEQ ID NO: 2 from position 21 to position 95 from the N-terminus.

2. Use of the nucleic acid molecule encoding the fusion protein of claim 1 in producing a protein HFBI having biological activity; The amino acid sequence of the protein HFBI is shown in SEQ ID NO: 2 from position 21 to position 95 from the N-terminus.

3. Use of a recombinant vector containing the nucleic acid molecule of claim 2 in producing a protein HFBI having biological activity; The amino acid sequence of the protein HFBI is shown in SEQ ID NO:2 from position 21 to position 95 from the N-terminus; The recombinant vector expresses the fusion protein described in claim 1; the original vector constituting the recombinant vector is an insect cell expression vector.

4. Use of a recombinant baculovirus containing the recombinant vector of claim 3 in producing a protein HFBI having biological activity; The amino acid sequence of the protein HFBI is shown in SEQ ID NO: 2 from position 21 to position 95 from the N-terminus.

5. A method for producing a protein HFBI having biological activity, comprising the steps of (1): infecting insect cells with the recombinant baculovirus of claim 4, and collecting supernatant 1; the supernatant 1 contains the protein HFBI having biological activity; The amino acid sequence of the protein HFBI is shown in SEQ ID NO: 2 from position 21 to position 95 from the N-terminus.

6. The method according to claim 5, characterized in that: The method also includes step (2): adding ammonium sulfate to the supernatant 1 collected in step (1), mixing, and obtaining a precipitation system; then stirring, centrifuging, and separating from top to bottom into three layers: a liquid layer, a protein aggregation layer, and a precipitation layer; collecting the protein aggregation layer and dissolving it with water to obtain a protein solution.

7. The method according to claim 6, characterized in that: In the precipitation system, the concentration of ammonium sulfate is 1-3 M; and the stirring time is 1-3 h.

8. The method according to claim 6, characterized in that: The method further comprises step (3): taking the protein solution obtained in step (2), treating it at 85°C-95°C for 20min-40min, then centrifuging it, and collecting supernatant 2; supernatant 2 contains the biologically active protein HFBI.

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