CN104195157A - High-efficiency recombination expression and purification method of biological active peptide in prokaryotic cells - Google Patents

Abstract

The invention discloses a high-efficiency recombinant expression and purification method of biologically active peptides in prokaryotic cells, including the construction of recombinant expression vector pHGB1-TEV, growth factor recombinant expression plasmids pHGB1-TEV-hEGF, pHGB1-TEV-mEGF and pHGB1-TEV- LL-37 and high-efficiency expression and purification of antimicrobial peptide LL-37 and human and mouse epidermal growth factors in Escherichia coli JM109 strain. The invention adopts modern biotechnology to realize the production of human epidermal growth factor and antibacterial peptide. The N-terminus of the recombinant protein expressed through this system has the sequence His ₆ -GB1-TEV. The six histidine tags facilitate the purification of the recombinant protein through the Ni-NTA column. Excision, so as to obtain the target biologically active peptide. Bioactive peptides have multiple functions and have a huge market demand. Using this technology to recombine, express and purify bioactive peptides has the advantages of high yield, high activity, simple process, and low production cost, and has high industrial development value.

Description

High-efficiency recombinant expression and purification method of biologically active peptides in prokaryotic cells

technical field

The invention relates to the efficient expression and purification of bioactive peptides such as antibacterial peptide LL-37 and epidermal growth factor in a prokaryotic expression system using fusion expression technology, and specifically relates to a high-efficiency recombinant expression and purification method of bioactive peptides in prokaryotic cells, belonging to biological engineering field.

Background technique

The antimicrobial peptide LL-37 is a cathelicidin antimicrobial peptide produced by the human natural immune system. Its precursor peptide hCAP-18 is mainly released by neutrophils, which is cleaved by serine protease to produce active LL-37, which has the ability to resist the invasion of foreign microorganisms, Eliminate various physiological functions such as mutant cells in the body. LL-37 is a 37-peptide starting with two leucines, with a molecular weight of 4.5 kDa and a facultative α-helix spatial structure. Since LL-37 contains more

Basic amino acids, with a positive charge on the surface of the molecule, kill bacteria by interacting with the negatively charged bacterial cell membrane.

Both human and mouse epidermal growth factors (EGF) contain 53 amino acids and three pairs of intramolecular disulfide bonds. EGF is a multifunctional cell growth factor. EGF can promote cell mitosis and the synthesis of sugar, protein, DNA, and RNA. Therefore, it has a wide range of effects on promoting the division and proliferation of epithelial cells. It is clinically associated with many diseases, such as immune skin diseases, Wound tissue repair and periodontitis, etc.

The traditional biological extraction and purification of bioactive peptides are complex, cumbersome and low yield. In every step of the chemical synthesis method of peptides, its by-products must be removed, so the longer the length of the amino acid sequence, the more complicated the chemical synthesis of the peptide, the more by-products, the more complicated the purification, and the more expensive the price. It is the most economical and effective method to express these proteins or polypeptides recombinantly by genetic engineering technology. Because many biologically active peptides derived from higher organisms have complex spatial structures, contain many disulfide bonds, or have many surface charges, it is difficult to directly express them in prokaryotic organisms. Exploring better recombinant expression systems to express and purify bioactive peptides has great theoretical and economic value.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, provide a highly efficient and stable recombinant expression and separation and purification technology of biologically active peptides, and realize the efficient recombinant expression and rapid separation of biologically active peptides in prokaryotic expression systems through fusion expression technology Purification greatly reduces the production cost of bioactive peptides.

The high-efficiency recombinant expression and purification method of bioactive peptides in prokaryotic cells provided by the present invention includes the following aspects:

(1) Construction of recombinant plasmid pHGB1-TEV

Plasmid pET-22b (+) was extracted, treated with restriction endonucleases Nde I and Bam HI, and recovered from the gel to obtain a large fragment (linear fragment of pET-22b (+) plasmid after double digestion);

The His ₆ -GB1-TEV (TEV proteinase restriction site) DNA coding sequence was synthesized, its nucleotide sequence is shown in SEQ ID NO.1, and its protein sequence is shown in Figure 2.

Use primers with Nde I and Bam HI restriction sites to amplify the His ₆ -GB1-TEV coding DNA fragment by PCR, Nde I and Bam HI double digestion treatment, gel recovery to obtain small fragments (with restriction endonucleases) The DNA fragment of the enzyme cleavage site (His ₆ -GB1-TEV coding sequence)).

Large fragments and small fragments were ligated with T4 DNA ligase at a ratio of 1:5; the ligated products were transformed into Escherichia coli DH5α competent, after ampicillin resistance screening, positive clones were picked, plasmids were extracted and subjected to restriction endonuclease Nde I And Bam HI double digestion identification, through DNA sequencing to determine the recombinant plasmid: pHGB1-TEV.

(2) Construction of recombinant plasmid pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF)

The recombinant plasmid pHGB1-TEV was extracted, digested with restriction endonucleases Bam H I and Xho I, electrophoresed on 1% agarose gel, and a large fragment was recovered from the gel (the linear fragment of pHGB1-TEV after double digestion).

Synthesized LL-37 (Gene ID: 820), hEGF (Gene ID: 1950) and mEGF (Gene ID: 13645) gene fragments, the nucleotide sequences of which are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO .4 shown. LL-37, hEGF and mEGF gene fragments were amplified by PCR using primers with Bam H I and Xho I restriction sites respectively; the PCR products were double digested with Bam H I and Xho I and ligated with large fragments; the ligated products were transformed into

Escherichia coli DH5α was competent, after ampicillin resistance screening, positive clones were picked, the plasmid was extracted and identified by restriction endonuclease Bam H I and Xho I double digestion, and the recombinant plasmid pHGB1-TEV-LL was confirmed by DNA sequencing -37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF)

(3) Expression and purification of LL-37 (or mEGF or hEGF)

The recombinant plasmid pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF) was transformed into Escherichia coli JM109 competent to obtain an engineered strain expressing LL-37 (or mEGF or hEGF).

Pick the monoclonal engineered strain of LL-37 (or mEGF or hEGF) in LB liquid medium and culture overnight at 37°C with shaking. Transfer the overnight culture to a new LB liquid medium containing ampicillin at a ratio of 1:100, culture with shaking at 37°C for about 3-4 hours, add IPTG to a final concentration of 0.2 mM when the _OD600 value is 0.6, and store at 22°C Induce overnight.

Collect the bacteria by centrifugation at 5000 rpm for 15 minutes, discard the supernatant and invert for 1 minute to drain the residual medium. Resuspend the pellet with bacteriostasis buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, pH 8.0), centrifuge at 5000 rpm for 10 minutes to collect the bacteria, and discard the supernatant. Add 50 mL of bacteriostasis buffer to the pellet and pipette evenly, add PMSF (phenylmethylsulfonyl fluoride) at a ratio of 1:200, and ultrasonically destruct the bacteria for 15-20 minutes until translucent. Centrifuge the bacterial disruption liquid at 15,000 rpm for 15 minutes, and take the supernatant. Purify on a Ni-NTA column, and slowly drop the sample to make the protein better bind to the column. After loading the sample, first wash 5 column volumes with bacteriostasis buffer. Then use elution buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, 250 mM imidazole, pH8.0) to elute, and detect the protein elution by 15% SDS-PAGE. The protein eluted with Ni-NTA was further purified by fast protein liquid chromatography (FPLC). To the purified fusion protein, add 100 μg of TEV enzyme (with His ₆ tag) and mix well at 4 °C for overnight enzyme digestion (ENLYFQ↓ G). The enzyme digestion buffer is: (50 mM Tris-HCl, 0.5 mM EDTA, 1mM DTT , pH 8.0) digested product slowly flowed through the Ni-NTA column, and the collected protein solution was the cleaved LL-37 (or mEGF or hEGF) without His ₆ tag.

Wherein, the TEV-specific cutting enzyme described in step (3) is prepared by the following method:

The recombinant expression plasmid pET-28b-TEV protease (preserved in our laboratory) was transformed into Escherichia coli JM109 competent to obtain an engineering strain expressing TEV protease. Pick a single clone in 10 mL of LB liquid medium containing kanamycin and culture overnight at 37°C with shaking. Transfer the overnight culture to a new 1L LB liquid medium containing kanamycin at a ratio of 1:100, culture with shaking at 37°C for about 3-4 hours, and add IPTG to a final concentration of 0.2 mM when the _OD600 value is 0.6 , induced overnight at 22°C.

Collect the bacteria by centrifugation at 5000 rpm for 15 minutes, discard the supernatant and invert for 1 minute to drain the residual medium. Resuspend the pellet with bacteriostasis buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, pH 8.0), centrifuge at 5000 rpm for 10 minutes to collect the bacteria, and discard the supernatant. Take 50 ml of bacteriostasis buffer and add it to the pellet and pipette evenly, add PMSF (phenylmethylsulfonyl fluoride) at a ratio of 1:200, ultrasonicate for 15-20 minutes to destruct the bacteria until translucent. Bacterial disruption solution was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was taken. Purify on a Ni-NTA column, and slowly drop the sample to make the protein better bind to the column. After sample loading, wash 5 column volumes with bacteriostasis buffer, then elute with elution buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, 250 mM imidazole, pH8.0), 15% SDS-PAGE Check protein elution. The protein eluted with Ni-NTA was further purified by fast protein liquid chromatography (FPLC).

Beneficial effects of the present invention:

The present invention adopts modern biotechnology to construct the prokaryotic recombinant expression carrier pHGB1-TEV, and clone and express LL-37, mEGF, hEGF and other bioactive peptides on this basis. In this fusion expression technology, the fused GB1 domain can greatly increase the expression level and water solubility of the recombinant protein, the His ₆ tag is convenient for purification, and the introduction of the TEV protease cleavage site makes these fusion expressed sequences very convenient to be excised. Using the invention to produce bioactive peptides is fast and efficient, has low cost, high yield and high activity, and has very broad market prospects.

Description of drawings

Figure 1 is a diagram showing the results of PCR amplification of the His ₆ -GB1-TEV coding sequence. In the figure, the left lane is the molecular weight marker, and the right lane is the His ₆ -GB1-TEV coding sequence.

Figure 2 is a schematic diagram of the protein sequence of the His ₆ -GB1-TEV coding sequence. The middle sequence in the figure is the GB1 coding sequence, and the front and rear sequences are the coding sequences of His ₆ and TEV enzyme recognition sites, respectively.

Fig. 3 is a construction map of the recombinant plasmid pHGB1-TEV.

Figure 4 is a construction map of the recombinant plasmid pHGB1-TEV-LL-37.

Detailed ways

The present invention is specifically described or further illustrated by the following examples, the purpose of which is to better understand the technical connotation of the present invention, but the protection scope of the present invention is not limited to the following implementation scope.

Example 1: Construction of recombinant expression vector: pHGB1-TEV

(1) Test material

Plasmids and strains: Plasmid pET-22b (+) and Escherichia coli DH5α were purchased from Novagen and Takara Biotechnology Co., Ltd., respectively.

Reagents: restriction endonucleases Nde I and Bam HI, and kanamycin were purchased from NEB Company.

Gene fragment: His ₆ -GB1-TEV coding DNA sequence and primers were synthesized by Shanghai Sangong.

(2) Implementation plan

A. Enzyme digestion of plasmid pET-22b (+):

Plasmid pET-22b (+) was extracted in large quantities. After purification, restriction endonucleases Nde I and Bam HI were added and digested at 37°C for 3 hours. The digested product was electrophoresed on 1% agarose gel, and the large fragment (pET -22b (+) plasmid linear fragment after double digestion).

B. Obtaining the restriction fragment of His ₆ -GB1-TEV coding sequence:

Using the synthetic His ₆ -GB1-TEV coding sequence (as shown in SEQ ID NO.1) as a template, design forward and reverse primers with Nde I and Bam HI restriction sites respectively. After PCR amplification (the amplification results are shown in Figure 1, the left lane is the molecular weight marker, and the right lane is the His ₆ -GB1-TEV coding sequence, as indicated by the arrow, the fragment length is 201bp.) Then use Nde I and Bam HI Double enzyme digestion treatment, gel recovery to obtain small fragments (DNA fragments with restriction endonuclease sites (His6-GB1-TEV coding sequence)) for use.

The primer sequences are as follows: the underlined position is the restriction site.

Forward primer F ( Nde I): CGGGTTAAC CATATG CACCATCATCATCATCACCA

Reverse primer R ( Bam HI): GTT GGATCC CTGGAAATACAGATTTTCTT

C. Construction of recombinant plasmid pHGB1-TEV:

Large fragments and small fragments were ligated with T4 DNA ligase at a ratio of 1:5; the ligated products were transformed into Escherichia coli DH5α competent, after screening for ampicillin resistance, positive clones were picked, plasmids were extracted and subjected to restriction endonuclease Nde I and Bam HI double digestion identification, through DNA sequencing to determine the recombinant plasmid: pHGB1-TEV.

The construction map of the recombinant plasmid pHGB1-TEV is shown in Figure 3.

Example 2: Construction of recombinant plasmid pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF)

(1) Test material

Plasmid and strain: Escherichia coli DH5α was purchased from Takara Biotechnology Co., Ltd.

Reagents: restriction endonucleases Bam H I and Xho I, and kanamycin were purchased from NEB Company.

Gene fragment: LL-37 (or hEGF or mEGF) coding DNA sequence (as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4) and primers were synthesized by Shanghai Sangon.

(2) Implementation plan

A． Enzyme digestion of plasmid pHGB1-TEV

A large amount of plasmid pHGB1-TEV was extracted, and after purification, restriction endonucleases Bam H I and Xho I were added to digest at 37°C for 3 hours. The digested product was electrophoresed on 1% agarose gel, and the large fragment (pHGB1-TEV linear fragment after double digestion).

B． Obtaining LL-37 (or hEGF or mEGF) gene restriction fragments

Synthesized LL-37 (Gene ID: 820), hEGF (Gene ID: 1950) and mEGF (Gene ID: 13645) gene fragments, the nucleotide sequences of which are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO .4 shown.

Using the synthetic LL-37 (or hEGF or mEGF) coding sequence as a template, design forward and reverse primers with Bam H I and Xho I restriction sites, respectively. After PCR amplification, double digestion with Bam HI and Xho I was performed, and small fragments were recovered from the gel for use.

Primer sequences for amplifying LL-37:

Forward primer F ( Bam HI): GTT GGATCC CTGCTGGGTGATTTCTTC

Reverse primer R ( Xho I): CCG CTCGAG CTAGGACTCTGTCCTGGGTA

Primer sequences for amplifying hEGF:

Forward primer F ( Bam HI): GTT GGATCC AATAGTGACTCTGAATGT

Reverse primer R ( Xho I): CCG CTCGAG CTAGCGCAGTTCCCACCACTT

Primer sequences for amplifying mEGFF:

Forward primer F ( Bam HI): GTT GGATCC AATAGTTATCCAGGATGC

Reverse primer R ( Xho I): CCG CTCGAG CTAACGCAGCTCCCACCATCG

C． Construction of recombinant plasmid pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF)

Large fragments and small fragments were ligated with T4 DNA ligase at a ratio of 1:5; the ligated products were transformed into Escherichia coli DH5α competent, after screening for ampicillin resistance, positive clones were picked, plasmids were extracted and subjected to restriction endonuclease Bam H I and Xho I double enzyme digestion identification, through DNA sequencing to determine the recombinant plasmid: pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF).

The construction of the recombinant plasmid takes pHGB1-TEV-LL-37 as an example, as shown in FIG. 4 .

Example 3: Expression and purification of LL-37 (or hEGF or mEGF)

(1) Test material

Plasmid and strain: Escherichia coli BL21 (DE3) was purchased from Takara Biotechnology Co., Ltd.

Reagents: Ampicillin and IPTG were purchased from NEB Company.

(2) Implementation plan

A． Inducible expression of LL-37 (or hEGF or mEGF) fusion protein

The recombinant plasmid pHGB1-TEV-LL-37 (or pHGB1-TEV-hEGF or pHGB1-TEV-mEGF) was transformed into Escherichia coli JM109 competent to obtain a fusion expression LL-37 (or mEGF or hEGF) engineering strain.

Pick the monoclonal engineered strain of LL-37 (or hEGF or mEGF) in LB liquid medium and culture overnight at 37°C with shaking. Transfer the overnight culture to a new 1L LB liquid medium containing ampicillin at a ratio of 1:100, culture with shaking at 37°C for about 3-4 hours, and add IPTG to a final concentration of 0.2mM when the _OD600 value is 0.6, 22 °C induction overnight.

B. Purification of LL-37 (or hEGF or mEGF)

Collect the bacteria by centrifugation at 5000 rpm for 15 minutes, discard the supernatant and invert for 1 minute to drain the residual medium. Resuspend the pellet with bacteriostasis buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, pH 8.0), centrifuge at 5000 rpm for 10 minutes to collect the bacteria, and discard the supernatant. Take 50ml of bacteriostasis buffer and add it to the precipitate and pipette evenly, add PMSF (phenylmethylsulfonyl fluoride) at a ratio of 1:200, and ultrasonicate for 15-20 minutes until translucent. Centrifuge the bacterial disruption liquid at 15,000 rpm for 15 minutes, and take the supernatant. Purify on a Ni-NTA column, and slowly drop the sample to make the protein better bind to the column. After loading the sample, first wash 5 column volumes with bacteriostasis buffer.

Then use elution buffer (50 mM NaH ₂ PO ₄ , 500 mM NaCl, 250 mM imidazole, pH8.0) to elute, and detect the protein elution by 15% SDS-PAGE. The protein eluted with Ni-NTA was further purified by fast protein liquid chromatography (FPLC). To the purified fusion protein, add 100 μg of TEV enzyme (with His tag) and mix well at 4°C for overnight enzyme digestion (ENLYFQ↓ G). The enzyme digestion buffer is: (50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT , pH 8.0)) and then slowly flow the digested product through the Ni-NTA column, and the collected protein solution is the digested LL-37 (or hEGF or mEGF) without His tag.

Embodiment 4: MTT method measures the activity of hEGF or mEGF protein after the purification

(1) Test material

Cells: Mouse embryonic fibroblasts (Balb/C 3T3 cells) were purchased from ATCC.

Reagent:

Add 10 ⁵ IU of penicillin and 10 ⁵ IU of streptomycin to 1000 mL of RPMI 1640 medium, then add NaHCO ₃

2.1g, dissolved, mixed, sterilized and filtered, stored at 4°C; take 4 mL of fetal bovine serum (FBS) and add 1000 mL of RPMI 1640 medium to obtain maintenance culture medium; take 100 mL of fetal bovine serum (FBS) and add 1000 mL of RPMI1640 Culture medium to obtain complete culture solution; PBS: weigh 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na ₂ HPO ₃ , 0.24 g of KH ₂ PO ₃ , add water to 1000 mL, and sterilize at 121°C for 15 minutes; thiazolyl blue (MTT) Solution: Dissolve 0.1 g of MTT powder in 20 mL of PBS, filter through a 0.22 μm filter membrane, and store in the dark at 4°C.

(2) Implementation plan

A. Take the recombinant human epidermal growth factor standard product and reconstitute it according to the instructions, and then dilute it with maintenance medium to contain 50 IU per 1 mL. In a 96-well cell culture plate, 4-fold serial dilutions were made, a total of 8 dilutions, and 2 controls were made for each concentration. Aseptic operation;

B. After the sample is reconstituted, it is diluted with maintenance medium. In a 96-well cell culture plate, 4-fold serial dilutions were made, a total of 8 dilutions, and 2 controls were made for each concentration. Aseptic operation;

C. The Balb/c 3T3 cell line is cultured with complete culture medium at 37°C, 5% CO ₂ , the control cell concentration is 1.0×10 ⁵ -5.0×10 ⁵ cells per 1 mL, and it is used 24-36 hours after passage in the determination of biological activity. Discard the culture medium in the culture flask, digest and collect the cells, use the complete culture medium to make a cell suspension containing 5.0×10 ⁴ -8.0×10 ⁴ cells per 1 mL, and inoculate in a 96-well cell culture plate. Well 100 μL. Incubate at 37°C, 5% CO ₂ for 15-24h. For the prepared cell culture plate, discard the maintenance solution, add standard solution and sample solution, 100 μL per well. Incubate at 37°C, 5% CO ₂ for 60-72 h. Add 20 μL of MTT solution to each well, and incubate at 37°C, 5% CO ₂ for 2-8 h. The above operations were carried out under sterile conditions. After discarding the liquid in the culture medium, add 100 μL of dimethyl sulfoxide (DMSO) to each well, mix well, and measure the absorbance at a wavelength of 570 nm on a microplate reader with 630 nm as the reference wavelength. Record the measurement results.

The activity assay of Balb/C 3T3 cells showed that the hEGF sample had higher biological activity than the standard product, and the detected activities of the hEGF and mEGF samples were 3×10 ⁵ U/mg and 3.5×10 ⁵ U/mg, respectively.

Embodiment 5: the antibacterial activity experiment of LL-37

(1) Test material

Cells: Salmonella typhi S.typhi, preserved by our laboratory.

Reagents: ordinary LB liquid medium, antimicrobial peptide LL-37, polymyxin (PxB).

(2) Implementation plan

Pick a single colony of Salmonella typhi wild strain from the LB plate and place it in 2 mL LB liquid medium, shake overnight at 37°C, then transfer to 20 mL LB liquid medium at a ratio of 1:100, add antimicrobial peptides LL-37 (final concentration 50 μg/mL) and 20 mL LB liquid medium added with the same concentration of polymyxin (PxB). Shake the culture at 37°C, measure the OD ₆₀₀ every 1 h, and draw the growth curve. The results showed that LL-37 expressed and purified by this method had higher antibacterial (antibacterial) activity than polymyxin (PxB).

SEQUENCE LISTING

the

<110> Jiangsu University

the

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Claims (8)

1. A recombinant plasmid pHGB1-TEV, characterized in that, said plasmid is made up of original plasmid pET-22b (+) and His ₆ -GB1-TEV DNA coding sequence. 2. a recombinant plasmid pHGB1-TEV-LL-37, is characterized in that, described plasmid is made up of the nucleotide sequence of recombinant plasmid pHGB1-TEV and LL-37 gene described in claim 1, is used for antimicrobial peptide LL -37 expression. 3. A recombinant plasmid pHGB1-TEV-hEGF, characterized in that the plasmid is composed of the recombinant plasmid pHGB1-TEV according to claim 1 and the nucleotide sequence of hEGF gene, and is used for the expression of hEGF. 4. A recombinant plasmid pHGB1-TEV-mEGF, characterized in that the plasmid is composed of the recombinant plasmid pHGB1-TEV according to claim 1 and the nucleotide sequence of the mEGF gene, and is used for the expression of mEGF. 5. A recombinant engineering bacterium comprising the recombinant plasmid as claimed in claim 2. 6. A recombinant engineering bacterium comprising the recombinant plasmid as claimed in claim 3. 7. A recombinant engineering bacterium comprising the recombinant plasmid as claimed in claim 4. 8. The purification method of biologically active peptides in prokaryotic cells is characterized in that, in the purification process, the TEV enzyme with the His ₆ tag is added, and the specific operation of the purification method is as follows: Pick the recombinant engineering bacterial strain as claimed in claim 5 or claim 6 or claim 7 in LB liquid culture medium, Shake overnight culture; transfer the overnight culture to a new LB liquid medium containing ampicillin, culture with shaking at 37°C for about 3-4 hours, add IPTG to a final concentration of 0.2 mM when the OD ₆₀₀ value is 0.6, and induce at 22°C Collect the bacteria by centrifugation overnight, discard the supernatant and invert to drain the remaining medium; resuspend the pellet with the bacteria-breaking buffer, collect the bacteria by centrifugation, and discard the supernatant; add the bacteria-breaking buffer to the pellet and pipette evenly, add 1:200 Phenylmethylsulfonyl fluoride, ultrasonically break the bacteria until translucent; centrifuge the broken bacteria, take the supernatant; purify on the Ni-NTA column, slowly drop the sample to make the protein better bind to the column; after loading the sample, use a broken The column volume was washed with bacteria buffer, and then eluted with elution buffer, and the elution of protein was detected by 15% SDS-PAGE; the protein eluted by Ni-NTA was further purified by fast protein liquid chromatography FPLC; the purified fusion protein , add the TEV enzyme with His ₆ tag and mix well at 4 °C for overnight digestion, and then slowly flow the digested product through the Ni-NTA column, _and collect the effluent protein solution, which is the excised LL-37 or mEGF or hEGF.