Methods for expressing antimicrobial peptides in prokaryotes

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to a method for prokaryotic expression of antimicrobial peptides. The method provided by this invention involves inserting an antimicrobial peptide gene into the 3' or 5' end of a fusion tag gene for fusion expression. The protein, forming numerous inclusion bodies, eliminates the original antimicrobial activity of the antimicrobial peptide, thus preventing the killing of host bacteria. The method provided by this invention allows for the large-scale preparation of antimicrobial peptides in a prokaryotic expression system. The method is simple and easy to implement, and the obtained antimicrobial peptides exhibit significant in vitro antibacterial effects after enzymatic digestion and purification.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for expressing antimicrobial peptides in prokaryotes. Background Technology

[0002] Currently, due to the improper use of traditional antibiotics in clinical treatment, pathogenic microorganisms such as bacteria and fungi are developing strong resistance to antibiotics at an alarming rate. In particular, the emergence of multidrug-resistant and super-resistant bacteria often leads to global epidemics within a very short period, threatening human life and causing enormous losses in both health and the economy. Antimicrobial peptides are a class of small-molecule polypeptides produced by organisms to defend against the invasion of exogenous pathogenic microorganisms. Compared with traditional antibiotics, they have advantages such as small molecular weight, broad antibacterial spectrum, strong thermal stability, and unique antibacterial mechanisms. Utilizing antimicrobial peptides to replace traditional antibiotics is of great significance to the development of modern medicine and animal husbandry, and holds the promise of completely solving the international problems of drug residues and bacterial resistance. To date, thousands of antimicrobial peptides have been isolated and identified. Antimicrobial peptides generally consist of 10-60 amino acid residues, with a net charge mostly between +2 and +6, exhibiting strong cationicity (pI 8.9-10.7), and a molecular weight of approximately 2000-7000 Da.

[0003] The sources and preparation of antimicrobial peptides can be broadly categorized into three routes: natural biological extraction, chemical synthesis, and genetic engineering. However, natural product extraction is easily limited by source availability, especially for antimicrobial peptides derived from rare and endangered plant and animal resources, where raw material availability is even more critical. Chemical synthesis has high production costs and requires the extensive use of various highly toxic reagents (hydrofluoric acid, potassium cyanide, etc.), and the resulting environmental pollution from waste is difficult to resolve. Genetic engineering and fermentation engineering methods are not limited by source availability and should be pollution-free; however, the antibacterial activity of antimicrobial peptides themselves makes prokaryotic expression technically challenging. During expression, they can be toxic to the host bacteria, and due to their small molecular weight, they are easily degraded by various endogenous proteases of the host bacteria. A conventional solution is co-expression with a fusion tag, but this presents several technical problems: the fusion tag protein is too large, the solubility-enhancing effect is poor, and the expression product has low cleavage efficiency. Even more seriously, the antimicrobial peptides cannot exhibit their original antibacterial activity after enzymatic digestion and purification. Although the expression is successful, the antimicrobial peptides lose their activity and still cannot be used for industrial applications.

[0004] Therefore, the present invention addresses the problem of finding suitable fusion tags for antimicrobial peptides that can achieve efficient and non-toxic expression of antimicrobial peptides in host bacteria, while also increasing yield, and obtaining high-purity antimicrobial peptides with good antimicrobial activity through targeted renaturation after selecting appropriate enzymatic digestion / cleavage methods. This invention explores fusion tags for various antimicrobial peptides and studies methods for efficient prokaryotic expression of antimicrobial peptides. Summary of the Invention

[0005] To solve the above-mentioned technical problems, this invention employs inclusion body renaturation technology of antimicrobial peptides to inhibit their toxic effects on host cells, enabling the antimicrobial peptides to be successfully expressed in large quantities in Escherichia coli. The resulting antimicrobial peptides still have good activity and exhibit significant in vitro antibacterial effects.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for prokaryotic expression of antimicrobial peptides, characterized in that the antimicrobial peptide gene is inserted into the 3' or 5' end of a fusion tag gene for fusion expression.

[0008] Preferably, the amino acid sequence of the antimicrobial peptide is as shown in SEQ ID No. 3 and / or SEQ ID No. 5.

[0009] Preferably, the fusion tag is β2 microglobulin, whose amino acid sequence is shown in SEQ ID No. 1.

[0010] Considering the current market demand for an efficient production process for highly active antimicrobial peptides suitable for modern industrial production systems, this invention provides a method for prokaryotic expression of antimicrobial peptides. This method involves inserting an antimicrobial peptide gene into the 3' or 5' end of a fusion tag gene for fusion expression. The prokaryotic expression method uses β2-microglobulin (its amino acid sequence is shown in SEQ ID No. 1) as an inclusion body fusion tag, which helps the antimicrobial peptide to be expressed in large quantities in *E. coli*. Simultaneously, this invention optimizes the β2-microglobulin gene for codons preferred by *E. coli*, and the optimized nucleotide sequence is shown in SEQ ID No. 2. This invention inserts the antimicrobial peptide gene into the 3' or 5' end of the β2-microglobulin gene, an auxiliary protein expression tag, for fusion expression. The β2-microglobulin gene, acting as an inclusion body fusion tag, enables the antimicrobial peptide to be expressed in large quantities in *E. coli* in an inclusion body state. The inclusion body state of the antimicrobial peptide does not directly kill the host bacteria, thus allowing for the continuous and large-scale production of fusion proteins containing the antimicrobial peptide, thereby increasing yield and efficiency.

[0011] Furthermore, the present invention preferably adds a histidine tag or other purification tag between the fusion tag gene and the antimicrobial peptide gene to help purify the antimicrobial peptide.

[0012] Furthermore, the present invention preferably adds a protease cleavage site between the fusion tag gene and the antimicrobial peptide gene to help purify the antimicrobial peptide.

[0013] Adding the above-mentioned enzyme cleavage sites and purification tags helps to separate and purify antimicrobial peptides. In the subsequent process, the antimicrobial peptides can be cleaved from the fusion protein and released for further purification through enzyme cleavage and refolding.

[0014] The method provided by this invention utilizes the Escherichia coli recombinant expression inclusion body system to prepare antimicrobial peptides on a large scale, which helps to solve the problem of drug resistance to traditional antibiotics and the threat of superbugs to humans. It is also suitable for industrial and large-scale production, and can truly be marketed to benefit mankind.

[0015] In a second aspect, the present invention provides a fusion protein having an amino acid sequence as shown in SEQ ID No. 7 or SEQ ID No. 9.

[0016] Thirdly, the present invention provides a polynucleotide whose nucleotide sequence is shown in SEQ ID No. 8 or SEQ ID No. 10.

[0017] Fourthly, the present invention provides an expression vector comprising the polynucleotide.

[0018] Fifthly, the present invention provides a host cell comprising the expression vector and expressing the polynucleotide.

[0019] Preferably, the host cell is Escherichia coli or yeast.

[0020] In a sixth aspect, the present invention provides the use of the polynucleotide, or the expression vector, or the host cell in the production of antimicrobial peptides.

[0021] Beneficial effects:

[0022] This invention provides a method for prokaryotic expression of antimicrobial peptides. β2-microglobulin is used as an inclusion body fusion tag, resulting in the formation of numerous inclusion body proteins during fusion expression. This fusion protein eliminates the original antimicrobial activity of the antimicrobial peptide during expression and is non-toxic to the host bacteria, thus achieving efficient expression within the host bacteria. Subsequently, a high concentration of hydroxylamine solution is added to the purified denatured inclusion body protein to lyse the target protein, releasing the free antimicrobial peptide. Dialysis refolding purification is then performed to activate the original antimicrobial effect of the antimicrobial peptide. The method provided by this invention enables the large-scale preparation of antimicrobial peptides in a prokaryotic expression system. The method is simple and easy to implement, and the obtained antimicrobial peptides retain their antimicrobial activity after enzymatic purification, exhibiting significant in vitro antibacterial effects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0024] Figure 1 A map and schematic diagram of the construction method for B2MG-HNG-GL18 into the pET-26b expression vector.

[0025] Figure 2 A map and schematic diagram of the construction method for B2MG-HNG-GK25 into the pET-26b expression vector.

[0026] Figure 3 The gel electrophoresis results for inclusion body proteins expressed by B2MG-HNG-GL18 in the examples are shown.

[0027] Figure 4 The gel electrophoresis results for inclusion body proteins expressed by B2MG-HNG-GK25 in the examples are shown.

[0028] Figure 5 The results of the antibacterial test (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) for the GL18 antimicrobial peptide prepared in the examples are shown.

[0029] Figure 6 The results of the antimicrobial test (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) for the GK25 antimicrobial peptide prepared in the examples are shown.

[0030] Figure 7 The results of the plate antimicrobial activity test of the antimicrobial peptides prepared in the examples are shown. Detailed Implementation

[0031] β2-microglobulin (also known as β2-microglobulin, β2-MG, or B2MG) is an endogenous low molecular weight serum protein that serves as a component of the HLA (human leukocyte antigen) molecule in the human body. It has important value in the diagnosis and monitoring of certain diseases, can reflect glomerular filtration indicators, is used for monitoring hematologic diseases, and can also be used to assess the activity of autoimmune diseases.

[0032] This invention creatively fuses β2-microglobulin with an antimicrobial peptide for expression, successfully achieving a fusion protein in an inclusion body state. This fusion protein does not possess the original antimicrobial peptide's antimicrobial activity, therefore it does not kill host bacteria. After purification, the target protein is lysed with a high concentration of hydroxylamine solution, releasing the individual antimicrobial peptide, which still exhibits good antibacterial effects. This method can be used for the large-scale preparation of various antimicrobial peptides in prokaryotic expression systems. The amino acid sequence of this preferred B2MG fusion tag is shown in SEQ ID No. 1, and its codon-optimized base sequence for expression in *E. coli* is shown in SEQ ID No. 2.

[0033] This invention also provides the sequences of two experimentally successful antimicrobial peptides, GL18 and GK25. Both antimicrobial peptides can be co-expressed with B2MG to form inclusion bodies, and highly active antimicrobial peptides can be obtained through enzymatic digestion and purification. The amino acid sequence of GL18 is shown in SEQ ID No. 3, and the base sequence is shown in SEQ ID No. 4. The amino acid sequence of GK25 is shown in SEQ ID No. 5, and the base sequence is shown in SEQ ID No. 6.

[0034] This invention also provides the sequences of fusion proteins containing purification tags and purification cleavage sites for B2MG+ antimicrobial peptides, namely B2MG-HNG-GL18 and B2MG-HNG-GK25, for prokaryotic expression of GL18 and GK25 antimicrobial peptides, respectively. Both genes contain six histone repeat tag sequences and an NG sequence for hydroxylamine cleavage. The amino acid sequence of the B2MG-HNG-GL18 fusion protein is shown in SEQ ID No. 7, and the base sequence is shown in SEQ ID No. 8. The amino acid sequence of the B2MG-HNG-GK25 fusion protein is shown in SEQ ID No. 9, and the base sequence is shown in SEQ ID No. 10.

[0035] This invention also provides a specific method for prokaryotic expression of antimicrobial peptides, the method comprising the following steps:

[0036] The genes for B2MG-HNG-GL18 or B2MG-HNG-GK25 are synthesized, and recombinant expression vectors, such as the commonly used PET series prokaryotic expression vectors, are constructed. These recombinant expression vectors are then transformed into expression strains, such as BL21, and IPTG-induced expression is performed. Cells expressing the fusion protein at high levels are collected by centrifugation. After lysis, the inclusion body fraction is collected by centrifugation, washed several times with buffer, and then dissolved in a protein denaturing solution, such as urea solution. The fusion protein is then purified using Ni column affinity chromatography to obtain the purified denatured protein B2MG-HNG-GL18 or B2MG-HNG-GK25.

[0037] The target protein is lysed by adding a high concentration of hydroxylamine solution in the denatured state, releasing individual antimicrobial peptides GL18 or GK25. The released antimicrobial peptides can be refolded using methods such as dialysis or limiting dilution. The refolded antimicrobial peptides can then be recovered from the mixture using conventional methods in the art, such as ion exchange chromatography, extraction, spray drying, evaporation, and precipitation. The purified peptides can be concentrated using the BCA method, purified using liquid chromatography, and their accurate molecular weight determined using mass spectrometry.

[0038] In this invention, the antimicrobial peptide fused with B2MG protein can be directly expressed as a fusion protein containing inclusion bodies. The specific steps can be performed using conventional methods in the art. For example, it can be produced by the following steps: (1) construction of Escherichia coli genetically engineered bacteria; (2) fermentation culture of Escherichia coli genetically engineered bacteria; (3) induction and expression of the fusion protein; and (4) purification of the fusion protein and cleavage of the antimicrobial peptide.

[0039] In step (1), the construction of Escherichia coli genetically engineered bacteria can be carried out as follows: the DNA fragment of B2MG-antimicrobial peptide fusion protein is codon optimized and spliced ​​and recombined using PCR to obtain the target gene fragment; the obtained target gene fragment is inserted into the PET-26b expression vector to obtain the recombinant expression plasmid; the recombinant expression plasmid is transformed into Escherichia coli competent cells BL21(DE3) and positive Escherichia coli genetically engineered bacteria are screened.

[0040] In steps (2) and (3), the fermentation culture of Escherichia coli and the induction and expression of B2MG-antimicrobial peptide fusion protein can be carried out as follows: select the preferred Escherichia coli genetically engineered bacteria from LB plates for single colony culture; inoculate the bacterial solution into 2×YT medium for amplification culture; add IPTG for induction and continue culture, and collect the bacterial cells by centrifugation.

[0041] In step (4), the purification and cleavage of the B2MG-antimicrobial peptide fusion protein can be performed as follows: Bacteria are resuspended in phosphate buffer, and inclusion bodies are collected by centrifugation after bacterial disruption. The inclusion bodies are dissolved, and the B2MG-antimicrobial peptide fusion protein is bound using an NI-NTA affinity column. After rinsing off other proteins, the target protein is eluted. The fusion protein is lysed using hydroxylamine to release the free antimicrobial peptide. The released antimicrobial peptide can be further purified and refolded by dialysis, and then its activity can be tested or it can be lyophilized for later use.

[0042] The antimicrobial peptides expressed in this invention can achieve efficient expression in prokaryotic expression systems through inclusion body renaturation technology. They can also be purified and renatured using simple methods, facilitating industrial scale-up. The obtained antimicrobial peptides exhibit good antimicrobial activity and significant in vitro antibacterial effects, with obvious inhibitory effects against Escherichia coli and Staphylococcus aureus.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0044] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0046] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0047] In this invention, the host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Escherichia coli. It should be understood that those skilled in the art can replace the aforementioned Escherichia coli strain with other expressing strains as the host cell.

[0048] In this invention, the specific sequences involved are as follows:

[0049] β2-microglobulin amino acid sequence beta-2-microglobulin (B2MG), SEQ ID No. 1:

[0050] MGIQRTPKIPPVPSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.

[0051] The amino acid sequence of β2-microglobulin (B2MG), SEQ ID No. 2:

[0052] ATGGGCATCCACGGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGT GGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATG.

[0053] GL18 amino acid sequence SEQ ID No. 3: GNNRPVYIPQPRPPHPRL.

[0054] GL18 base sequence SEQ ID No. 4:

[0055] GGCAATAATCGGCCAGTATATATTCCACAACCAAGACCCCCTCATCCGCGTCTA。

[0056] GK25 amino acid sequence, SEQ ID No.5:

[0057] GLLSNVAGLLKQFAKGGVNAVLNPK。

[0058] GK25 base sequence SEQ ID No.6:

[0059] GGACTCCTTTCTAATGTCGCAGGTCTTTTGAAACAATTTGCAAAAGGTGGTGTAAATGCTGTATTGAACCCAAAA。

[0060] Fusion protein B2MG-HNG-GL18 amino acid sequence SEQ ID No.7:

[0061] MGIQRTPKIPVPSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMPKSGSHHHHHHNGNNRPVYIPQPRPPHPRL。

[0062] Fusion protein B2MG-HNG-GL18 base sequence SEQ ID No.8:

[0063] ATGGGCATCCAGCGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGCCAAAGTCTGGTTCTCATCACCACCATCATCATAACGGCAATAATCGGCCAGTATATATTCCACAACCAAGACCCCCTCATCCGCGTCTA。

[0064] The amino acid sequence of B2MG-HNG-GK25 is SEQ ID No. 9:

[0065] MGIQRTPKIPPVPSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMPKSGSHHHHHHNGLLSNVAGLLKQFAKGGVNAVLNPK.

[0066] B2MG-HNG-GK25 base sequence SEQ ID No. 10:

[0067] ATGGGCATCCACGGTACTCCAAAGATTCCGGTTCCCTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTAC ACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGCCAAAGTCTGGTTCTCATCACCACCATCATCATAACGGACTCCTTTCTAATGTCGCAGGTCTTTTGAAACAATTTGCAAAAGGTGGTGTAAATGCTGTATTGAACCCAAAA.

[0068] Example 1

[0069] This embodiment provides a method for the preparation and purification of GL18 antimicrobial peptide.

[0070] The amino acid sequence of the B2MG-GL18 antimicrobial peptide fusion protein B2MG-HNG-GL18 used in this embodiment is shown in SEQ ID No. 7; the corresponding base sequence is shown in SEQ ID No. 8, and the codons have been optimized for Escherichia coli.

[0071] In this embodiment, the full-length B2MG-HNG-GL18 gene sequence is 393 bp. The gene fragment was synthesized by Shanghai Huajin Biotechnology Co., Ltd., and the synthesized gene fragment was inserted into the PET-26b expression vector through the restriction enzyme sites of BamHI (NEB, catalog number: R0136L) and XhoI (NEB, catalog number: R0146L).

[0072] In this embodiment, the successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3) (Merck). The specific process is as follows:

[0073] 1: Take 1 µl of the plasmid and place it in 100 µl of E. coli competent cells BL21(DE3), and incubate on ice for 30 min.

[0074] 2: Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.

[0075] 3: Add 600µl of non-resistant LB to the mixture and incubate at 37°C and 220rpm for 1h.

[0076] 4: Take 200µl of the bacterial suspension and spread it evenly on an LB agar plate containing ampicillin resistance (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar, 100µg / ml kanamycin).

[0077] 5: Invert the plate and incubate it in a 37°C incubator for about 20 hours until clearly visible colonies grow.

[0078] In this embodiment, single colonies were picked from transformed LB plates and cultured in 10 ml LB medium (containing 100 µg / ml kanamycin) at 37°C and 220 rpm for 12-16 h. Then, they were transferred at a 1:100 ratio to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) for expansion culture at 37°C and 220 rpm for approximately 3 h. When the OD600 of the bacterial culture reached 0.4-0.6, 0.5 mM IPTG (Sigma, catalog number: I5502-1G) was added to induce expression. The induction conditions were 18°C ​​and 180 rpm for 20 h. Finally, the bacterial cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next purification step.

[0079] In this embodiment, the bacterial pellet was resuspended in approximately 50 ml (1 L) of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride). After sterilization using an autoclave (Xinzhi Biotechnology), the pellet was centrifuged at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0080] In this embodiment, the inclusion bodies were washed twice with 30 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), centrifuged at 13000 rpm for 30 min, and the washed inclusion bodies were collected. The inclusion bodies were then fully dissolved in 20 ml of denaturing buffer (8 M Urea, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). The mixture was centrifuged at 13000 rpm for 30 min, and the fusion protein in the supernatant was collected.

[0081] In this embodiment, the fusion protein was purified using a Ni column. The Ni-NTA (Qiagen, catalog number 30210) affinity column was equilibrated with binding buffer (8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). The fusion protein supernatant was then added and incubated at 4°C for 0.5–1 h to ensure complete binding of the recombinant protein to the column. Impurities were then washed with 200 ml of wash buffer containing 10 mM imidazole (Sigma) (10mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). Finally, the fusion protein was eluted with 20 ml of elution buffer containing 250 mM imidazole (250mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). The concentration and purity of the product were analyzed by SDS-PAGE electrophoresis. The specific process is as follows: Take 40µl of protein solution, add 10µl of 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ water for 10min, then add 10µl to each well of SDS-PAGE protein gel, run at 80V for 2h, stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20min, and then destain with protein destaining solution (10% acetic acid, 5% ethanol).

[0082] In this embodiment, the fusion protein was cleaved using hydroxylamine lysis to release the free GL18 antimicrobial peptide. The B2MG-HNG-GL18 fusion protein solution was mixed with hydroxylamine lysis buffer (4M hydroxylamine, pH 9.0) at a 1:1 volume ratio, and the pH was adjusted to 9.0. The reaction was then carried out in a closed, shaken environment at 45°C for approximately 4-6 hours until complete. The hydroxylamine solution specifically cleaves the Asn-Gly sites within the protein under weakly alkaline conditions, effectively releasing the GL18 antimicrobial peptide from the fusion protein. The progress of the enzyme cleavage reaction was monitored by SDS-PAGE electrophoresis.

[0083] The GL18 antimicrobial peptide released in this embodiment can be refolded and purified by dialysis, and then its activity can be tested or it can be lyophilized for later use. The specific process is as follows: The GL18 antimicrobial peptide is transferred to a dialysis bag with a 1 kDa molecular weight cutoff, placed in 100 times the volume of distilled water, and dialyzed at 4°C for 24 hours with continuous stirring. The distilled water can be changed every 4 hours. The solution in the dialysis bag is then collected, centrifuged, and the supernatant is taken, which is the purified GL18 antimicrobial peptide.

[0084] Example 2

[0085] This embodiment provides a method for the preparation and purification of GK25 antimicrobial peptide.

[0086] The amino acid sequence of the B2MG-GK25 antimicrobial peptide fusion protein B2MG-HNG-GK25 used in this embodiment is shown in SEQ ID No. 9, and its corresponding base sequence is shown in SEQ ID No. 10. The codons have been optimized for Escherichia coli.

[0087] In this embodiment, the full-length B2MG-HNG-GK25 gene sequence is 414 bp. Based on the optimized base sequence shown in SEQ ID No. 10, the gene fragment was synthesized by Shanghai Huajin Biotechnology Co., Ltd., and the synthesized gene fragment was inserted into the PET-26b expression vector through the restriction enzyme sites of BamHI (NEB, catalog number: R0136L) and XhoI (NEB, catalog number: R0146L).

[0088] In this embodiment, the successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3) (Merck). The specific process is as follows:

[0089] 1: Take 1 µl of the plasmid and place it in 100 µl of E. coli competent cells BL21(DE3), and incubate on ice for 30 min.

[0090] 2: Heat shock the mixture in a 42°C water bath for 90 seconds, then quickly place it on ice and let it stand for 2 minutes.

[0091] 3: Add 600µl of non-resistant LB to the mixture and incubate at 37°C and 220rpm for 1h.

[0092] 4: Take 200µl of the bacterial suspension and spread it evenly on an LB agar plate containing kanamycin sulfate resistance (10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 15g / L agar, 100µg / ml kanamycin).

[0093] 5: Invert the plate and incubate it in a 37°C incubator for about 20 hours until clearly visible colonies grow.

[0094] In this embodiment, single colonies were picked from transformed LB plates and cultured in 10 ml LB medium (containing 100 µg / ml kanamycin) at 37°C and 220 rpm for 12-16 h. Then, they were transferred at a 1:100 ratio to 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) for expansion culture at 37°C and 220 rpm for approximately 3 h. When the OD600 of the bacterial culture reached 0.4-0.6, 0.5 mM IPTG (Sigma, catalog number: I5502-1G) was added to induce expression. The induction conditions were 18°C ​​and 180 rpm for 20 h. Finally, the bacterial cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next purification step.

[0095] In this embodiment, the bacterial pellet was resuspended in approximately 50 ml (1 L) of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride). After sterilization using an autoclave (Xinzhi Biotechnology), the pellet was centrifuged at 13,000 rpm for 30 min to fully separate the soluble protein from the inclusion bodies.

[0096] In this embodiment, the inclusion bodies were washed twice with 30 ml of phosphate buffer (pH 7.8) (40 mM sodium dihydrogen phosphate, 500 mM sodium chloride), centrifuged at 13000 rpm for 30 min, and the washed inclusion bodies were collected. The inclusion bodies were then fully dissolved in 20 ml of denaturing buffer (8 M Urea, 40 mM NaH2PO3, 500 mM NaCl, pH 7.8). The mixture was centrifuged at 13000 rpm for 30 min, and the fusion protein in the supernatant was collected.

[0097] In this embodiment, the fusion protein was purified using a Ni column. The Ni-NTA (Qiagen, catalog number 30210) affinity column was equilibrated with binding buffer (8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). The fusion protein supernatant was then added and incubated at 4°C for 0.5–1 h to ensure complete binding of the recombinant protein to the column. Impurities were then washed with 200 ml of wash buffer containing 10 mM imidazole (Sigma) (10mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). Finally, the fusion protein was eluted with 20 ml of elution buffer containing 250 mM imidazole (250mM imidazole, 8M Urea, 40mM NaH2PO3, 500mM NaCl, pH 7.8). The concentration and purity of the product were analyzed by SDS-PAGE electrophoresis. The specific process is as follows: Take 40µl of protein solution, add 10µl of 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ water for 10min, then add 10µl to each well of SDS-PAGE protein gel, run at 80V for 2h, stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20min, and then destain with protein destaining solution (10% acetic acid, 5% ethanol).

[0098] In this embodiment, the fusion protein was cleaved using hydroxylamine lysis to release the free GK25 antimicrobial peptide. The B2MG-HNG-GK25 fusion protein solution was mixed with hydroxylamine lysis buffer (4M hydroxylamine, pH 9.0) at a 1:1 volume ratio, and the pH was adjusted to 9.0. The reaction was then carried out in a closed, shaken environment at 45°C for approximately 4-6 hours until complete. The hydroxylamine solution specifically cleaves the Asn-Gly sites within the protein under weakly alkaline conditions, effectively releasing the GK25 antimicrobial peptide from the fusion protein. The progress of the enzyme cleavage reaction was monitored by SDS-PAGE electrophoresis.

[0099] The GK25 antimicrobial peptide released in this embodiment can be refolded and purified by dialysis, and then its activity can be tested or it can be lyophilized for later use. The specific process is as follows: The GK25 antimicrobial peptide is transferred to a 1 kDa molecular weight cutoff dialysis bag and placed in 100 times the volume of dialysis fluid in distilled water. Dialysis is performed at 4°C for 24 hours with continuous stirring, and the distilled water can be changed every 4 hours. The solution in the dialysis bag is then collected, centrifuged, and the supernatant is taken as the purified GK25 antimicrobial peptide.

[0100] Figures 3-4Gel electrophoresis results for inclusion body proteins expressed by B2MG-HNG-GL18 and B2MG-HNG-GK25 in the examples are presented respectively. The electrophoresis results show that both corresponding inclusion body proteins were expressed.

[0101] Experimental Example 1

[0102] Because the antimicrobial peptides inhibit antimicrobial activity during inclusion body expression, their antimicrobial activity often cannot be activated during subsequent refolding and purification processes. This invention successfully verified that GL18 and GK25 retain good antimicrobial activity after the above steps, and showed excellent results in inhibition experiments against *Escherichia coli* and *Staphylococcus aureus*. These verification experiments further demonstrate the superiority and feasibility of this invention.

[0103] This experiment tests the antibacterial properties of the GL18 antimicrobial peptide prepared in Example 1 and the GK25 antimicrobial peptide prepared in Example 2.

[0104] The experimental procedure for this example is as follows:

[0105] (1) After thawing the frozen strain, inoculate it into 100 μl of solid culture medium, incubate at 37°C overnight, pick a single colony, select one single colony and inoculate it into a 20 ml liquid culture medium (LB) Erlenmeyer flask, place it on a shaker and rotate at 180 rpm, incubate at 37°C for 16 hours, and use it as a bacterial storage solution.

[0106] (2) Blank control group: 1 ml of bacterial stock solution was taken and added to 5 ml of liquid culture medium LB. The mixture was mixed evenly and incubated in a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0107] (3) Antimicrobial peptide (GL18 group, GK25 group) experimental group: 1 ml of bacterial stock solution was taken, and the corresponding antimicrobial peptide was added according to the final concentration of 50 ug / ml. The mixture was mixed evenly, and the mixture was incubated in a shaker at 37℃ for 4 h. The OD value at 600 nm was measured.

[0108] (4) Antibiotic control group: 1 ml of bacterial stock solution was taken and antibiotics were added at a final concentration of 50 ug / ml. The mixture was mixed evenly and incubated in a shaker at 37°C for 4 h. The OD value at 600 nm was measured.

[0109] Inhibition rate formula = (OD value of blank group bacterial solution - OD value of experimental group bacterial solution) / OD value of blank group bacterial solution.

[0110] The plate antibacterial activity test was conducted as follows: Several plates were prepared according to the E. coli culture criteria, and the sample locations were clearly marked with streaks. Water was added as a blank group, antimicrobial peptides as the experimental group, and kanamycin as the control group. The plates were incubated overnight. Finally, obvious plaques were observed for the antimicrobial peptides GL18 and GK25, indicating good antibacterial effects.

[0111] Figure 5 The results of the antibacterial test (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) for the GL18 antimicrobial peptide obtained in Example 1 are shown. Figure 5 The results show that at a concentration of 50 ug / ml, GL18 inhibited Escherichia coli by 97% and Staphylococcus aureus by 92.8%.

[0112] Figure 6 The results of the antibacterial test (Escherichia coli and Staphylococcus aureus, with kanamycin as the control) for the GK25 antimicrobial peptide obtained in Example 2 are shown. Figure 6 The results show that at a concentration of 50 ug / ml, GK25 inhibited Escherichia coli by 97% and Staphylococcus aureus by 96.4%.

[0113] Figure 7 The images show plate antimicrobial assay results for the GL18 antimicrobial peptide obtained in Example 1 and the GK25 antimicrobial peptide obtained in Example 2. Figure 7 As can be seen, both GL18 and GK25 showed large plaques against Escherichia coli, similar to the effect of kanamycin.

[0114] The above experiments successfully verified that the GL18 and GK25 antimicrobial peptides prepared in this invention retain good antimicrobial activity after denaturation and renaturation, and exhibit excellent effects in inhibition experiments against Escherichia coli and Staphylococcus aureus, as well as good antimicrobial effects against other strains. These verification experiments fully demonstrate the industrial applicability of this invention, possessing significant industrial and commercial value, and contributing to the large-scale production and preparation of antimicrobial peptides.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for expressing antimicrobial peptides in prokaryotes, characterized in that, The antimicrobial peptide gene is inserted into the 3' or 5' end of the fusion tag gene for fusion expression; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID No. 3 or SEQ ID No. 5; the fusion tag is β2 microglobulin, whose amino acid sequence is shown in SEQ ID No. 1; a purification tag and a protease cleavage site are added between the fusion tag gene and the antimicrobial peptide gene, and the fusion protein expressed is B2MG-HNG-GL18 or B2MG-HNG-GK25; the amino acid sequence of B2MG-HNG-GL18 is shown in SEQ ID No. 7; the amino acid sequence of B2MG-HNG-GK25 is shown in SEQ ID No.

9.

2. A fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID No. 7 or SEQ ID No.

9.

3. A polynucleotide, characterized in that, Its nucleotide sequence is shown in SEQ ID No. 8 or SEQ ID No.

10.

4. An expression carrier, characterized in that, It contains the polynucleotide of claim 3.

5. A host cell, characterized in that, It comprises the expression vector of claim 4 and expresses the polynucleotide of claim 3.

6. The host cell according to claim 5, characterized in that, The host cell is either Escherichia coli or yeast.

7. The use of the polynucleotide of claim 3, or the expression vector of claim 4, or the host cell of claim 5 or 6 in the production of antimicrobial peptides; wherein the antimicrobial peptide is GL18 or GK25; the amino acid sequence of GL18 is shown in SEQ ID No. 3; and the amino acid sequence of GK25 is shown in SEQ ID No. 5.

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