A method for producing toxic proteins and its application

Abstract

This invention discloses a method for producing toxic proteins and its applications, belonging to the fields of genetic engineering and biotechnology. The method includes: constructing a fusion expression vector containing a domain capable of mediating biomolecular phase separation and an antimicrobial peptide gene; transforming the fusion expression vector into *Corynebacterium glutamicum*, culturing the transformed *Corynebacterium glutamicum* to express the fusion protein and form a membraneless organelle; collecting the cultured bacterial cells and extracting the antimicrobial peptide. This invention utilizes the principle of biomolecular phase separation, isolating the antimicrobial peptide from the host cell by forming a membraneless organelle, effectively increasing the expression level of the antimicrobial peptide in *Corynebacterium glutamicum*. This method can be applied to the production of various antimicrobial peptides.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology, and specifically relates to a method for producing toxic proteins and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of short-chain polypeptides naturally produced by organisms, typically composed of 12 to 50 amino acids, and play a key role in the innate immune system. These peptides possess broad-spectrum antimicrobial activity, effectively combating bacteria, fungi, and viruses, primarily by disrupting the integrity of microbial cell membranes, interfering with cellular metabolic processes, or inhibiting DNA / RNA synthesis.

[0003] Due to their advantages such as rapid bactericidal action and low induction of drug resistance, antimicrobial peptides have shown broad application prospects in the pharmaceutical, agricultural, and food industries. Especially against the backdrop of increasingly serious antibiotic resistance problems, they are considered important candidates to replace or supplement traditional antibiotics. Traditionally, the production of antimicrobial peptides has mainly relied on chemical synthesis and natural extraction. Chemical synthesis methods are suitable for small-scale production with high purity requirements, but are costly; natural extraction faces problems such as low yield and unstable purity.

[0004] In recent years, advancements in biosynthesis and recombinant expression technologies have provided new avenues for the production of antimicrobial peptides. Microbial systems such as Escherichia coli, yeast, and insect cells have been used to express antimicrobial peptides.

[0005] However, high expression of antimicrobial peptides in these expression systems may disrupt host cell membrane properties and induce host cell toxicity. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for producing toxic proteins.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for producing toxic proteins, comprising,

[0010] Construct a fusion expression vector containing a domain capable of mediating phase separation of biomolecules and an antimicrobial peptide gene;

[0011] The fusion expression vector was transformed into Corynebacterium glutamicum, and the transformed Corynebacterium glutamicum was cultured to express the fusion protein and form membraneless organelles.

[0012] Collect cultured bacterial cells and extract antimicrobial peptides.

[0013] As a preferred embodiment of the method described in this invention, the domain comprising the ability to mediate phase separation of biomolecules is an intrinsically disordered protein region, a low-complexity sequence, or a domain having multivalent interactions.

[0014] In a preferred embodiment of the method described in this invention, the structural domain capable of mediating phase separation of biomolecules is an RGG structural domain.

[0015] In a preferred embodiment of the method described in this invention, the amino acid sequence of the RGG domain is shown in SEQ1, and the nucleotide sequence is shown in SEQ2.

[0016] In a preferred embodiment of the method described in this invention, the antimicrobial peptide gene includes a melitin peptide gene and a lactoferrin B gene.

[0017] In a preferred embodiment of the method of the present invention, the amino acid sequence of the bee venom peptide gene is shown in SEQ5, and the nucleotide sequence is shown in SEQ6; the amino acid sequence of the lactoferrin B gene is shown in SEQ7, and the nucleotide sequence is shown in SEQ8.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for producing toxic proteins for use in the production of antimicrobial peptides, wherein: it includes isolating the antimicrobial peptides from the host cells by forming membraneless organelles through biomolecular phase separation.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for increasing the expression level of antimicrobial peptides in Corynebacterium glutamicum using biomolecular phase separation, comprising,

[0020] The antimicrobial peptide gene is fused with a domain gene capable of mediating phase separation of biomolecules for expression.

[0021] The domain that mediates the phase separation of biomolecules is the RGG domain.

[0022] Beneficial effects of this invention:

[0023] (1) This invention has made a breakthrough in solving the toxicity problem in the production of antimicrobial peptides: by forming membraneless organelles, the toxic effects of antimicrobial peptides on host cells are effectively isolated, overcoming the main bottleneck in traditional methods.

[0024] (2) This invention significantly improves the yield of antimicrobial peptides: By using biomolecular phase separation technology, the expression level of antimicrobial peptides in Corynebacterium glutamicum is greatly improved, laying the foundation for industrial production.

[0025] (3) This invention has wide applicability: This method can be applied to the production of various antimicrobial peptides, such as bee venom peptide and lactoferrin B, and has broad application prospects.

[0026] (4) This invention provides a new approach to the production of toxic proteins: This invention is not only applicable to antimicrobial peptides, but also provides an innovative technical platform for the production of other cytotoxic proteins in microorganisms; the method has a simple operation process, is easy to implement on an industrial scale, and has good industrialization potential.

[0027] (5) Promote the research and application of antimicrobial peptides: By providing an efficient production method, this invention is expected to accelerate the research and application of antimicrobial peptides in the fields of medicine and food; by innovatively applying the principle of biomolecular phase separation to the production of toxic proteins (antimicrobial peptides), this invention solves the technical problems that have long plagued this field and lays the foundation for the development and application of related products. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0029] Figure 1 This is a fluorescence microscope image of membrane-free organelles mediated by liquid-liquid phase separation in an embodiment of the present invention.

[0030] Figure 2 This is an SDS-PAGE analysis diagram of the antimicrobial peptide in an embodiment of the present invention.

[0031] Figure 3 This is a fluorescence microscopy image of the antimicrobial peptide fused with EGFP at the C-terminus in an embodiment of the present invention.

[0032] Figure 4 This is a schematic diagram illustrating the principle of using phase separation to form membraneless organelles to achieve large-scale expression of antimicrobial peptides in an embodiment of the present invention. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Example 1

[0037] Construction of membrane-free organelles mediated by liquid-liquid phase separation in Corynebacterium glutamicum:

[0038] The expression vector RGG-EGFP was constructed based on the RGG domain, which can mediate liquid-liquid phase separation, and the reporter gene.

[0039] The gene encoding the RGG domain was synthesized by Anshengda Biotechnology Co., Ltd. after codon optimization based on Corynebacterium glutamicum. The optimized RGG nucleotide sequence is shown in SEQ2.

[0040] RGG-EGFP and EGFP (EGFP amino acid sequence shown in SEQ3, nucleotide sequence shown in SEQ4) were ligated to the Corynebacterium glutamicum / Escherichia coli shuttle plasmid pXMJ19 vector backbone (GenBank: AJ133195.1) via Hind3 and EcoR1, respectively, to obtain expression vectors RGG-EGFP-P19 and EGFP-P19.

[0041] The specific steps are as follows: digest the pXMJ19 backbone vector and the above fragment with Hind3 and EcoR1, purify the digested vector and fragment, mix the pXMJ19 backbone vector and fragment, and perform the ligation reaction using TakaRa's Solution I ligase.

[0042] Refer to Tables 1 and 2 for the enzyme digestion and ligation system and reaction conditions.

[0043] The ligation product was transferred into competent E. coli cells, gently mixed, and incubated on ice for 10 min. It was then heat-shocked at 42°C for 90 s, incubated on ice for 5 min, and finally transferred to LB recovery medium at 37°C and 100 rpm. -1 After culturing for 1 hour, the bacterial culture was centrifuged and resuspended in 100 μL of culture medium and spread onto chloramphenicol-resistant LB plates. The culture was then incubated at 37°C for 16 hours.

[0044] Single colonies grown on the plate were verified by PCR, and positive clones were selected for sequencing verification, finally obtaining the corresponding expression vectors RGG-EGFP-P19 and EGFP-P19.

[0045] Table 1. Double enzyme digestion reaction system

[0046] Element Added amount Enzyme 1 5μL enzyme 2 5μL Buffer 10μL Expression carrier / fragment 2-4μg sterile water To bring the volume up to 100 μL

[0047] Reaction conditions: 37℃, 60min.

[0048] The glue was recycled to obtain a linearized carrier and fragments with the same viscous ends, and the linkage system is shown in Table 2.

[0049] Table 2 Connection Reaction System

[0050] Element Volume (μL) Enzyme digestion vector 2 Fragments after enzyme digestion 3 Solution I 5 Total volume 10

[0051] The expression plasmid described above was transformed into Corynebacterium glutamicum CGMCC 1.15647 using high-voltage electroporation.

[0052] First, take the competent cells of Corynebacterium glutamicum stored in a -80℃ freezer, place them on ice to thaw slowly, add 5μL of sterile water with a concentration of not less than 300ng / μL to dissolve the plasmid, gently pipette to mix, and then place them on ice for about 10 minutes.

[0053] The mixed bacterial culture was then pumped into a clean, pre-cooled (-20°C) electroporator (1 mm, Bio-rad), and the culture was clicked twice using the bacterial click mode. The clicked culture was immediately transferred to LBHIS recovery medium and incubated at 46°C for 6 minutes. The transformed culture was then placed at 30°C and 100 rpm. -1 Incubate in a shaker for 1-1.5 hours for recovery culture;

[0054] After the recovery culture is completed, take about 200 μL of bacterial culture and spread it on a pre-prepared LBHIS solid plate containing 10 mg / L chloramphenicol using a spreader.

[0055] After culturing at 30℃ for 48 hours, the correct single clones were selected, and three clones were selected for each expression plasmid. Each clone was inoculated into 10 mL of LBB medium and then cultured at 30℃ and 220 rpm for 12 hours. Wild-type Corynebacterium glutamicum CGMCC 1.15647 was inoculated simultaneously as a control.

[0056] Next, the strains cultured overnight were transferred to bottles containing 10 mL of LBB liquid medium at an inoculation rate of 2%. After culturing for 24 hours at 30°C and 220 rpm, an appropriate amount of bacterial solution was taken, fixed with fixative, and observed under a fluorescence microscope.

[0057] The results are as follows Figure 1 The results showed that the fluorescent proteins of the RGG-EGFP-P19 group were mainly located at both ends of the bacteria, while the fluorescent proteins of the EGFP-P19 group were evenly distributed in the cytoplasm; the RGG domain successfully mediated the generation of liquid-liquid phase-separated membraneless organelles in Corynebacterium glutamicum.

[0058] Example 2

[0059] Liquid-liquid phase separation successfully mediated the expression of melitoxin and lactoferrin B:

[0060] Melittin, a multifunctional bioactive peptide derived from bee venom, has attracted considerable attention due to its broad-spectrum biological properties. It not only possesses potent antibacterial, anti-inflammatory, and analgesic effects but also exhibits potential antitumor activity. Furthermore, melittin plays a crucial role in regulating the immune system, making it an ideal candidate for developing novel therapeutics.

[0061] Lactoferricin B is another protein with multiple biological functions, mainly found in the milk of mammals. It is renowned for its exceptional iron-binding capacity, playing a crucial role in the regulation of iron metabolism in the human body. In addition, lactoferricin B exhibits significant antibacterial and antiviral activity, effectively modulates the immune system, and possesses powerful antioxidant capabilities. These properties make it highly promising for applications in nutritional supplementation and medicine.

[0062] In this experiment, liquid-liquid phase separation technology was applied to the production of Melittin and Lactoferricin B. The specific implementation method is as follows:

[0063] Gene optimization and synthesis: The encoding genes of Melittin and Lactoferricin B were optimized according to the codons of Corynebacterium glutamicum and synthesized by Anshengda Biotechnology Co., Ltd. The optimized nucleotide sequences of Melittin and Lactoferricin B are shown in SEQ6 and SEQ8, respectively.

[0064] Expression vector construction: RGG-Melittin, RGG-Lactoferricin B, Melittin and Lactoferricin B were ligated to the pXMJ19 vector backbone of Corynebacterium glutamicum / Escherichia coli shuttle plasmid via Hind3 and EcoR1 restriction endonuclease sites, respectively.

[0065] The experimental procedures were performed according to Example 1, and the expression vectors RGG-Melittin-P19, RGG-Lactoferricin-P19, Melittin-P19 and Lactoferricin-P19 were finally obtained.

[0066] Transformation and culture: The above expression vector was transformed into the Corynebacterium glutamicum expression strain CGMCC1.15647 by electroporation. Correct single clones were picked and inoculated into 10 mL LBB medium, and then transferred to fresh 10 mL LBB medium at an inoculation rate of 2%.

[0067] After culturing at 220 rpm and 30°C for 24 hours, the bacterial cells were collected by centrifugation at 10 OD.

[0068] Protein expression detection: After ultrasonic disruption of bacterial cells, the supernatant was collected by centrifugation and the expression of the above proteins was detected by 12% SDS-PAGE.

[0069] like Figure 2 As shown, when antimicrobial peptides are expressed alone, the bands are faint or not observed at the expected molecular weight, while the RGG-fused separated group shows obvious protein bands at the expected protein size, indicating that the fusion of the RGG domain significantly improves the expression of antimicrobial peptides.

[0070] Liquid-liquid phase separation observation: Further EGFP was fused to the C-terminus of the antimicrobial peptide molecule to verify that the increased protein expression was due to the formation of membraneless organelles during phase separation. The antimicrobial peptide expression vectors RGG-Melittin and RGG-Lactoferricin were linearized using EcoR1, and the EGFP fragment was inserted into the EcoR1 site using homologous recombination. These plasmids were transformed into Corynebacterium glutamicum, and the formation of membraneless organelles was observed using fluorescence microscopy. The results are shown in the figure. Figure 3 As shown, after the C-terminus of the antimicrobial peptide expression vector was fused with EGFP, membrane-free organelles formed by liquid-liquid phase separation were observed.

[0071] For details on the principle of using phase separation to form membraneless organelles to achieve large-scale expression of antimicrobial peptides, please refer to [link to relevant documentation]. Figure 4 It can be seen that non-membrane organelles can confine antimicrobial peptides to a certain area, preventing them from damaging the cell membrane and causing host death; non-membrane organelles can isolate biochemical reactions, reducing the impact of antimicrobial peptides on cell wall synthesis and protein synthesis; and non-membrane organelles can mitigate the effect of antimicrobial peptides binding to DNA / RNA.

[0072] The above results demonstrate that liquid-liquid phase separation, by encapsulating antimicrobial peptides in droplets, can effectively isolate their toxic effects on host cells. This discovery provides a novel solution to the current difficulties in the recombinant expression of antimicrobial peptides and is expected to promote the large-scale production and application of antimicrobial peptides.

[0073] In summary, *Corynebacterium glutamicum* was selected as the chassis cell for producing antimicrobial peptides in this invention. *Corynebacterium glutamicum* is a Gram-positive bacterium renowned for its widespread use in amino acid production. In recent years, its potential as a protein expression system has also attracted increasing attention. Compared to traditional protein expression systems such as *Escherichia coli*, *Corynebacterium glutamicum* has significant advantages, including strong secretion capacity, low extracellular protease activity, and the absence of endotoxin contamination. This invention aims to establish a novel method for expressing toxic antimicrobial peptides using liquid-liquid phase separation technology in *Corynebacterium glutamicum*. This innovative production method not only holds promise for overcoming current technical bottlenecks in antimicrobial peptide production but may also provide new insights for the production of other cytotoxic bioactive molecules.

[0074] SEQ1: RGG amino acid sequence;

[0075] SEQ2: RGG nucleotide sequence;

[0076] SEQ3: EGFP amino acid sequence;

[0077] SEQ4: EGFP nucleotide sequence;

[0078] SEQ5: Amino acid sequence of bee venom peptide;

[0079] SEQ6: Methionine sequence of bee venom peptide;

[0080] SEQ7: Lactoferrin B amino acid sequence;

[0081] SEQ 8: Lactoferrin B nucleotide sequence;

[0082] The specific sequence is as follows:

[0083] SEQ1:

[0084] MAVQQQLHPQAVMIVEVVQVVAATAEVAVTPVEAEVVVTIAAITITAM IAITEVVPVATVAIATTKTADTTVAAVVVATAATTTTAVAVEVATTAKIAVM VALPTSLAAVTTIAMKAPITVAPVALTTTIAAITAVSLWASVPCLKSSPLDCQH*

[0085] SEQ2:

[0086] ATGCCACGCGAATCCAACCAGTCCAACAACGGCGGTTCCGGTAACGCAGCCTTGAACCGTGGTGGTCGCTACGTTCCACCACACTTGCGCGGTGGTGATGGCGGTGCAGCAGCAGCTGCATCCGCAGGCGGTGATGATCGTCGAGGTGGTGCAGGTGGTGGCGGCTACCGCCGAGGTGGCGGTAACTCCGGTGGAGGCGGAGGTGGTGGTTACGATCGCGGCTATAACGATAACCGCGATGATCGCGATAACCGAGGTGGTTCCGGTGGCTACGGTCGCGATCGCAACTACGAAGACCGCGGATACAACGGTGGCGGCGGTGGTGGTGGCAACCGCGGCTACAACAACAACCGCGGTGGCGGTGGAGGTGGCTACAACCGCCAAGATCGCGGTGATGGTGGCTCTTCCAACTTCTCTCGCGGCGGTTACAACAATCGCGATGAAGGCTCCGATAACCGTGGCTCCGGTCGCTCTTACAACAACGATCGCCGCGATAACGGCGGT;

[0087] SEQ3:

[0088] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH*

[0089] SEQ4:

[0090] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCACCATCACCATCACCATTAA

[0091] SEQ5:

[0092] MGIGAVLKVLTTGLPALISWIKRKRQQHHHHHH*;

[0093] SEQ6:

[0094] ATGGGCATCGGTGCCGTGCTTAAAGTCCTCACCACTGGACTGCCAG CATTGATTTCCTGGATCAAGCGCAAGCGTCAGCAA;

[0095] SEQ7:

[0096] MFKCRRWQWRMKKLGAPSITCVRRAFHHHHHH*;

[0097] SEQ8:

[0098] ATGTTCAAATGTCGGCGCTGGCAGTGGCGCATGAAGAAGCTGGGCGCACCATCCATCACCTGCGTGCGTCGAGCCTTTCACCATCACCATCACCATTAA

[0099] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the present invention.

Claims

1. A method for producing toxic proteins, characterized in that: The invention includes constructing a fusion expression vector comprising a domain capable of mediating phase separation of biomolecules and an antimicrobial peptide gene; wherein the domain capable of mediating phase separation of biomolecules is an RGG domain, the amino acid sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.2; the antimicrobial peptide gene is a melittin gene or a lactoferrin B gene, the amino acid sequence of which is shown in SEQ ID NO.5 and the nucleotide sequence of which is shown in SEQ ID NO.6, and the amino acid sequence of which is shown in SEQ ID NO.7 and the nucleotide sequence of which is shown in SEQ ID NO.8; The fusion expression vector was transformed into Corynebacterium glutamicum, and the transformed Corynebacterium glutamicum was cultured to express the fusion protein and form membraneless organelles. Collect cultured bacterial cells and extract antimicrobial peptides.

2. The application of the method of claim 1 in the production of antimicrobial peptides.

3. The application as described in claim 2, characterized in that: This includes isolating antimicrobial peptides from host cells through membrane-free organelles formed by biomolecular phase separation.

4. A method for increasing the expression level of antimicrobial peptides in Corynebacterium glutamicum using biomolecular phase separation, characterized in that: include, The antimicrobial peptide gene was fused with a domain gene capable of mediating phase separation of biomolecules for expression. The domain capable of mediating biomolecule phase separation is an RGG domain, the amino acid sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.2; the antimicrobial peptide gene is a melittin gene or a lactoferrin B gene, the amino acid sequence of which is shown in SEQ ID NO.5 and the nucleotide sequence of which is shown in SEQ ID NO.6, and the amino acid sequence of which is shown in SEQ ID NO.7 and the nucleotide sequence of which is shown in SEQ ID NO.8.

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