Method for producing toxic protein and application thereof

Abstract

The invention discloses a method for producing toxic protein and application thereof, and belongs to the technical field of genetic engineering and biologication.The method comprises the steps that a fusion expression vector containing a structural domain capable of mediating biomolecule phase separation and an antibacterial peptide gene is constructed; transforming the fusion expression vector into corynebacterium glutamicum, and culturing the transformed corynebacterium glutamicum to express a fusion protein to form a membrane-free organelle; and collecting the cultured thalli and extracting the antibacterial peptide. According to the invention, a biomolecular phase separation principle is utilized, and membrane-free organelles are formed to isolate the toxic effect of the antibacterial peptide on host cells, so that the expression quantity of the antibacterial peptide in corynebacterium glutamicum is effectively improved. The method can be applied to production of various antibacterial peptides.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biotechnology, and specifically relates to a method for producing toxic protein and application thereof. Background Art

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

[0003] Due to the advantages of rapid bactericidal activity and low induction of drug resistance, antimicrobial peptides have shown broad application prospects in the pharmaceutical, agricultural and food industries. Especially in the context of the increasingly serious problem of antibiotic resistance, they are regarded as important candidates to replace or supplement traditional antibiotics. Traditionally, the production of antimicrobial peptides mainly relies on chemical synthesis and natural extraction. Chemical synthesis methods are suitable for small-scale and high-purity production, but the cost is relatively high; natural extraction faces problems such as low yield and unstable purity.

[0004] In recent years, the development of biosynthesis and recombinant expression technology has provided new ways 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, large amounts of antimicrobial peptides expressed 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 summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

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

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

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for producing toxic protein, comprising:

[0010] Constructing a fusion expression vector comprising a domain capable of mediating biomolecular phase separation and an antimicrobial peptide gene;

[0011] transforming the fusion expression vector into Corynebacterium glutamicum, culturing the transformed Corynebacterium glutamicum to express the fusion protein and form membraneless organelles;

[0012] The cultured bacteria were collected and the antimicrobial peptides were extracted.

[0013] As a preferred embodiment of the method of the present invention, the structural domain capable of mediating biomolecule phase separation is an intrinsically disordered protein region, a low complexity sequence, or a structural domain with multivalent interactions.

[0014] As a preferred embodiment of the method of the present invention, the structural domain capable of mediating biomolecule phase separation is an RGG structural domain.

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

[0016] As a preferred embodiment of the method of the present invention, the antimicrobial peptide genes include melittin gene and lactoferrin B gene.

[0017] As a preferred embodiment of the method of the present invention, the amino acid sequence of the melittin 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 object of the present invention is to overcome the deficiencies in the prior art and provide a method for producing toxic proteins for use in the production of antimicrobial peptides, which includes isolating the toxic effects of antimicrobial peptides on host cells through membrane-less organelles formed by biomolecular phase separation.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for increasing the expression of antimicrobial peptides in Corynebacterium glutamicum by using biomolecule phase separation, comprising:

[0020] The antimicrobial peptide gene is fused with the domain gene that can mediate phase separation of biomolecules;

[0021] The structural domain capable of mediating biomolecule phase separation is an RGG structural domain.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention has solved the toxicity problem in the production of antimicrobial peptides in a breakthrough way: by forming membrane-less organelles, the toxic effects of antimicrobial peptides on host cells are effectively isolated, overcoming the main bottleneck in traditional methods.

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

[0025] (3) The present invention has wide applicability: the 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) The present invention provides a new idea for the production of toxic proteins: the present 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 and 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, the present invention is expected to accelerate the research and application of antimicrobial peptides in the fields of medicine and food. The present invention innovatively applies the principle of biomolecular phase separation to the production of toxic proteins (antimicrobial peptides), thereby solving the technical problems that have long plagued this field and laying the foundation for the development and application of related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 This is a fluorescence microscopy image of a membraneless organelle 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 peptides in the examples of the present invention.

[0031] Figure 3 This is a fluorescence microscope observation image of the antimicrobial peptide after the C-terminus of EGFP is fused in the example of the present invention.

[0032] Figure 4 This is a schematic diagram of 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 DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0036] Example 1

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

[0038] An expression vector RGG-EGFP was constructed based on the RGG domain capable of mediating liquid-liquid phase separation and a reporter gene.

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

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

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

[0042] For the enzyme digestion and ligation systems and reaction conditions, refer to Table 1 and Table 2;

[0043] The ligation product was transferred into the competent E. coli, mixed gently, and placed on ice for 10 min, heat-shocked at 42°C for 90 s, placed on ice for 5 min, and then transferred into LB recovery medium at 37°C, 100 r·min -1 , cultured for 1 h, centrifuged and resuspended with 100 μL of culture medium, spread on chloramphenicol-resistant LB plates, and cultured at 37 °C for 16 h;

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

[0045] Table 1 Double enzyme digestion reaction system

[0046] Element Addition amount Enzyme 1 5μL Enzyme 2 5μL Buffer 10μL Expression vector / fragment 2-4 μg Sterile water Make up to 100 μL

[0047] Reaction conditions: 37°C, 60 min.

[0048] The linearized vector and fragments with the same sticky ends were recovered from the gel, and the connection system was shown in Table 2.

[0049] Table 2 Ligation reaction system

[0050] Element Volume (μL) Vector after enzyme digestion 2 Enzyme digested fragments 3 SolutionI 5 Total volume 10

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

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

[0053] Then, the mixed bacterial solution was transferred into a clean and -20℃ precooled electric transfer cup (1mm, Bio-rad), and the bacterial click mode was selected to click twice. The clicked bacterial solution was immediately transferred to LBHIS recovery medium, and then placed in a 46℃ water bath for 6 minutes. The transformed bacterial solution was then placed at 30℃ and 100r / min. -1 Incubate in a shaker for 1-1.5 h for recovery culture;

[0054] After the recovery culture is completed, take about 200 μL of the bacterial solution and spread it on the LBHIS solid plate containing 10 mg / L chloramphenicol prepared in advance using a spreading rod;

[0055] After culturing at 30°C for 48 hours, the correct single clones were selected, and 3 clones were selected for each expression plasmid, respectively inoculated into 10 mL of LBB medium, and then cultured at 30°C and 220 rpm for 12 hours. At the same time, the wild-type Corynebacterium glutamicum CGMCC 1.15647 was inoculated as a control;

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

[0057] The results are as follows Figure 1 It was shown that the fluorescent proteins of the RGG-EGFP-P19 group were mainly localized at the two 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 formation of liquid-liquid phase separation membraneless organelles in Corynebacterium glutamicum.

[0058] Example 2

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

[0060] Melittin is a multifunctional bioactive peptide derived from bee venom that has attracted much attention due to its broad spectrum of biological properties. It not only has powerful antibacterial, anti-inflammatory and analgesic effects, but also exhibits potential anti-tumor activity. In addition, melittin also plays an important role in regulating the immune system, making it an ideal candidate for the development of new therapeutic drugs.

[0061] Lactoferricin B is another protein with multiple biological functions, mainly found in the milk of mammals. It is known for its excellent iron ion binding ability and plays a key role in the regulation of human iron metabolism. In addition, lactoferricin B has significant antibacterial and antiviral activities, can effectively regulate the immune system, and has strong antioxidant capacity. These properties make it have broad prospects in the fields of nutritional supplementation and medical applications.

[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 genes encoding 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 referred to SEQ6 and SEQ8, respectively.

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

[0065] The experimental operation was carried out with reference to Example 1, and finally the expression vectors RGG-Melittin-P19, RGG-Lactoferricin-P19, Melittin-P19 and Lactoferricin-P19 were obtained.

[0066] Transformation and culture: The above expression vector was transferred into the Corynebacterium glutamicum expression strain CGMCC1.15647 by electroporation, and the correct single clone was picked and inoculated into 10 mL LBB medium, and then transferred to fresh 10 mL LBB medium at a 2% inoculum volume;

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

[0068] Protein expression detection: After the bacteria were disrupted by ultrasound, the supernatant was obtained by centrifugation and the expression of the above proteins was detected using 12% SDS-PAGE.

[0069] like Figure 2 As shown, the antimicrobial peptide expressed alone had a faint band or no band observed at the expected molecular weight, while the RGG fusion phase separation group observed a clear protein band at the expected protein size, indicating that the expression of the antimicrobial peptide was significantly improved after the fusion of the RGG domain.

[0070] Liquid-liquid phase separation observation: EGFP was further fused to the C-terminus of the antimicrobial peptide molecule to verify that the increase in protein expression was due to the membraneless organelles formed by phase separation. After the antimicrobial peptide expression vectors RGG-Melittin and RGG-Lactoferricin were linearized using EcoR1, the EGFP fragment was inserted into the EcoR1 site using homologous recombination. The above plasmids were transformed into Corynebacterium glutamicum, and the formation of membraneless organelles was observed by 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-less organelles formed by liquid-liquid phase separation were observed.

[0071] For the specific principle of using phase separation to form membraneless organelles to achieve large-scale expression of antimicrobial peptides, see Figure 4 It can be seen that membraneless organelles can restrict antimicrobial peptides to a certain area, preventing them from destroying the cell membrane and causing host death; membraneless organelles can isolate biochemical reactions and reduce the effects of antimicrobial peptides on cell wall synthesis and protein synthesis; membraneless organelles reduce the effects of antimicrobial peptides binding to DNA / RNA.

[0072] The above results prove that liquid-liquid phase separation can effectively isolate the toxic effects of antimicrobial peptides on host cells by encapsulating them in droplets. This discovery provides a new 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] To sum up, in the present invention, Corynebacterium glutamicum was selected as the chassis cell for producing antimicrobial peptides. Corynebacterium glutamicum is a gram-positive bacterium, which is well-known for its wide application in amino acid production. In recent years, it has also received increasing attention as a potential of a protein expression system. Compared with traditional protein expression systems such as Escherichia coli, Corynebacterium glutamicum has significant advantages, including powerful secretion capacity, low extracellular protease activity and the characteristic of no endotoxin pollution. The present invention is intended to establish a novel method for expressing toxic protein antimicrobial peptides in conjunction with liquid-liquid phase separation technology in Corynebacterium glutamicum. This innovative production method is not only expected to solve the technical bottleneck faced in the current antimicrobial peptide production, but also may provide new ideas 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 melittin;

[0079] SEQ6: Melittin nucleotide sequence;

[0080] SEQ7: Lactoferrin B amino acid sequence;

[0081] SEQ8: 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 rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for producing a toxic protein, characterized in that: include, Constructing a fusion expression vector comprising 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 membraneless organelles; The cultured bacteria were collected and the antimicrobial peptides were extracted.

2. The method according to claim 1, characterized in that: The structural domain capable of mediating biomolecule phase separation is an intrinsically disordered protein region, a low-complexity sequence, or a structural domain with multivalent interactions.

3. The method according to claim 1 or 2, characterized in that: The structural domain capable of mediating biomolecule phase separation is an RGG structural domain.

4. The method according to claim 3, characterized in that: The amino acid sequence of the RGG domain is shown in SEQ1, and the nucleotide sequence is shown in SEQ2.

5. The method according to claim 1, characterized in that: The antimicrobial peptide genes include melittin gene and lactoferrin B gene.

6. The method according to claim 5, characterized in that: The amino acid sequence of the melittin 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.

7. Use of the method according to any one of claims 1 to 6 in the production of antimicrobial peptides.

8. The use according to claim 7, characterized in that: Including, the toxic effects of antimicrobial peptides on host cells are isolated by membraneless organelles formed by biomolecular phase separation.

9. A method for increasing the expression of antimicrobial peptides in Corynebacterium glutamicum by using biomolecular phase separation, characterized in that: include, The antimicrobial peptide gene is fused with a domain gene capable of mediating phase separation of biomolecules and expressed.

10. The use according to claim 9, characterized in that: The structural domain capable of mediating biomolecule phase separation is an RGG structural domain.

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