A method for producing recombinant human-derived proteins in industrial microorganisms

Abstract

The application discloses a method for producing recombinant human proteins in industrial microorganisms, comprising the following steps: introducing a gene coding alpha synuclein into corynebacterium glutamicum by genetic engineering technology to realize expression of the alpha synuclein; and isolating the alpha synuclein in liquid droplet-like membraneless organelles by liquid-liquid phase separation technology to further improve protein yield. Compared with the most widely used Escherichia coli expression system, the method has the following advantages: the method uses corynebacterium glutamicum as a new type of chassis cell, and overcomes problems such as endotoxin pollution and insoluble inclusion body formation in a traditional Escherichia coli expression system; the method significantly improves the yield of alpha synuclein by applying liquid-liquid phase separation technology; the method is not only suitable for the production of alpha synuclein, but also can be popularized to other difficult-to-express proteins, and provides a new technical path for the field of biological pharmacy.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology, and specifically relates to a method for producing recombinant human proteins in industrial microorganisms. Background Technology

[0002] Recombinant human proteins have wide applications in biomedical research, drug development, disease diagnosis, and treatment. With the development of biotechnology, the market demand for high-quality, low-cost, and safe recombinant human proteins is increasing. Currently, the industrial production of recombinant human proteins mainly relies on several major expression systems. The *E. coli* expression system has the advantages of rapid growth and low culture cost, but it suffers from endotoxin contamination and inclusion body formation. The yeast expression system, as a eukaryotic expression system, allows for post-translational modification, but it is prone to over-glycosylation, affecting protein function. While mammalian cell expression systems can obtain products with complete post-translational modifications and structures closest to the natural protein, they have high culture costs and long production cycles.

[0003] Corynebacterium glutamicum, a Gram-positive bacterium recognized as GRAS (Generally Recognized As Safe), exhibits unique advantages in recombinant protein production due to its lack of endotoxin contamination, strong protein secretion capacity, mature fermentation process, and clear genetic background. In recent years, its potential as a highly efficient prokaryotic recombinant protein expression host has attracted widespread attention.

[0004] This study focuses on α-synuclein, a key protein in the nervous system. Widely expressed in presynaptic nerve endings, α-synuclein participates in neuronal function regulation and synaptic plasticity. It is closely associated with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and can serve as an important biomarker for disease diagnosis. It holds significant value in basic research, drug development, and diagnostic applications.

[0005] Existing technologies face numerous challenges in the production of α-synaptic proteins. Traditional expression systems suffer from safety risks and unstable product quality. Furthermore, the tendency of α-synaptic proteins to aggregate and their toxicity to host cells also pose challenges to their production. 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 recombinant human proteins in industrial microorganisms.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for producing recombinant human protein in industrial microorganisms, comprising,

[0010] Construct a fusion expression vector containing the RGG domain and codon-optimized human α-synaptic protein gene;

[0011] The fusion expression vector was transformed into Corynebacterium glutamicum;

[0012] Transformed Corynebacterium glutamicum was cultured at 30–37°C and 200–250 rpm to induce the expression of the fusion protein and the formation of a dynamically reversible droplet-like structure within the cell.

[0013] Collect cultured bacterial cells and extract human α-synaptic protein.

[0014] In a preferred embodiment of the method described in this invention, the amino acid sequence of the RGG domain is SEQ ID NO:3.

[0015] In a preferred embodiment of the method described in this invention, the amino acid sequence of the human α-synaptic protein is SEQ ID NO:1, and its codon-optimized nucleic acid sequence is SEQ ID NO:2.

[0016] As a preferred embodiment of the method described in this invention, the Corynebacterium glutamicum includes strain CGMCC1.15647.

[0017] In a preferred embodiment of the method described in this invention, the culture is carried out in LBB medium for 18–30 hours.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for producing recombinant human proteins in industrial microorganisms, with applications in the production of human α-synaptic proteins.

[0019] As a preferred embodiment of the application described in this invention, the method increases the expression level of human α-synaptic protein in Corynebacterium glutamicum.

[0020] Another object of the present invention is to overcome the shortcomings of the prior art and provide a fusion expression vector for producing human α-synaptic protein, comprising,

[0021] It contains genes with RGG domains and human α-synaptic protein genes;

[0022] Contains a 6×His tag sequence;

[0023] The human α-synaptic protein gene has undergone codon optimization.

[0024] As a preferred embodiment of the fusion expression vector of the present invention, wherein the amino acid sequence of the RGG domain is SEQ ID NO:3.

[0025] Beneficial effects of this invention:

[0026] (1) Application of novel chassis cells: This invention is the first to apply Corynebacterium glutamicum to the production of α-synaptic protein, providing a new chassis cell option for protein expression.

[0027] (2) No endotoxin contamination: Corynebacterium glutamicum does not produce endotoxins, ensuring the high safety of the α-synaptic protein produced, which is particularly suitable for biomedical and clinical applications.

[0028] (3) High protein yield: By applying liquid-liquid phase separation technology, the present invention significantly increases the yield of α-synaptic protein.

[0029] (4) Wide range of applications: This method is not only applicable to the production of α-synaptic proteins, but can also be extended to the expression of other challenging proteins. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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:

[0031] Figure 1 This is an SDS-PAGE analysis diagram of α-synuclein in an embodiment of the present invention.

[0032] Figure 2 This is a grayscale analysis diagram of recombinant bacterial growth and SDS-PAGE in an embodiment of the present invention.

[0033] Figure 3 This is a fluorescence microscope image of membrane-free organelles mediated by liquid-liquid phase separation in an embodiment of the present invention.

[0034] Figure 4 This is a schematic diagram of the method in an embodiment of the present invention. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Example 1

[0039] Construction and expression of Corynebacterium glutamicum α-synaptic protein expression vector:

[0040] The amino acid sequence of human α-synuclein (GenBank: AGJ51950.1) was optimized based on the codon preference of Corynebacterium glutamicum, and a 6×His tag was added to the C-terminus (for subsequent purification). The optimized gene sequence will be submitted to Anshengda Biotechnology Co., Ltd. for gene synthesis. The protein sequence and the optimized gene sequence are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0041] α-synuclein was ligated to the backbone of the Corynebacterium glutamicum / Escherichia coli shuttle plasmid pXMJ19 (Biovector, BiovectorpXMJ19) via Hind3 and EcoR1 restriction endonuclease sites.

[0042] The specific steps are as follows: The pXMJ19 backbone vector and the above fragment are digested with Hind3 and EcoR1, the digested vector and fragment are purified, the pXMJ19 backbone vector and fragment are mixed, and the ligation reaction is performed using TakaRa's Solution I ligase.

[0043] Refer to Tables 1 and 2 for the enzyme digestion and ligation system and reaction conditions.

[0044] Table 1. Double enzyme digestion reaction system

[0045] 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

[0046] Reaction conditions: 37℃, 60min.

[0047] Table 2 Connection Reaction System

[0048] Element Volume (μL) Enzyme digested vector 2 Fragments after enzyme digestion 3 Solution I 5 Total volume 10

[0049] 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.

[0050] PCR was used to verify the single colonies that grew on the plate.

[0051] Positive clones were selected for sequencing verification, and the expression vector α-synuclein-P19 was finally obtained.

[0052] The above expression vector was transformed into the Corynebacterium glutamicum expression strain CGMCC 1.15647 by electroporation.

[0053] Select the correct single clones and inoculate them into 10 mL of LBB medium. Then, transfer 2% of the inoculum to fresh 10 mL of LBB medium. The LBB medium formula is as follows:

[0054] Component mass concentration

[0055] Tryptone 10g / L

[0056] Yeast extract 5g / L

[0057] NaCl (sodium chloride) 10g / L

[0058] Brain and heart extract 10g / L

[0059] Add distilled water to a final volume of 1L.

[0060] After culturing at 220 rpm and 30°C for 24 hours, the bacterial cells were collected by centrifugation at 10 OD.

[0061] 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; see schematic diagram of the method. Figure 4 .

[0062] like Figure 1 As shown, a distinct protein band was observed at the expected protein molecular weight, indicating that α-synaptic protein was successfully expressed in Corynebacterium glutamicum with a molecular weight of approximately 20 kDa.

[0063] Example 2

[0064] Liquid-liquid phase separation technology further enhanced the expression of α-synaptic protein:

[0065] In cells, overexpression and accumulation of α-synaptic proteins can be toxic to cells;

[0066] In Corynebacterium glutamicum, to avoid the potential toxicity of α-synaptic protein aggregation, this invention explores the effectiveness of liquid-liquid phase separation technology-mediated enhancement of α-synaptic protein expression in membrane-free organelles.

[0067] The RGG domain was optimized according to the Corynebacterium glutamicum codon (see SEQ ID NO:3 for the optimized sequence) and synthesized by Anshengda Biotechnology Co., Ltd.

[0068] The α-synuclein-P19 vector constructed in Example 1 was linearized using HindIII restriction enzyme, and the RGG domain was inserted into the N-terminus of the α-synuclein protein by homologous recombination to obtain the fusion expression vector RGG-α-synuclein-P19.

[0069] The correctly sequenced vector was transferred into the Corynebacterium glutamicum expression strain CGMCC 1.15647 by electroporation.

[0070] Select the correct single clones and inoculate them into 10 mL of LBB medium, then transfer them to fresh 10 mL of LBB medium at a 2% inoculation rate.

[0071] After culturing at 220 rpm and 30°C for 24 hours, the bacterial cells were collected by centrifugation at 10 OD.

[0072] After ultrasonic disruption of the bacterial cells, the supernatant was collected by centrifugation, and the expression of the above proteins was detected by 12% SDS-PAGE.

[0073] like Figure 1 As shown, the histone fused with RGG phase was successfully expressed with a molecular weight of approximately 38 kDa; further grayscale analysis of the protein bands was performed using ImageJ.

[0074] Figure 2 The results showed that phase separation significantly increased the expression level of α-synuclein protein. The protein yield of the phase-separated group was 1.48 times that of the non-phase-separated control group. Furthermore, the OD of the RGG-fused phase-separated group at the end of shake-flask fermentation was 10.25, which was close to the control group's 10.55 and significantly higher than that of the non-RGG-fused experimental group (8.55). This indicates that liquid-liquid phase separation-mediated non-membrane organelles reduced the toxic effects of α-synuclein intracellular accumulation on cells to some extent.

[0075] Liquid-liquid phase separation observation: Further, EGFP was fused to the C-terminus of the target protein α-synuclein to construct the phase-separated expression vector RGG-α-synuclein-EGFP and the non-phase-separated vector α-synuclein-EGFP. These plasmids were then transformed into Corynebacterium glutamicum, and the formation of membrane-free organelles was observed using a fluorescence microscope. Figure 3 As shown, the non-separated experimental group α-synuclein-EGFP exhibited a uniformly distributed fluorescence signal in the cytoplasm, and no obvious aggregate formation was observed.

[0076] In contrast, RGG-α-synuclein-EGFP, fused with the RGG domain, formed distinct spherical or hemispherical droplet-like structures in Corynebacterium glutamicum, primarily distributed in the polar regions of the cell. This characteristic distribution pattern is highly consistent with RGG-mediated liquid-liquid phase separation observed in Escherichia coli, indicating that the RGG domain successfully mediated the formation of membraneless organelles of the target protein α-synuclein in the host cell.

[0077] In summary, the results demonstrate that liquid-liquid phase separation, by encapsulating α-synuclein in droplets, avoids the potential toxicity of protein aggregation to host cells and increases the expression yield of α-synuclein. This discovery provides a novel solution for the recombinant expression of α-synuclein, and is expected to promote the large-scale production and application of α-synuclein.

[0078] This invention proposes an innovative use of Corynebacterium glutamicum as an expression system to ensure product safety; it also introduces liquid-liquid phase separation (LLPS) technology, which effectively reduces protein aggregation, lowers cytotoxicity, and increases expression yield by isolating the target protein in droplet-shaped membraneless organelles.

[0079] SEQ ID NO:1, α-synuclein protein sequence:

[0080] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAHHHHHH

[0081] SEQ ID NO:2, Codon-optimized α-synuclein gene coding sequence:

[0082] ATGGACGTGTTCATGAAGGGCCTGTCCAAGGCAAAGGAAGGCGTGGTGGCAGCAGCAGAAAAGACCAAGCAGGGCGTGGCAGAAGCCGCCGGCAAGACCAAGGAAGGCGTCCTGTACGTGGGCTCCAAGACCAAGGAGGGCGTGGTGCACGGCGTGGCCACCGTTGCTGAAAAGACCAAAGAACAGGTGACCAACGTGGGCGGCGCAGTGGTGACCGGCGTGACTGCCGTGGCACAGAAGACCGTGGAAGGCGCCGGCTCCATCGCAGCAGCCACCGGTTTCGTGAAGAAGGACCAGCTGGGCAAGAACGAAGAAGGCGCCCCACAGGAAGGCATCCTGGAAGACATGCCAGTGGACCCAGATAACGAAGCCTACGAAATGCCATCCGAAGAAGGCTACCAGGATTACGAACCAGAAGCACACCATCACCATCACCATTAA

[0083] SEQ ID NO:3, RGG domain-encoding gene sequence:

[0084] ATGCCACGCGAATCCAACCAGTCCAACAACGGCGGTTCCGGTAACGCAGCCTTGAACCGTGGTGGTCGCTACGTTCCACCACACTTGCGCGGTGGTGATGGCGGTGCAGCAGCAGCTGCATCCGCA GGCGGTGATGATCGTCGAGGTGGTGCAGGTGGTGGCGGCTACCGCCGAGGTGGCGGTAACTCCGGTGGAGGCGGAGGTGGTGGTTACGATCGCGGCTATAACGATAACCGCGATGATCGCGATAAC CGAGGTGGTTCCGGTGGCTACGGTCGCGATCGCAACTACGAAGACCGCGGATACAACGGTGGCGGCGGTGGTGGTGGCAACCGCGGCTACAACAACAACCGCGGTGGCGGTGGAGGTGGCTACAAC CGCCAAGATCGCGGTGATGGTGGCTCTTCCAACTTCTCTCGCGGCGGTTACAACAATCGCGATGAAGGCTCCGATAACCGTGGCTCCGGTCGCTCTTACAACAACGATCGCCGCGATAACGGCGGT

[0085] This invention discloses a highly efficient method for producing recombinant proteins based on Corynebacterium glutamicum, cleverly utilizing liquid-liquid phase separation technology to overcome existing technological bottlenecks. To achieve efficient expression of α-synaptic proteins, this invention first optimizes the gene sequence codons to better suit the Corynebacterium glutamicum expression system. More importantly, this invention innovatively constructs a fusion protein containing an RGG domain, utilizing its phase separation-mediated properties to guide the target protein to spontaneously form stable droplet-like structures within the cell. This "droplet factory" not only effectively reduces abnormal protein aggregation but also significantly reduces toxicity to host cells.

[0086] Compared to traditional expression systems, the Corynebacterium glutamicum platform used in this invention naturally avoids the risk of endotoxin contamination, laying the foundation for simplifying downstream purification processes and reducing production costs. The introduction of liquid-liquid phase separation technology provides a novel approach to solving the challenging problems of α-synaptic protein's easy aggregation and high toxicity, making the production process more efficient and controllable. The technical solution established in this invention not only opens up new avenues for the large-scale production of α-synaptic protein but also provides important theoretical and technical references for the industrial production of other recombinant proteins with similar characteristics (such as proteins that are prone to aggregation or have cytotoxicity), possessing broad application prospects and market value.

[0087] 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 or substitutions should be covered within the scope of the present invention.

Claims

1. A method for producing recombinant human protein in industrial microorganisms, characterized in that: include, Construct a fusion expression vector containing the RGG domain and codon-optimized human α-synaptic protein gene; The fusion expression vector was transformed into Corynebacterium glutamicum; Transformed Corynebacterium glutamicum was cultured at 30-37℃ and 200-250 rpm to express the fusion protein and form a dynamically reversible droplet structure within the cell. Collect cultured bacterial cells and extract human α-synaptic protein; The amino acid sequence of the RGG domain is SEQ ID NO:3; The amino acid sequence of the human α-synaptic protein is SEQ ID NO:1, and its codon-optimized nucleic acid sequence is SEQ ID NO:

2.

2. The method as described in claim 1, characterized in that: The Corynebacterium glutamicum includes strain CGMCC 1.15647.

3. The method as described in claim 1, characterized in that: The culture was carried out in LBB medium for 18–30 h.

4. The application of the method according to any one of claims 1 to 3 in the production of human α-synaptic protein.

5. The application as described in claim 4, characterized in that: The method improves the expression level of human α-synaptic protein in Corynebacterium glutamicum.

6. A fusion expression vector for producing human α-synaptic protein, characterized in that: include, It contains genes with RGG domains and human α-synaptic protein genes; Contains a 6×His tag sequence; The human α-synaptic protein gene has undergone codon optimization; The amino acid sequence of the RGG domain is SEQ ID NO:3; The amino acid sequence of the human α-synaptic protein is SEQ ID NO:1, and its codon-optimized nucleic acid sequence is SEQ ID NO:2.

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