Method for producing recombinant human protein in industrial microorganisms

Abstract

The invention discloses a method for producing recombinant human-derived protein in industrial microorganisms, which comprises the following steps: introducing a gene for coding alpha synaptic protein into corynebacterium glutamicum by utilizing a genetic engineering technology to realize expression of the alpha synaptic protein; in order to avoid toxicity to host cells after a large amount of alpha synaptic proteins are expressed and gathered, the alpha synaptic proteins are isolated in droplet-shaped membrane-free organelles by using a liquid-liquid phase separation technology, so that the protein yield is further improved. Compared with the most widely used Escherichia coli expression system production mode, the method provided by the invention has the following advantages: corynebacterium glutamicum is adopted as a novel chassis cell, so that the problems of endotoxin pollution, insoluble inclusion body formation and the like in the traditional Escherichia coli expression system are solved; by applying a liquid-liquid phase separation technology, the yield of alpha synapsin is remarkably improved; the method is not only suitable for production of alpha synapsin, but also can be popularized to other proteins which are difficult to express, and provides a new technical path for the field of biological pharmacy.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biotechnology, and specifically relates to a method for producing recombinant human protein in industrial microorganisms. Background Art

[0002] Recombinant human proteins have a wide range of applications in biomedical research, drug development, disease diagnosis and treatment, and other fields. With the development of biotechnology, the market demand for high-quality, low-cost, and highly safe recombinant human proteins is growing. At present, the industrial production of recombinant human proteins mainly relies on several major expression systems. The Escherichia coli expression system has the advantages of rapid growth and low culture cost, but there are problems of endotoxin contamination and easy formation of inclusion bodies; the yeast expression system, as a eukaryotic expression system, can perform post-translational modifications, but it is prone to excessive glycosylation, which affects protein function; although the mammalian cell expression system can obtain products with complete post-translational modifications and structures closest to nature, the culture cost is high and the production cycle is long.

[0003] Corynebacterium glutamicum is a Gram-positive bacterium that is generally recognized as safe (GRAS). It has unique advantages in the field of recombinant protein production due to its lack of endotoxin contamination, strong protein secretion ability, mature fermentation process and clear genetic background. In recent years, its potential as an efficient prokaryotic recombinant protein expression host has attracted widespread attention.

[0004] The research object is α-synuclein, a key protein in the nervous system. This protein is widely expressed in presynaptic nerve endings and is involved in the regulation of neuronal function and synaptic plasticity. α-synuclein is closely related to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and can be used as an important biomarker for disease diagnosis. It has important value in basic research, drug development and diagnostic applications.

[0005] Existing technologies face many challenges in the production process of α-synaptic protein. Traditional expression systems have safety risks and unstable product quality. At the same time, α-synaptic protein is prone to aggregation and is toxic to host cells, which also poses challenges to its production. 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 object of the present invention is to overcome the deficiencies in the prior art and provide a method for producing recombinant human proteins in industrial microorganisms.

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

[0010] Constructing a fusion expression vector containing the RGG domain and the codon-optimized human α-synapsin gene;

[0011] Transforming the fusion expression vector into Corynebacterium glutamicum;

[0012] Cultivating the transformed Corynebacterium glutamicum at 30-37° C. and 200-250 rpm to express the fusion protein and form a dynamic and reversible droplet-like structure in the cell;

[0013] The cultured bacteria were collected and human α-synaptophysin was extracted.

[0014] As a preferred embodiment of the method of the present invention, the amino acid sequence of the RGG domain is SEQ ID NO:3.

[0015] As a preferred embodiment of the method of the present invention, the amino acid sequence of the human α-synaptophysin is SEQ ID NO: 1, and the codon-optimized nucleic acid sequence thereof is SEQ ID NO: 2.

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

[0017] As a preferred embodiment of the method of the present invention, the culturing is carried out in LBB medium for 18 to 30 hours.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for producing recombinant human proteins in industrial microorganisms and its application in the production of human α-synaptophysin.

[0019] As a preferred embodiment of the application of the present invention, the method increases the expression level of human α-synaptophysin in Corynebacterium glutamicum.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide a fusion expression vector for producing human α-synapsin, comprising:

[0021] Contains RGG domain gene and human alpha-synapsin gene;

[0022] Contains 6×His tag sequence;

[0023] The human α-synapsin gene is codon-optimized.

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

[0025] Beneficial effects of the present invention:

[0026] (1) Application of novel chassis cells: The present 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 endotoxin, which ensures the high safety of the produced α-synaptophysin, making it 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-synaptophysin.

[0029] (4) Broad application potential: This method is not only applicable to the production of α-synaptobrevin, but can also be extended to the expression of other challenging proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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:

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

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

[0033] Figure 3 This is a fluorescence microscopy image of a membraneless organelle mediated by liquid-liquid phase separation in an embodiment of the present invention.

[0034] Figure 4 Schematic diagram of the method in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[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 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.

[0038] Example 1

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

[0040] The amino acid sequence of human α-synuclein (α-synuclein) (GenBank: AGJ51950.1) was optimized with reference to 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 for gene synthesis. The protein sequence and the optimized gene sequence refer to SEQ ID NO: 1 and SEQ ID NO: 2;

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

[0042] The specific steps are as follows: double-digest the pXMJ19 backbone vector and the above-mentioned fragment with Hind3 and EcoR1, purify the digested vector and fragment, mix the pXMJ19 backbone vector and fragment, and use TakaRa's Solution I ligase to perform a ligation reaction.

[0043] Please refer to Table 1 and Table 2 for the enzyme digestion and ligation system and reaction condition settings.

[0044] Table 1 Double enzyme digestion reaction system

[0045] Element Addition amount Enzyme 1 5μL Enzyme 2 5μL Buffer 10μL Expression vector / fragment 2-4 μg Sterile water Make up to 100 μL

[0046] Reaction conditions: 37°C, 60 min.

[0047] Table 2 Ligation reaction system

[0048] Element Volume (μL) Enzyme-cut vector 2 Enzyme digested fragments 3 SolutionI 5 Total volume 10

[0049] 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;

[0050] Single colonies grown on the plates were verified by PCR;

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

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

[0053] Pick the correct single clone and inoculate it into 10mL LBB medium, then transfer it into fresh 10mL LBB medium at a 2% inoculation volume. The LB medium formula is:

[0054] Component mass concentration

[0055] Tryptone 10g / L

[0056] Yeast extract 5g / L

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

[0058] Brain heart extract 10g / L

[0059] Add distilled water to make up to 1L.

[0060] After culturing at 220 rpm and 30°C for 24 h, 10 OD of bacteria were collected by centrifugation;

[0061] After ultrasonic disruption of the cells, the supernatant was centrifuged and the expression of the above proteins was detected using 12% SDS-PAGE. Figure 4 .

[0062] like Figure 1 As shown, a clear protein band was observed at the expected protein molecular weight, and α-synaptophysin was successfully expressed in Corynebacterium glutamicum with a molecular weight of about 20 kDa.

[0063] Example 2

[0064] Liquid-liquid phase separation technology further improves alpha-synaptophysin expression:

[0065] In cells, overexpression and aggregation of α-synapsin can be toxic to cells;

[0066] In order to avoid the potential toxicity caused by the aggregation of α-synaptophysin in Corynebacterium glutamicum, the present invention explores the effectiveness of membraneless organelles mediated by liquid-liquid phase separation technology in increasing the expression of α-synaptophysin.

[0067] The RGG domain was optimized according to the codons of Corynebacterium glutamicum (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 with HindIII endonuclease, and the RGG domain was inserted into the N-terminus of α-synuclein by homologous recombination to obtain a fusion expression vector RGG-α-synuclein-P19.

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

[0070] Pick the correct single clone and inoculate it into 10mL LBB medium, then transfer it into fresh 10mL LBB medium at a 2% inoculation volume;

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

[0072] After the bacteria were disrupted by ultrasound, 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 fusion RGG phase separation histone was successfully expressed with a molecular weight of approximately 38 kDa; ImageJ was further used to perform grayscale analysis of the protein bands.

[0074] Figure 2 The results showed that phase separation significantly increased the expression level of α-synuclein protein. The protein yield of the phase separation group was 1.48 times that of the non-phase separation control group. In addition, the OD of the RGG fusion phase separation group at the end of shake flask fermentation was 10.25, which was close to 10.55 of the control group and significantly higher than the experimental group without RGG fusion (8.55), indicating that the membraneless organelles mediated by liquid-liquid phase separation reduced the toxic effects of α-synuclein intracellular aggregation on cells to a certain extent.

[0075] Liquid-liquid phase separation observation: EGFP was further fused to the C-terminus of the target protein α-synuclein to construct a phase separation expression vector RGG-α-synuclein-EGFP and a non-phase separation vector α-synuclein-EGFP. The above plasmids were transformed into Corynebacterium glutamicum, and the formation of membraneless organelles was observed under a fluorescence microscope. Figure 3 As shown, in the experimental group without phase separation, α-synuclein-EGFP showed 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 obvious spherical or hemispherical droplet-like structures in Corynebacterium glutamicum, which were mainly distributed in the polar regions of the cells. This characteristic distribution pattern is highly consistent with the RGG-mediated liquid-liquid phase separation phenomenon observed in Escherichia coli by previous researchers, indicating that the RGG domain successfully mediated the formation of membrane-less organelles of the target protein α-synuclein in host cells.

[0077] In summary, the above results prove that liquid-liquid phase separation can avoid the potential toxicity of protein aggregation to host cells by encapsulating α-synuclein in droplets, thereby increasing the expression yield of α-synuclein. This discovery provides a new solution for the recombinant expression of α-synuclein, which is expected to promote the large-scale production and application of α-synuclein.

[0078] The present invention proposes the innovative use of Corynebacterium glutamicum as an expression system to ensure product safety; the liquid-liquid phase separation (LLPS) technology is introduced to effectively reduce protein aggregation, lower cytotoxicity and increase 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] The present invention discloses a highly efficient method for producing recombinant proteins based on Corynebacterium glutamicum, which cleverly utilizes liquid-liquid phase separation technology to overcome the bottleneck of the prior art; in order to achieve efficient expression of α-synaptophysin, the present invention first performs codon optimization on its gene sequence to make it more suitable for the expression system of Corynebacterium glutamicum. More importantly, the present invention innovatively constructs a fusion protein containing an RGG domain, and utilizes its mediated phase separation characteristics to guide the target protein to spontaneously form a stable droplet-shaped structure in the cell. This "droplet factory" not only effectively reduces the abnormal aggregation of proteins, but also significantly reduces the toxicity to host cells.

[0086] Compared with the traditional expression system, the Corynebacterium glutamicum platform adopted in the present invention naturally avoids the risk of endotoxin contamination, laying the foundation for the simplification of downstream purification process and the reduction of production cost. The introduction of liquid-liquid phase separation technology provides a new idea for solving the challenging problems such as the easy aggregation and high toxicity of α-synaptic protein, making the production process more efficient and controllable. The technical scheme established by the present invention not only opens up a new way for the large-scale production of α-synaptic protein, but also provides important theoretical reference and technical reference for the industrial production of other recombinant proteins with similar characteristics (such as proteins that are easy to aggregate or have cytotoxicity), and has 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 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 can 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 recombinant human protein in industrial microorganisms, characterized in that: include, Constructing a fusion expression vector containing the RGG domain and the codon-optimized human α-synapsin gene; Transforming the fusion expression vector into Corynebacterium glutamicum; Cultivating the transformed Corynebacterium glutamicum at 30-37° C. and 200-250 rpm to express the fusion protein and form a dynamic and reversible droplet-like structure in the cell; The cultured bacteria were collected and human α-synaptophysin was extracted.

2. The method according to claim 1, characterized in that: The amino acid sequence of the RGG domain is SEQ ID NO:

3.

3. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the human alpha-synaptophysin is SEQ ID NO: 1, and the codon-optimized nucleic acid sequence is SEQ ID NO:

2.

4. The method according to claim 3, characterized in that: The Corynebacterium glutamicum includes the CGMCC 1.15647 strain.

5. The method according to claim 1 or 4, characterized in that: The culturing is carried out in LBB medium for 18 to 30 hours.

6. Use of the method according to any one of claims 1 to 5 in the production of human α-synapsin.

7. The use according to claim 6, characterized in that: The method increases the expression level of human alpha synaptophysin in Corynebacterium glutamicum.

8. A fusion expression vector for producing human α-synaptophysin, characterized in that: include, Contains RGG domain gene and human alpha-synapsin gene; Contains 6×His tag sequence; The human α-synapsin gene is codon-optimized.

9. The fusion expression vector according to claim 8, characterized in that: The amino acid sequence of the RGG domain is SEQ ID NO:3.

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