Method for improving expression quantity of recombinant staphylococcus aureus protein A

Abstract

The invention provides a method for improving the expression quantity of recombinant staphylococcus aureus protein A. The method comprises the following steps: inoculating a recombinant staphylococcus aureus protein A strain into a YPD culture medium for activation; sequentially inoculating the activated strains into a BMGY composite culture medium containing coffee seed polyphenol and juglone and a BMMY composite culture medium containing myrobalan tannin and neoandrographolide for amplification and induction culture, finally harvesting cells, and measuring the expression quantity of the recombinant staphylococcus aureus protein A. Aiming at the strain growth characteristics in the culture process of the recombinant staphylococcus aureus protein A, an innovative optimization strategy is provided on the basis of BMGY and BMMY culture media commonly used in a yeast expression system: in different stages of strain amplification and induction, an amplification culture medium and an induction culture medium which are specially matched are respectively used, and according to the strain requirements of each stage, the culture medium and the induction culture medium are used for amplification and induction; different natural compounds are added. According to the staged and differentiated culture mode, proliferation of the strain can be remarkably promoted, the yield of the recombinant staphylococcus aureus protein A can be effectively increased, the problem that the expression quantity of the recombinant staphylococcus aureus protein A is low at present is successfully solved, and the expression quantity is remarkably increased.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation and protein expression, and particularly to a method for increasing the expression level of recombinant Staphylococcus aureus protein A. Background Art

[0002] Recombinant Staphylococcus aureus protein A (hereinafter referred to as protein A) is a tool protein of great value in the fields of biotechnology and biomedicine. It can specifically bind to the Fc regions of various mammalian immunoglobulins, and this property enables it to play a key role in many fields such as antibody purification, immunoassay, diagnostic reagent development, and biopharmaceuticals.

[0003] Protein A was initially isolated from the cell wall of Staphylococcus aureus and has many important biological functions. In the field of immunology, it can specifically bind to the Fc segments of various mammalian immunoglobulins, such as human IgG1, IgG2, IgG4, etc., and this binding does not affect the binding of the Fab segment of the antibody to the antigen, and can be used in immunoprecipitation experiments to isolate and identify antigens; it can also activate the complement system, promote phagocytosis of pathogens by phagocytes, and regulate the activation and proliferation of B cells, thereby regulating the immune response. At the same time, it can act as a mimic antigen to induce an immune response in the body and interact with the T cell surface receptor to participate in cell-mediated immunity. In terms of the bacterial pathogenesis mechanism, protein A can bind to the host immunoglobulin to form a complex, masking the bacterial antigen epitope to evade host immune surveillance, and can also bind to the host cell surface receptor to promote bacterial adhesion and invasion, such as binding to the platelet receptor to facilitate bacterial colonization and diffusion in the blood vessels. In biotechnological applications, relying on its specific binding property to the Fc segment of immunoglobulins, it is widely used for antibody purification, efficiently separating and purifying antibodies from complex biological samples by affinity chromatography; it is also used in immunoassays, such as as a detection reagent in experiments such as ELISA and Western blot, enhancing the detection sensitivity and specificity.

[0004] Low expression levels of Protein A have caused a series of problems in both scientific research and production. In the field of scientific research, for basic biological research to deeply explore the structure, function, mechanism of action of Protein A and its interactions with other molecules, a large amount of high-purity Protein A is required for various experiments. However, the low expression level makes it extremely difficult to obtain sufficient Protein A, resulting in the inability to carry out experiments as planned and severely delaying the research progress. For example, in the experiment of Protein A crystal structure analysis, it is difficult to successfully cultivate crystals due to insufficient protein quantity. At the same time, the low expression level limits the sample size, making it difficult to conduct in-depth research that requires a large number of samples. For experiments such as the determination of the affinity between Protein A and ligand molecules and kinetic studies, comprehensive concentration gradient experiments and multi-parameter analyses cannot be carried out, affecting the in-depth understanding of its biological characteristics. In addition, the low expression level limits the production of Protein A per unit time, prolonging the production cycle, reducing efficiency, affecting the enterprise's market supply capacity and economic benefits, and increasing production risks such as bacterial contamination. Therefore, it is crucial to improve the expression level of Protein A. Summary of the Invention

[0005] In view of this, the present invention proposes a method for improving the expression level of recombinant Staphylococcus aureus Protein A, achieving the purpose of increasing the expression level of recombinant Staphylococcus aureus Protein A.

[0006] The technical solution of the present invention is realized as follows: The present invention provides a method for improving the expression level of recombinant Staphylococcus aureus Protein A, comprising the following steps:

[0007] Inoculate the recombinant Staphylococcus aureus Protein A strain in YPD medium for activation, and then sequentially inoculate the activated strain into BMGY complex medium containing small fruit coffee seed polyphenols and juglone, and BMMY complex medium containing chebulinic acid and neoandrographolide for amplification and induction culture. Finally, harvest the cells and measure the expression level of recombinant Staphylococcus aureus Protein A.

[0008] On the basis of the above technical solution, preferably, the components of the BMGY complex medium are: yeast extract 8 - 12 g / L, peptone 18 - 22 g / L, glycerol 0.5 - 1.5 v / v%, YBM 1.2 - 1.4 g / L, sodium hydrogen phosphate - citrate buffer 8 - 12 v / v%, biotin 0.6 - 1.0 μ / L, small fruit coffee seed polyphenols 1 - 30 μM, juglone 1 - 10 μM, and finally made up to 1 L with deionized water.

[0009] When preparing the BMGY complex medium, first add yeast extract, peptone, glycerol and part of deionized water, mix well, sterilize by high temperature and high pressure (105°C, 20 min), and then mix in sterile YNB, sodium hydrogen phosphate - citrate buffer, biotin, small fruit coffee seed polyphenols and juglone, and finally make up the volume.

[0010] On the basis of the above technical solutions, preferably, the components of the BMMY complex medium are: 8 - 12 g / L of yeast extract, 18 - 22 g / L of peptone, 0.5 - 1.5 v / v% of glycerol, 1.2 - 1.4 g / L of YBM, 8 - 12 v / v% of disodium hydrogen phosphate - citric acid buffer, 5 v / v% of methanol, 0.8 μ / L of biotin, 1 - 50 μM of chebulinic acid, 1 - 10 μM of neoandrographolide, and finally made up to 1 L with deionized water.

[0011] When preparing the BMMY complex medium, first add yeast extract, peptone, glycerol and part of deionized water, mix well, sterilize by high temperature and high pressure (105 °C, 20 min), and then mix in sterile YNB, disodium hydrogen phosphate - citric acid buffer, methanol, biotin, chebulinic acid and neoandrographolide, and finally make up the volume.

[0012] When preparing YNB in the present invention, first prepare a 10 - fold solution (10×YNB), specifically: 1.2 - 1.4 g of solid YNB is dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4 °C, and diluted when in use.

[0013] When preparing biotin in the present invention, first prepare a 500 - fold solution (500×B), specifically: 15 - 25 mg of biotin is dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4 °C, and diluted when in use.

[0014] Small - fruit coffee seed polyphenols, juglone, chebulinic acid and neoandrographolide are respectively filtered and sterilized through a 0.22 - um filter membrane and then added to the medium.

[0015] On the basis of the above technical solutions, preferably, the YPD medium contains 8 - 12 g / L of yeast extract, 18 - 22 g / L of peptone, and 18 - 22 g / L of glucose.

[0016] On the basis of the above technical solutions, preferably, when the OD of the recombinant Staphylococcus aureus protein A bacterial liquid 600 reaches 0.6 - 1.2, transfer the bacterial liquid to the BMGY complex medium for amplification culture.

[0017] On the basis of the above technical solutions, preferably, when the OD of the recombinant Staphylococcus aureus protein A bacterial liquid 600 reaches 0.6 - 0.8, transfer the bacterial liquid to the BMMY complex medium for induction culture.

[0018] On the basis of the above technical solutions, preferably, during the activation culture, the inoculation amount of the strain is 1%, and the culture conditions are 28 - 32 °C, shaking in a shaker at 210 - 230 r / min for 14 - 18 h.

[0019] On the basis of the above technical scheme, preferably, during the amplification culture, the inoculation amount of the strain is 1%, the culture conditions are 28-32°C, and the shaking culture is carried out at 210-230r / min.

[0020] On the basis of the above technical scheme, preferably, during activation culture, the inoculation amount of the strain is 1%, the culture conditions are 18-23° C., and shaking on a shaker at 210-230 r / min for 70-75 h.

[0021] On the basis of the above technical solution, preferably, the pH value of the disodium hydrogen phosphate-citric acid buffer is 3-5.

[0022] The method of increasing the expression amount of recombinant Staphylococcus aureus protein A of the present invention has the following beneficial effects compared with the prior art:

[0023] (1) The present invention aims at the growth characteristics of the strains during the cultivation of recombinant Staphylococcus aureus protein A, and proposes an innovative optimization strategy based on the BMGY and BMMY culture media commonly used in yeast expression systems: at different stages of strain amplification and induction, specially adapted amplification medium and induction medium are used respectively, and different natural compounds are added according to the strain requirements at each stage. This phased and differentiated cultivation method can not only significantly promote the proliferation of strains, but also effectively increase the yield of recombinant Staphylococcus aureus protein A, successfully solving the current problem of low expression of recombinant Staphylococcus aureus protein A and achieving a significant increase in expression.

[0024] (2) During the expansion period of culture, the present invention adds small-fruit coffee seed polyphenols and juglone to the culture medium, which can regulate the expression of cell cycle-related proteins, accelerate cell division and proliferation, and significantly improve the cell growth rate and final cell density in the nutrient-rich BMGY culture medium, thereby laying a solid cellular foundation for the subsequent protein A expression.

[0025] (3) In the induction stage of Staphylococcus aureus protein A, the present invention adds terminalia tannin and neoandrographolide to the culture medium, which can enhance the induction effect of methanol, upregulate the expression level of related genes, and then promote the synthesis of protein A. At the same time, it helps to improve the protein folding environment in the cell, can interact with the molecular chaperone in the cell, enhance its function, and improve the folding efficiency and quality of protein A. Terminalia tannin and neoandrographolide can reduce the stress response of the cell, protect the cell from stress damage, maintain the activity and stability of the cell during the induced expression process, and ensure the continuous and efficient expression of protein A.

[0026] (4) According to the characteristics of the growth stage of Staphylococcus aureus protein A, natural compounds are added to the BMGY and BMMY media, which not only ensures the rapid and stable doubling of the protein A bacterial liquid cells, but also improves the expression level of protein A. Brief Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Comparison chart of the expression levels of Staphylococcus aureus protein A in Example 1 and Comparative Examples 1-3;

[0029] Figure 2 Comparison chart of the expression levels of Staphylococcus aureus protein A in Comparative Example 1 and Comparative Examples 4-6;

[0030] Figure 3 Comparison chart of the expression levels of Staphylococcus aureus protein A in Comparative Example 2 and Comparative Examples 7-9;

[0031] Figure 4 Comparison chart of the expression levels of Staphylococcus aureus protein A in Comparative Examples 1-2 and Comparative Examples 10-13. Detailed Embodiments

[0032] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0033] The recombinant Staphylococcus aureus protein A described in the present invention is derived from Wuhan Wonder Biotechnology Co., Ltd.; yeast extract and peptone are purchased from OXOID Company; the YNB medium is purchased from Solarbio Company; the methanol is purchased from Sinopharm Chemical Reagent Co., Ltd.; the glycerol is purchased from Macklin Co., Ltd.; the biotin is purchased from Biosharp Company; the polyphenols from Coffea arabica seeds and juglone are purchased from Shanghai Jingke Chemical Technology Co., Ltd.; the chebulinic acid and neoandrographolide are purchased from Nanjing Qingyun Biotechnology Co., Ltd.

[0034] Example 1

[0035] The method for increasing the expression level of Staphylococcus aureus protein A in this embodiment includes the following steps:

[0036] (1) Strain activation stage: Take out the cryopreserved recombinant Staphylococcus aureus protein A strain from the -80°C refrigerator, inoculate it into the YPD medium at a ratio of 1%, and culture it at 30°C with shaking at 220 r / min for 16 h.

[0037] The YPD medium (100 mL) contains 1 g of yeast extract, 2 g of peptone, and 2 g of glucose.

[0038] (2) Cell amplification stage: When the OD600nm of the recombinant Staphylococcus aureus protein A bacterial solution reaches 0.8, transfer the bacterial solution to the BMGY complex medium and culture it at 30°C with shaking at 220 r / min.

[0039] The formula of the BMGY medium is that 100 mL of BMGY contains 1 g of yeast extract, 2 g of peptone, 1 mL of glycerol, 79 mL of deionized water. After autoclaving (105°C, 20 min), it is mixed with 10 mL of sterile 10×YNB, 10 mL of sodium hydrogen phosphate-citric acid buffer solution with pH 4, 0.2 mL of 500×B. Polyphenols from Coffea arabica seeds are filtered and sterilized through a 0.22 μm filter membrane and added to the BMGY medium to a final concentration of 10 μM. Juglone is filtered and sterilized through a 0.22 μm filter membrane and added to the BMGY medium to a final concentration of 5 μM.

[0040] The formula of 10×YNB is 13.4 g of YNB solid dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4°C. The formula of 500×B is 20 mg of biotin dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4°C.

[0041] (3) Cell induction stage: When the OD 600 of the recombinant Staphylococcus aureus protein A bacterial solution reaches 0.7, transfer the bacterial solution to the BMMY complex medium and culture it at 20°C with shaking at 220 r / min for 72 h. Centrifuge at 8000 rpm for 5 min to harvest the supernatant, and measure the expression level of recombinant Staphylococcus aureus protein A in the supernatant by the Bradford method.

[0042] The MMY medium formulation is that 100 mL of BMMY contains 1 g of yeast extract, 2 g of peptone, 1 mL of glycerol, 80 mL of deionized water, which is autoclaved (105 °C, 20 min) and then mixed with 10 mL of sterile 10×YNB (same as the amplification stage), 10 mL of sodium hydrogen phosphate-citrate buffer at pH 4, 0.5 mL of methanol, 0.2 mL of 500×B (same as the amplification stage). Chebulinic acid is filtered and sterilized through a 0.22-μm filter membrane and added to the BMMY medium to a final concentration of 20 μM, and neoandrographolide is filtered and sterilized through a 0.22-μm filter membrane and added to the BMMY medium to a final concentration of 5 μM.

[0043] Example 2

[0044] The method for increasing the expression level of Staphylococcus aureus protein A in this example includes the following steps:

[0045] (1) Strain activation stage: Take out the cryopreserved recombinant Staphylococcus aureus protein A strain from the -80 °C refrigerator and inoculate it into YPD medium for cultivation at a ratio of 1%, and shake it on a shaker at 28 °C and 210 r / min for 18 h.

[0046] The YPD medium (100 mL) contains 0.8 g of yeast extract, 1.8 g of peptone, and 1.8 g of glucose.

[0047] (2) Cell amplification stage: When the OD600nm of the recombinant Staphylococcus aureus protein A bacterial solution reaches 1.2, transfer the bacterial solution to BMGY complex medium and shake it for cultivation at 28 °C and 210 r / min.

[0048] The BMGY medium formulation is that 100 mL of BMGY contains 0.8 g of yeast extract, 1.8 g of peptone, 0.5 mL of glycerol, 79 mL of deionized water, which is autoclaved (105 °C, 20 min) and then mixed with 10 mL of sterile 10×YNB, 8 mL of sodium hydrogen phosphate-citrate buffer at pH 3, 0.2 mL of 500×B. Polyphenols from Coffea arabica seeds are filtered and sterilized through a 0.22-μm filter membrane and added to the BMGY medium to a final concentration of 1 μM, and juglone is filtered and sterilized through a 0.22-μm filter membrane and added to the BMGY medium to a final concentration of 10 μM.

[0049] The 10×YNB formulation is 1.2 g of YNB solid dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4 °C. The 500×B formulation is 15 mg of biotin dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4 °C.

[0050] (3) Cell induction stage: When the OD of the recombinant Staphylococcus aureus protein A bacterial solution 600When it reaches 0.8, transfer the bacterial solution to BMMY complex medium, shake it at 18 °C and 210 r / min on a shaker for 70 h, centrifuge at 8000 rpm for 5 min to harvest the supernatant, and measure the expression level of recombinant Staphylococcus aureus protein A in the supernatant by the Bradford method.

[0051] The formula of MMY medium is that 100 mL of BMMY contains 1 g of yeast extract, 2 g of peptone, 1 mL of glycerol, 80 mL of deionized water. After autoclaving (105 °C, 20 min), mix it with 10 mL of sterile 10×YNB (same as the amplification stage), 8 mL of sodium hydrogen phosphate-citrate buffer solution with pH 4, 0.5 mL of methanol, 0.2 mL of 500×B (same as the amplification stage). Chebulinic acid is filtered and sterilized through a 0.22 μm filter membrane and then added to the BMMY medium to a final concentration of 1 μM. Andrographolide is filtered and sterilized through a 0.22 μm filter membrane and then added to the BMMY medium to a final concentration of 10 μM.

[0052] Example 3

[0053] The method for increasing the expression level of Staphylococcus aureus protein A in this example includes the following steps:

[0054] (1) Strain activation stage: Take out the frozen recombinant Staphylococcus aureus protein A strain from the -80 °C refrigerator, inoculate it in YPD medium at a ratio of 1%, and culture it at 32 °C with shaking at 230 r / min for 14 h.

[0055] The YPD medium (100 mL) contains 1.2 g of yeast extract, 2.2 g of peptone, and 2.2 g of glucose.

[0056] (2) Cell amplification stage: When the OD600nm of the recombinant Staphylococcus aureus protein A bacterial solution reaches 0.8, transfer the bacterial solution to BMGY complex medium and culture it with shaking at 32 °C and 230 r / min.

[0057] The formula of the BMGY medium is that 100 mL of BMGY contains 1.2 g of yeast extract, 2.2 g of peptone, 1.5 mL of glycerol, 79 mL of deionized water. After autoclaving (105 °C, 20 min), mix it with 10 mL of sterile 10×YNB, 12 mL of sodium hydrogen phosphate-citrate buffer solution with pH 5, 0.2 mL of 500×B. Polyphenols from Coffea arabica seeds are filtered and sterilized through a 0.22 μm filter membrane and then added to the BMGY medium to a final concentration of 30 μM. Juglone is filtered and sterilized through a 0.22 μm filter membrane and then added to the BMGY medium to a final concentration of 1 μM.

[0058] The 10×YNB formulation is 1.4 g of YNB solid dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4°C. The 500×B formulation is 25 mg of biotin dissolved in 100 mL of distilled water, filtered and sterilized, and stored at 4°C.

[0059] (3) Cell induction stage: When the OD of the recombinant Staphylococcus aureus protein A bacterial solution 600 reaches 0.6, transfer the bacterial solution to BMMY complex medium and shake it at 23°C and 230 r / min for 75 h. Centrifuge at 8000 rpm for 5 min to harvest the supernatant, and measure the expression level of recombinant Staphylococcus aureus protein A in the supernatant by the Bradford method.

[0060] The MMY medium formulation is that 100 mL of BMMY contains 1 g of yeast extract, 2 g of peptone, 1 mL of glycerol, 80 mL of deionized water. After autoclaving (105°C, 20 min), mix it with 10 mL of sterile 10×YNB (same as the amplification stage), 12 mL of sodium hydrogen phosphate-citrate buffer at pH 4, 0.5 mL of methanol, 0.2 mL of 500×B (same as the amplification stage). Chebulinic acid is filtered and sterilized through a 0.22 μm filter membrane and added to the BMMY medium to a final concentration of 50 μM. Neoandrographolide is filtered and sterilized through a 0.22 μm filter membrane and added to the BMMY medium to a final concentration of 1 μM.

[0061] Example 4

[0062] The difference between Example 4 and Example 1 is that the final concentration of juglone in the BMGY medium is 10 μM, and the rest is the same as Example 1.

[0063] Example 5

[0064] The difference between Example 5 and Example 1 is that the final concentration of polyphenols from Coffea arabica seeds in the BMGY medium is 30 μM, and the rest is the same as Example 1.

[0065] Example 6

[0066] The difference between Example 6 and Example 1 is that the final concentration of chebulinic acid in the BMMY medium is 50 μM, and the rest is the same as Example 1.

[0067] Example 7

[0068] The difference between Example 7 and Example 1 is that the final concentration of neoandrographolide in the BMMY medium is 10 μM, and the rest is the same as Example 1.

[0069] Table 1 Expression levels of recombinant Staphylococcus aureus protein A measured in each example

[0070] Expression level (g / L) Example 1 5.6 Example 2 5.5 Example 3 5.7 Example 4 6.0 Example 5 5.9 Example 6 6.3 Example 7 6.1

[0071] As shown in Table 1, the expression level of recombinant Staphylococcus aureus protein A cultured using the embodiment of the present invention can reach 5.5 - 6.3 g / L. When adding polyphenols from Coffea arabica seeds, juglone, chebulinic acid, and neoandrographolide within a limited range, the expression level of recombinant Staphylococcus aureus protein A increases slightly. The expression level in Example 6 is the highest, reaching 6.3 g / L.

[0072] Comparative Example 1

[0073] Compared with Example 1, Comparative Example 1 lacks the cell induction stage, and the rest is the same as Example 1.

[0074] Comparative Example 2

[0075] Compared with Example 1, Comparative Example 2 lacks the cell amplification stage, and the rest is the same as Example 1.

[0076] Comparative Example 3

[0077] Compared with Example 1, in Comparative Example 3, the BMGY complex medium lacks polyphenols from Coffea arabica seeds and juglone, and the BMMY complex medium lacks chebulinic acid and neoandrographolide, and the rest is the same as Example 1.

[0078] Figure 1 The figure shows the comparison of the expression levels of Staphylococcus aureus protein A in Example 1 and Comparative Examples 1 - 3. It can be seen that the expression level of the segmented culture in Example 1 (5.6 g / L) is much higher than the expression levels of the whole stages in Comparative Example 1 (2.4 g / L) and Comparative Example 2 (2.5 g / L). Compared with Comparative Example 3 (1.2 g / L), when using the segmented culture in Example 1, polyphenols from Coffea arabica seeds and juglone are added to the BMGY medium, and chebulinic acid and neoandrographolide are added to the BMMY medium. The combined effect of the four natural compounds increases the expression level of protein A by 3 times.

[0079] Comparative Example 4

[0080] Compared with Comparative Example 1, in the cell amplification stage of Comparative Example 4, the BMGY complex medium lacks polyphenols from Coffea arabica seeds, and the rest is the same as Comparative Example 1.

[0081] Comparative Example 5

[0082] Compared with Comparative Example 1, in the cell amplification stage of Comparative Example 5, the BMGY complex medium lacks juglone, and the rest is the same as Comparative Example 1.

[0083] Comparative Example 6

[0084] Compared with Comparative Example 1, in the cell amplification stage of Comparative Example 6, the BMGY complex medium lacks polyphenols from Coffea arabica seeds and juglone, and the rest is the same as Comparative Example 1.

[0085] Figure 2The protein A expression of Staphylococcus aureus in Comparative Examples 1 and 4-6, the protein A expression of Comparative Example 4 is 1.7 g / L, the protein A expression of Comparative Example 5 is 1.8 g / L, and the protein A expression of Comparative Example 6 is 1.3 g / L. It can be seen that the BMGY composite medium adds small fruit coffee seed polyphenols and / or juglone, and the protein A expression is greatly improved. This is because: during the amplification period of culture, the small fruit coffee seed polyphenols and juglone added to the culture medium can regulate the expression of cell cycle-related proteins, accelerate cell division and proliferation, and significantly improve the cell growth rate and final cell density in the nutrient-rich BMGY medium, laying a solid cell foundation for the subsequent protein A expression.

[0086] Comparative Example 7

[0087] Compared with Comparative Example 2, in Comparative Example 7, the BMMY composite culture medium lacked terminalia chebula tannin during the cell induction stage, and the rest was the same as Comparative Example 2.

[0088] Comparative Example 8

[0089] Compared with Comparative Example 2, in Comparative Example 8, the BMMY composite culture medium lacked neoandrographolide during the cell induction stage, and the rest was the same as Comparative Example 2.

[0090] Comparative Example 9

[0091] Compared with Comparative Example 2, in Comparative Example 9, the BMMY composite culture medium lacked terminalia chebula tannin and neoandrographolide during the cell induction stage, and the rest was the same as Comparative Example 2.

[0092] Figure 3 The expression of protein A of Staphylococcus aureus in Comparative Example 2 and Comparative Examples 7-9 is 2.5 g / L in protein A of Comparative Example 2, 1.6 g / L in protein A of Comparative Example 7, 1.9 g / L in protein A of Comparative Example 8, and 1.3 g / L in protein A of Comparative Example 9. It can be seen that the expression of protein A in Comparative Example 2 is significantly better than that in Comparative Example 7-9, indicating that the addition of terminalia tannin or / and neoandrographolide to the BMMY composite culture medium can greatly increase the expression of protein A. This is because: in the induction stage of Staphylococcus aureus protein A, terminalia tannin and neoandrographolide added to the culture medium can enhance the induction effect of methanol, upregulate the expression level of related genes, and thus promote the synthesis of protein A. At the same time, it helps to improve the protein folding environment in cells, interact with molecular chaperones in cells, enhance their functions, and improve the folding efficiency and quality of protein A. In addition, terminalia chebula tannins and neoandrographolide, with their anti-inflammatory and antioxidant properties, can reduce the stress response of cells, protect cells from stress damage, maintain the activity and stability of cells during the induced expression process, and ensure the continuous and efficient expression of protein A.

[0093] Comparative Example 10

[0094] Compared with Comparative Example 1, in the cell expansion stage, the dosage of polyphenols from Coffea arabica seeds in the BMGY complex medium exceeded the specified range, specifically 50 μM, and the rest was the same as Comparative Example 1.

[0095] Comparative Example 11

[0096] Compared with Comparative Example 1, in the cell expansion stage, the dosage of juglone in the BMGY complex medium exceeded the specified range, specifically 30 μM, and the rest was the same as Comparative Example 1.

[0097] Comparative Example 12

[0098] Compared with Comparative Example 2, in the cell induction stage, the dosage of chebulinic acid in the BMMY complex medium exceeded the specified range, specifically 70 μM, and the rest was the same as Comparative Example 2.

[0099] Comparative Example 13

[0100] Compared with Comparative Example 2, in the cell induction stage, the dosage of neoandrographolide in the BMMY complex medium exceeded the specified range, specifically 30 μM, and the rest was the same as Comparative Example 2.

[0101] Figure 4 As can be seen from the above, when the dosages of polyphenols from Coffea arabica seeds, juglone, chebulinic acid, and neoandrographolide exceeded the specified range, the expression level of recombinant Staphylococcus aureus protein A was instead reduced. This is because: excessive polyphenols from Coffea arabica seeds over-activated cell cycle regulatory proteins (such as the CDK / Cyclin complex), resulting in an abnormally accelerated cell division rate and premature consumption of intracellular ATP reserves; and high-concentration polyphenols would have a strong hydrophobic interaction with the phospholipid bilayer of the cell membrane, reducing membrane fluidity. Excessive juglone competitively bound to mitochondrial complex III (ubiquinone-cytochrome c reductase), reducing the efficiency of electron transfer in the respiratory chain; when the concentration of quinone substances exceeded the standard, excessive hydroxyl radicals were generated through the Fenton reaction, leading to an increase in the content of the cell oxidative stress marker MDA. Excessive tannin concentration irreversibly bound to the β subunit of the 26S proteasome, reducing the efficiency of misfolded protein clearance and triggering endoplasmic reticulum stress (activation of the IRE1α pathway); excessive tannin also led to the accumulation of formic acid, a metabolite of methanol, inhibiting the activity of the AOX1 promoter. High-concentration lactone competitively inhibited the ATP-binding domain of Hsp70, reducing the protein folding efficiency; exceeding the standard concentration of lactone led to abnormal distribution of sterol components in the cell membrane, blocking the transmembrane transport of methanol. Therefore, each compound has a promoting effect on protein A expression within a specific concentration range, but exceeding this range will trigger complex physiological negative effects, ultimately resulting in a significant decrease in protein expression.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for increasing the expression of recombinant Staphylococcus aureus protein A, characterized in that: The following steps are involved: The recombinant Staphylococcus aureus protein A strain was inoculated into YPD medium for activation, and then the activated strain was inoculated into BMGY composite medium containing small-fruit coffee seed polyphenols and juglone, and BMMY composite medium containing terminalia chebula tannin and neoandrographolide for amplification and induction culture. Finally, the cells were harvested and the expression level of recombinant Staphylococcus aureus protein A was determined.

2. A method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: The components of the BMGY composite culture medium are: 8-12 g / L yeast extract, 18-22 g / L peptone, 0.5-1.5 v / v% glycerol, 1.2-1.4 g / L YBM, 8-12 v / v% disodium hydrogen phosphate-citric acid buffer, 0.6-1.0 μ / L biotin, 1-30 μM small fruit coffee seed polyphenols, and 1-10 μM juglone. Finally, the volume is fixed to 1 L with deionized water.

3. A method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: The components of the BMMY composite culture medium are: yeast extract 8-12 g / L, peptone 18-22 g / L, glycerol 0.5-1.5 v / v%, YBM 1.2-1.4 g / L, disodium hydrogen phosphate-citric acid buffer 8-12 v / v%, methanol v / v 5%, 0.8 μ / L biotin, terminalia chebula tannin 1-50 μM, and neoandrographolide 1-10 μM. Finally, the volume is adjusted to 1 L with deionized water.

4. A method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: The YPD culture medium contains 8-12 g / L of yeast extract, 18-22 g / L of peptone, and 18-22 g / L of glucose.

5. The method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: When the OD of recombinant Staphylococcus aureus protein A solution was 600 When the concentration reaches 0.6-1.2, the bacterial solution is transferred to BMGY compound medium for expansion culture.

6. A method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: When the OD of recombinant Staphylococcus aureus protein A solution was 600 When the concentration reaches 0.6-0.8, the bacterial solution is transferred to BMMY compound medium for induction culture.

7. A method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: During activation culture, the inoculation amount of the strain is 1%, the culture conditions are 28-32°C, and shaking at 210-230 r / min for 14-18 hours.

8. The method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: During the amplification culture, the inoculation amount of the strain is 1%, the culture conditions are 28-32° C., and the shaking culture is carried out at 210-230 r / min.

9. The method for increasing the expression of recombinant Staphylococcus aureus protein A according to claim 1, characterized in that: During activation culture, the inoculation amount of the strain is 1%, the culture conditions are 18-23°C, and shaking at 210-230 r / min for 70-75 hours.

10. A method for increasing the expression level of recombinant Staphylococcus aureus protein A according to claim 2 or 3, characterized in that: The pH value of the disodium hydrogen phosphate-citric acid buffer is 3-5.

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