Fermentation medium and fermentation process for preparing recombinant protein through high-density fermentation
By using a specific fermentation medium and process conditions, the problem of low expression levels in β2 microglobulin fermentation was solved, achieving high-density fermentation and high expression levels, which is suitable for the efficient preparation of recombinant proteins.
Patent Information
- Application Number
- CN202511553611.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-01-09
AI Technical Summary
Existing fermentation methods for preparing β2 microglobulin suffer from problems such as low expression levels and low fermentation density, which limit its large-scale production and application.
A fermentation medium and fermentation process containing specific components, including glycerol, potassium dihydrogen phosphate, diammonium hydrogen phosphate, citric acid, magnesium ions, trace element solution, biotin, and antifoaming agent, were used for high-density fermentation of recombinant engineered bacteria. By adjusting fermentation conditions such as pH, aeration rate, stirring speed, and dissolved oxygen maintenance, and in combination with the use of inducers, high expression of recombinant proteins was achieved.
It significantly improved the cell fermentation density and target protein expression level of recombinant engineered bacteria, with an OD600 fermentation density of over 200 and a target protein expression level of over 13 g/L, solving the problem of low expression level in existing technologies.
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Figure CN121294584A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of molecular biology and microbial engineering technology, and particularly relates to a fermentation medium and a fermentation process for preparing recombinant proteins by high-density fermentation. BACKGROUND
[0002] With the rapid development of biotechnology, using microorganisms (such as Escherichia coli, yeast, etc.) as cell factories, and producing important value proteins (such as vaccine antigens, antibodies, enzyme preparations, therapeutic proteins, etc.) by high-density fermentation technology has become the core of modern biological pharmaceuticals and industrial biotechnology. High-density fermentation aims to obtain extremely high cell density, thereby greatly improving the unit volume yield of target proteins, reducing the cost of downstream separation and purification, and having extremely high economic value.
[0003] Beta 2-microglobulin (β2M) is a low molecular weight protein that widely exists in human body fluids, especially in kidney diseases and certain tumors. The detection of β2-microglobulin has important significance for the diagnosis and monitoring of chronic kidney disease, multiple myeloma and other diseases. Currently, the preparation of β2-microglobulin mainly relies on chemical synthesis and biological fermentation method, among which the biological fermentation method gradually becomes the mainstream preparation process due to its low cost and high yield.
[0004] However, the existing β2-microglobulin fermentation preparation method has the problems of low expression amount and low fermentation density, which limits its large-scale production and application. Therefore, developing a high-density fermentation medium and fermentation process to prepare β2-microglobulin to realize high-density fermentation and high expression amount of β2-microglobulin has important industrial application prospects. SUMMARY
[0005] The present application aims to provide a fermentation medium for high-density fermentation of recombinant engineering bacteria to prepare recombinant proteins, which comprises or consists of the following components: glycerol 10-40 g / L, potassium dihydrogen phosphate 10-15 g / L, diammonium hydrogen phosphate 2-10 g / L, citric acid monohydrate 0.5-3 g / L, magnesium ions 2.03-12.2 mmol / L, L-serine 0.1-0.6 g / L, leucine 0.1-0.5 g / L, vitamin B1 0.001-0.01 g / L, trace element solution 1-10 mL / L, biotin 0.02-0.06 mg / L, kanamycin sulfate 10-100 mg / L, and defoaming agent 0.1 ml / L.
[0006] The trace element solution comprises or consists of the following components: iron ions 40.8-81.7 mmol / L, boric acid 0.1-1 g / L, manganese ions 5.05-15.16 mmol / L, cobalt ions 0.420-4.20 mmol / L, sodium ions 0.413-4.13 mmol / L, copper ions 0.587-5.87 mmol / L, zinc ions 2.28-22.8 mmol / L, calcium ions 0.901-9.01 mmol / L, and EDTA 0.1-1 g / L.
[0007] Further, the antifoam agent comprises antifoam 204 or Defoamer.
[0008] Further, the magnesium ion source is magnesium sulfate heptahydrate, the calcium ion source is anhydrous calcium chloride, the iron ion source is ferric citrate, the manganese ion source is manganese chloride tetrahydrate, the cobalt ion source is cobalt chloride hexahydrate, the sodium ion source is sodium molybdate dihydrate, the copper ion source is copper chloride dihydrate, and the zinc ion source is zinc acetate dihydrate.
[0009] The fermentation medium of the present application can be used for high-density fermentation preparation of various recombinant proteins, for example, the recombinant protein is β2 microglobulin.
[0010] The present application also provides a method for high-density fermentation preparation of recombinant proteins, which comprises the following steps:
[0011] (1) inoculating activated bacteria solution of recombinant engineering bacteria expressing recombinant proteins into the fermentation medium as described herein;
[0012] (2) fermenting and culturing the recombinant engineering bacteria under culture conditions suitable for the growth of the recombinant engineering bacteria until the OD 600nm value is 90-120;
[0013] (3) adding an inducer to induce expression of the recombinant proteins;
[0014] (4) continuing fermentation and culture until the growth of the recombinant engineering bacteria enters a plateau phase, ending the fermentation, and obtaining the recombinant engineering bacteria expressing the recombinant proteins.
[0015] Further, the recombinant engineering bacteria expressing the recombinant proteins are recombinant engineering bacteria transformed by an inducible expression vector encoding the recombinant proteins.
[0016] Further, the recombinant engineering bacteria are recombinant Escherichia coli.
[0017] The method of the present application can be used for high-density fermentation preparation of various recombinant proteins, for example, the recombinant protein is β2 microglobulin.
[0018] Further, the protein sequence of the beta 2 microglobulin is shown as SEQ ID NO. 1.
[0019] Further, the nucleic acid sequence encoding the beta 2 microglobulin is shown as SEQ ID NO. 2.
[0020] Further, the nucleic acid sequence encoding the beta 2 microglobulin is a codon-optimized nucleic acid sequence suitable for expression in the recombinant engineering bacteria.
[0021] Further, the inducible expression vector is a pET-28a(+) vector.
[0022] Further, the OD 600nm value of the activated bacteria solution is ≥3.0.
[0023] Further, the preparation method of the activated bacteria solution is well known to those skilled in the art. As an example, the activated bacteria solution can be prepared as follows: primary bacteria activation: inoculate the recombinant engineering bacteria expressing beta 2 microglobulin into LB medium, incubate at a constant temperature with shaking, incubation temperature 36.0-37.0℃, rotation speed 200-300 rpm, incubate for 12-16 h, until the OD 600nm value of the bacteria reaches ≥3.0, to obtain the primary bacteria culture solution; secondary bacteria activation: inoculate the well-cultured primary bacteria culture solution into LB medium, inoculation ratio 1:50-200, incubate at a constant temperature with shaking, incubation temperature 36.0-37.0℃, rotation speed 200-300 rpm, incubate for 12-16 h, until the OD 600nm value of the bacteria reaches ≥3.0, to obtain the secondary bacteria culture solution, which is the activated bacteria solution.
[0024] Further, the LB medium comprises or consists of the following components: 10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L NaCl.
[0025] Further, the inoculation ratio of the activated bacteria solution in the fermentation medium is 1:20 (v / v).
[0026] Further, the inoculation of the activated bacteria solution is carried out under the following conditions: the pH of the fermentation medium is 6.5-7.0, the aeration amount is 1-3 VVM, the dissolved oxygen is 100%, and the stirring speed is 200-1500 rpm.
[0027] Further, the culture conditions suitable for the growth of the recombinant engineering bacteria include: temperature 30-37℃, pH 6.5-7.0, initial aeration amount 0.5-3 VVM, dissolved oxygen 20-45%, initial tank pressure 0.045-0.055 MPa, and stirring speed 200-1500 rpm.
[0028] Further, in the fermentation process, the dissolved oxygen is maintained at 20-45% by adjusting the stirring speed or supplying oxygen. For example, the stirring speed can be first adjusted in the range of 200-1500 rpm to maintain the dissolved oxygen, and when the stirring speed reaches 1000 rpm, the dissolved oxygen is maintained by supplying oxygen.
[0029] Further, in the fermentation process of step (2), the addition of the feed medium is adjusted by monitoring the dissolved oxygen, wherein when the dissolved oxygen rapidly rises (at this time, it usually indicates that the nutrients are depleted, the cell metabolism is blocked, resulting in a decrease in oxygen demand, and the DO in the solution rises), the feed medium is added.
[0030] Further, the feed medium comprises or consists of: glycerol 600-850 g / L, magnesium ions 2.03-12.2 mmol / L, vitamin B1 0.001-0.01 g / L, proteose peptone 40-80 g / L, trace element solution 5-10 ml / L, and kanamycin sulfate 10-100 mg / L.
[0031] The person skilled in the art knows the method of maintaining normal DO by adding the feed medium and can determine the amount of feed medium added according to the actual situation. Generally speaking, when the DO rises significantly, the fixed feed can be supplemented with 10-30 g / L of glycerol. After the feed is supplemented, the DO is maintained normally, and when the DO rises rapidly next time, the feed is added again.
[0032] Further, in the fermentation process, ammonia water (e.g., 28% ammonia water) and phosphoric acid are used to adjust the pH.
[0033] Further, the inducer is IPTG or lactose.
[0034] Further, the concentration of IPTG added is 0.1-1 mM.
[0035] Further, the OD 600nm of the fermentation broth at the end of fermentation is above 200, even as high as above 240.
[0036] Further, the method further comprises the steps of collecting and lysing the recombinant engineering bacteria cells expressing the recombinant protein, and purifying the recombinant protein from the supernatant.
[0037] Advantages of the present application
[0038] The fermentation medium and the fermentation method can significantly improve the fermentation density of the recombinant engineering bacteria, the OD600 fermentation density can reach more than 200, even up to more than 240, and can significantly improve the fermentation expression of the bacteria, and the expression amount of the target protein can reach more than 13 g / L. BRIEF DESCRIPTION OF DRAWINGS
[0039] Figure 1 The change curve of the fermentation density of the bacteria and the expression amount of the target protein in the fermentation process of the application is shown. DETAILED DESCRIPTION
[0040] The application will be further described below in combination with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field, wherein
[0041] The fermentation medium: glycerol 30 g / L, potassium dihydrogen phosphate 13.3 g / L, diammonium hydrogen phosphate 4 g / L, citric acid monohydrate 1.7 g / L, magnesium sulfate heptahydrate 1.2 g / L, L-serine 0.5 g / L, leucine 0.2 g / L, vitamin B1 0.005 g / L, trace element solution 1 mL / L, biotin 0.03 mg / L, kanamycin sulfate 50 mg / L and antifoam 204 0.1 ml / L;
[0042] The composition of the trace element solution includes: ferric citrate 10 g / L, boric acid 0.3 g / L, manganese chloride tetrahydrate 1.5 g / L, cobalt chloride hexahydrate 0.25 g / L, sodium molybdate dihydrate 0.25 g / L, copper chloride dihydrate 0.15 g / L, zinc acetate dihydrate 1.3 g / L, boric acid 0.3 g / L, calcium chloride 0.4 g / L and EDTA 0.84 g / L.
[0043] The feed medium: glycerol 750 g / L, magnesium sulfate heptahydrate 20 g / L, vitamin B1 0.05 g / L, protein peptone 70 g / L, trace elements 5 ml / L and kanamycin sulfate 100 mg / L.
[0044] The LB medium: protein peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.
[0045] The above medium (except for trace elements, magnesium sulfate, vitamin B1 and various amino acids, which need to be filtered and sterilized) and the remaining components are subjected to high-pressure sterilization.
[0046] Example: High-density culture of beta 2 microglobulin
[0047] 1. Construction of recombinant E. coli
[0048] The protein sequence of the gene fragment of β2 microglobulin is shown in SEQ ID NO. 1, and the nucleic acid sequence after codon optimization of E. coli is shown in SEQ ID NO. 2;
[0049] The optimized nucleic acid fragment is inserted between the Ndel and Xhol enzyme cutting sites of the prokaryotic expression plasmid pET-28a(+), and a recombinant expression plasmid is constructed, which is completed by General Biotech Co., Ltd.;
[0050] The recombinant expression plasmid is transformed into E. coli expression host BL21 (DE3) by heat shock method (ice bath for 30 min, 90°C heat shock for 40 s, ice bath for 3 min), and positive clones are screened by LB (peptone 10 g, yeast powder 5 g, NaCl 10 g, 1 L) plate containing 100 mg / ml kanamycin antibiotic, then the single colony is cultured in LB medium, and then sent to the company for sequencing verification. The correct one is the recombinant E. coli expressing β2 microglobulin. Then use 50% glycerol to preserve bacteria.
[0051] 2. Strain activation
[0052] Primary strain culture: Take 10 μl of frozen recombinant E. coli bacteria and inoculate into 5 ml of LB medium, and incubate at 37.0°C with constant shaking at 220 rpm for 15 h, until the OD600nm value of the strain reaches ≥3.0, to obtain the primary strain culture;
[0053] Secondary strain culture: The well-cultured primary strain culture is inoculated into 300 ml of LB medium at a ratio of 1:100, and the seed tank is cultured at 37.0°C with constant shaking at 220 rpm for 15 h, until the OD600nm value of the strain reaches ≥3.0, to obtain the secondary activated strain culture.
[0054] 3. Fermentation culture
[0055] Activated strain inoculation: After adjusting the pH of the fermentation medium to 6.8 with NaOH, calibrating the pH electrode, sterilizing, and aerating at 1 VVM and 200 rpm for 1-2 h, the DO is calibrated to 100%, and then the secondary activated strain culture is aseptically inoculated into the fermentation medium at a ratio of 1:20, and the fermentation tank is cultured;
[0056] Initial culture conditions: temperature 37.0℃, pH 6.80±0.20, initial rotation speed 200 rpm, initial aeration amount 1VVM, initial tank pressure 0.050±0.005 MPa; DO is controlled at 30%, initially maintain DO by increasing rotation speed, when rotation speed reaches 1000 rpm, start aeration to maintain DO at about 30%.
[0057] Fermentation process control: during the fermentation culture process, the feed is adjusted by DO feedback, when the DO rises rapidly, the feed medium is added, when the OD600nm value is about 90 / 120, IPTG (0.5mM) is added for induction culture, and the fermentation is stopped when the induction reaches the plateau. The pH is adjusted by 28% ammonia and phosphoric acid during the process.
[0058] The cell density and target protein expression amount in the fermentation process were monitored, and OD600 was detected by Nanodrop every 1-2h, briefly, 1ml of bacteria was taken, centrifuged at 8000rpm for 3min, the supernatant was discarded, the bacteria were washed twice with 500ul of PBS, then the bacteria were resuspended with 500ul of PBS, ultrasonic crushing was carried out on ice bath, ultrasonic crushing for 3s and stop for 5s, cumulative ultrasonic crushing for 10-30min, 12000rpm 4℃ centrifugation for 10min, the supernatant was taken for ELISA detection of protein content. The results are shown in Figure 1 It can be seen that the fermentation method provided by the present application can significantly improve the cell fermentation density of the recombinant engineering bacteria, wherein the OD600 fermentation density can reach more than 200, and can significantly improve the cell fermentation expression amount, wherein the target protein expression amount is more than 13 g / L.
[0059] 4. Protein acquisition and purification:
[0060] 1) Collect bacteria: centrifuge the E. coli culture solution. Culture conditions: 37℃; culture time 20h; culture volume 2L; centrifugation conditions: 4℃, 8000rpm, 8min. Bacterial weight collected: 11.94g
[0061] 2) After completion, the slurry is dissolved with 20Mm PB-0.1M Nacl solution, and the dissolution volume is 100ml. After dissolution, homogenization is carried out, the homogenization pressure is 800Bar, the time is 5min, and the homogenization temperature is 6℃.
[0062] 3) Centrifuge the homogenate (homogenate volume is 100ml) at 12000rpm, 4℃, for 30min. After centrifugation, the precipitate is taken.
[0063] 4) First wash of the bacteria: Resuspend the bacteria pellet (100ml volume) in 20mM PB-0.1M Nacl-1% triton X-100 solution and continue stirring for 5 minutes. Homogenize the bacteria at 300 bar, 4°C for 5 minutes to release the inclusion bodies. Centrifuge the homogenate at 10000 rpm for 10 minutes at 4°C. Resuspend the pellet in 20mM PB-0.1M Nacl-1% triton X-100 solution (100ml volume) and continue stirring for 5 minutes. Homogenize the bacteria at 300 bar, 4°C for 5 minutes to release the inclusion bodies. Centrifuge the homogenate at 10000 rpm for 10 minutes at 4°C. The pellet obtained contains the inclusion bodies. Solution pH: 7.45 Conductivity: 11.97ms / cm;
[0064] 5) Second wash of the bacteria: Resuspend the pellet obtained in step (4) in 20mM PB-0.1M Nacl-0.5% sodium deoxycholate solution (100ml volume) and continue stirring for 5 minutes. Centrifuge the solution at 12000 rpm for 10 minutes at 4°C. Resuspend the pellet in 20mM PB-0.1M Nacl-0.5% sodium deoxycholate solution (100ml volume) and continue stirring for 5 minutes. Centrifuge the solution at 12000 rpm for 16 minutes at 4°C. The pellet obtained contains the inclusion bodies. Solution pH: 7.51 Conductivity: 12.27ms / cm. The second centrifugation did not work properly;
[0065] 6) First denaturation: Resuspend the pellet obtained in step (5) in 20mM PB-0.1M Nacl-2M urea solution (100ml volume) and continue stirring for 5 minutes. Centrifuge the solution at 10000 rpm for 5 minutes at 4°C. Solution pH: 7.51 Conductivity: 10.37ms / cm;
[0066] 7) Denaturation: Resuspend the pellet in 20mM PB-0.1M Nacl-8M urea solution (100ml volume) and let it stand for 5 minutes. Centrifuge the solution at 10000 rpm for 5 minutes at 4°C. Take the supernatant and use it as the sample for loading. The concentration of the target protein in the supernatant is 1.1603mg / ml;
[0067] 8) Purification: Filter the sample to be purified using a 0.45um needle filter. Purify the protein using a Ni column according to the protocol in Table 1 below to obtain the purified beta 2 microglobulin.
[0068] Table 1: Ni column protein purification protocol
[0069] Step Solution Equilibrium A1 : 20 Mm PB - 0.1 M Nacl - 8 M Urea solution Load A1 : Wash denatured protein solution Elution A1 : 20 Mm PB - 0.1 M Nacl - 8 M Urea solution Elution A1 : 20 Mm PB - 0.1 M Nacl - 8 M Urea solution B1 : 20 Mm PB - 0.1 M Nacl - 8 M Urea - 0.5 M Imidazole solution (0-100%)
[0070] It should be noted that the preferred embodiments of the present application are described in the specification and the drawings of this patent application and are illustrative of the various ways in which the principles of the application can be employed. Numerous modifications will be apparent to those skilled in the art in view of the foregoing description, and are intended to fall within the scope of the application. Accordingly, the particular embodiments discussed above are illustrative only and are not meant to limit the scope of the present application in any manner, and various implementations thereof can include substitutions, eliminations, additions and / or modifications of the features illustrated, described or otherwise implied above.
Claims
1. A fermentation medium for high-density fermentation of recombinant engineered bacteria to prepare recombinant proteins, characterized in that, The fermentation medium comprises: 10-40 g / L glycerol, 10-15 g / L potassium dihydrogen phosphate, 2-10 g / L diammonium hydrogen phosphate, 0.5-3 g / L citric acid monohydrate, 2.03-12.2 mmol / L magnesium ions, 0.1-0.6 g / L L-serine, 0.1-0.5 g / L leucine, 0.001-0.01 g / L vitamin B1, 1-10 mL / L trace element solution, 0.02-0.06 mg / L biotin, 10-100 mg / L kanamycin sulfate, and 0.1 mL / L antifoaming agent. The trace element solution contains: iron ions 40.8-81.7 mmol / L, boric acid 0.1-1 g / L, manganese ions 5.05-15.16 mmol / L, cobalt ions 0.420-4.20 mmol / L, sodium ions 0.413-4.13 mmol / L, copper ions 0.587-5.87 mmol / L, zinc ions 2.28-22.8 mmol / L, calcium ions 0.901-9.01 mmol / L, and EDTA 0.1-1 g / L.
2. The fermentation medium according to claim 1, characterized in that, The recombinant protein is β2 microglobulin.
3. The fermentation medium according to claim 1, characterized in that, The defoamer is either antifoam 204 or defoamer; Furthermore, the magnesium ion source is magnesium sulfate heptahydrate, the calcium ion source is anhydrous calcium chloride, the iron ion source is ferric citrate, the manganese ion source is manganese chloride tetrahydrate, the cobalt ion source is cobalt chloride hexahydrate, the sodium ion source is sodium molybdate dihydrate, the copper ion source is copper chloride dihydrate, and the zinc ion source is zinc acetate dihydrate.
4. A method for preparing recombinant proteins through high-density fermentation, characterized in that, Includes the following steps: (1) Inoculate the activated bacterial culture of the recombinant engineered bacteria expressing the recombinant protein into the fermentation medium according to any one of claims 1-3; (2) Ferment and culture the recombinant engineered bacteria under suitable culture conditions until OD 600nm The value is 90-120; (3) Add an inducer to induce the expression of the recombinant protein; (4) Continue fermentation culture until the growth of recombinant engineered bacteria enters the plateau phase, then end fermentation to obtain recombinant engineered bacteria cells expressing recombinant proteins.
5. The method according to claim 4, characterized in that, The recombinant engineered bacteria expressing the recombinant protein are recombinant engineered bacteria transformed by an inducible expression vector encoding the recombinant protein; Furthermore, the recombinant engineered bacteria is recombinant Escherichia coli; Furthermore, the recombinant protein is β2 microglobulin; Furthermore, the protein sequence of the β2 microglobulin is shown in SEQ ID NO.1; Furthermore, the nucleic acid sequence encoding the β2 microglobulin is shown in SEQ ID NO.2; Furthermore, the inducible expression vector is the pET-28a(+) vector.
6. The method according to claim 4, characterized in that, The OD of the activated bacterial solution 600nm Value ≥ 3.0; Furthermore, the inoculation ratio of the activated bacterial solution in the fermentation medium is 1:20; Furthermore, the inoculation of the activated bacterial solution is carried out under the following conditions: the pH of the fermentation medium is 6.5-7.0, the aeration rate is 1-3 VVM, the dissolved oxygen is 100%, and the stirring speed is 200-1500 rpm.
7. The method according to claim 4, characterized in that, The culture conditions suitable for the growth of the recombinant engineered bacteria include: temperature of 30-37℃, pH of 6.5-7.0, initial aeration rate of 0.5-3 VVM, dissolved oxygen of 20-45%, initial tank pressure of 0.045-0.055 MPa, and stirring speed of 200-1500 rpm. Furthermore, during the fermentation process, dissolved oxygen is maintained at 20-45% by adjusting the stirring speed or by introducing oxygen. Furthermore, during the fermentation process in step (2), the addition of feed medium is adjusted by monitoring dissolved oxygen, wherein feed medium is added when dissolved oxygen rises rapidly; Furthermore, the supplemental culture medium comprises: 600-850 g / L glycerol, 2.03-12.2 mmol / L magnesium ions, 0.001-0.01 g / L vitamin B1, 40-80 g / L peptone, 5-10 ml / L trace element solution, and 10-100 mg / L kanamycin sulfate; Furthermore, during the fermentation process, ammonia and phosphoric acid are used to adjust the pH.
8. The method according to claim 4, characterized in that, The inducer is IPTG or lactose; Furthermore, the concentration of IPTG added was 0.1-1 mM.
9. The method according to claim 4, characterized in that, OD of fermentation broth at the end of fermentation 600nm The value reaches 200 or above.
10. The method according to claim 4, characterized in that, The method also includes the steps of collecting and lysing recombinant engineered bacterial cells expressing recombinant proteins and purifying the recombinant proteins from the supernatant.