Culture medium and method for producing oct4 protein by fermentation using the same

Abstract

The application discloses a culture medium and a method for producing Oct4 protein by fermentation using the culture medium. The culture medium comprises a fermentation culture medium and a feeding culture medium, and the method uses DO-stat for intermittent automatic feeding. The culture medium is beneficial to the fermentation growth of the bacteria, and the OD value can still increase after the bacteria are cultured for 16 hours, and the OD value can reach above 200 in the subsequent fermentation process. The method can effectively control the concentration of the carbon source in the fermentation liquor, thereby reducing the influence of the accumulation of acetic acid in the fermentation process on the growth of the bacteria and the expression of the target protein, and significantly improving the purity of the Oct4 protein.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial fermentation technology, and to a culture medium and a method for producing Oct4 protein by fermentation using the culture medium and recombinant Escherichia coli. Background Technology

[0002] Octamer-binding protein 4 (Oct4, also known as POU5F1) is an important transcription factor for maintaining stem cell pluripotency and self-renewal. It is mainly expressed in embryonic stem cells, germ cells, and undifferentiated embryonic tumors. By binding to the regulatory regions of target genes, it selectively inhibits the expression of differentiation genes or promotes the expression of pluripotency genes. Oct4 is encoded by the Pou5F1 gene (Gene ID: 5460), which has several different isoforms due to differential splicing: Oct4A (360 amino acid residues, molecular weight 39 kDa), Oct4B (190 amino acid residues, molecular weight 19 kDa), and Oct4B1 (265 amino acid residues, molecular weight 30 kDa). Only Oct4A can enter the nucleus and bind to the promoters of Oct4-regulated genes, regulating the expression of a series of genes that maintain stem cell pluripotency and self-renewal.

[0003] Oct4 is one of the three major transcription factors that regulate pluripotent embryonic stem cells (ESCs). It can induce in situ epigenetic reprogramming of cancer cells within tumor tissues into transiently induced pluripotent stem cells (tiPSCs), thereby restoring their differentiation potential. Utilizing the synergistic effect of Oct4 protein and two other proteins to transform cancer cells into normal tissue cells is a novel mechanism for cancer treatment, which is less harmful to the human body compared to traditional treatments such as chemotherapy, radiotherapy, and immunotherapy.

[0004] However, the current prokaryotic expression system produces low yields of Oct4 protein, making industrial-scale production impossible. Summary of the Invention

[0005] To address the problem of low yield of Oct4 protein in industrial prokaryotic production in existing technologies, this invention provides a culture medium and a method for producing Oct4 protein by fermentation using the culture medium and recombinant Escherichia coli.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a fermentation culture medium comprising yeast powder, peptone, corn steep liquor powder, soybean protein powder, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, magnesium sulfate, calcium chloride, ammonium sulfate, sodium chloride, an antifoaming agent, and a trace element stock solution; wherein the yeast powder is 6 g / L, the peptone is 12 g / L, the corn steep liquor powder is 2 g / L, the soybean protein powder is 2 g / L, the dipotassium hydrogen phosphate is 8.2 g / L, the potassium dihydrogen phosphate is 1.15 g / L, the sodium bicarbonate is 0.5 g / L, the magnesium sulfate is 0.196 g / L, the calcium chloride is 0.5 g / L, the ammonium sulfate is 3 g / L, the sodium chloride is 5 g / L, the antifoaming agent is 0.1 ml / L, and the trace element stock solution is 0.4 ml / L.

[0008] It contains one or more of glucose, glycerol, lactose, and sucrose; the concentration is 32 g / L; and the pH is 6.5-7.0.

[0009] In this invention, the fermentation medium includes one or more of the following conditions:

[0010] The peptone includes one or more of the preferred selections: soybean peptone, tryptone, and yeast peptone.

[0011] The defoamer is FoamdOCTor F2875 Pennwhite.

[0012] In this invention, the trace element mother liquor in the fermentation culture medium includes one or more of FeSO4·7H2O, CoCl2·6H2O, H3BO3, NaMoO4·2H2O, ZnSO4·7H2O, CuCl2·2H2O, MnSO4·H2O, CaCl2, and MgSO4.

[0013] In this invention, the trace element mother liquor is preferably FeSO4·7H2O, CoCl2·6H2O, H3BO3, NaMoO4·2H2O, ZnSO4·7H2O, CuCl2·2H2O, MnSO4·H2O, CaCl2, and MgSO4.

[0014] The present invention also provides a fed culture medium comprising yeast extract, peptone, corn steep liquor powder, soybean protein powder, (NH4)2SO4, and MgSO4;

[0015] And, one or more of glucose, glycerol, lactose, and sucrose; concentration of 20 g / L; pH is natural pH.

[0016] In this invention, the peptone in the supplemental culture medium is preferably one or more of soybean peptone, tryptone, and yeast peptone.

[0017] This invention also provides a method for fermenting and producing Oct4 protein, wherein recombinant *E. coli* is cultured using the aforementioned fermentation medium, and fed the medium according to the dissolved oxygen (DO) value during the culture process; the recombinant *E. coli* expresses the Oct4 protein with the amino acid sequence shown in SEQ ID NO:2, wherein...

[0018] Before Oct4 protein induction: feed culture medium was added when the DO value was controlled at 25-35%; after Oct4 protein induction: feed culture medium was added when the DO value was controlled at 10-20%.

[0019] In this invention, the fermentation conditions are selected from one or more of the following:

[0020] Aeration rate: 0.5-2 vvm; stirring speed: 50-800 rpm;

[0021] The fermentation temperature was 37℃;

[0022] Preferably, before Oct4 protein induction: the fed medium is added at an average flow rate of 0.6 ml / L / min while controlling the DO value to 25-35%; and / or, when the OD value in the fermentation broth is... 600 When the pH value is 30-60, preferably 45-55, an inducing agent is added; the inducing agent is, for example, IPTG 0.8-1.2 mmol / L; and / or, the pH of the fermentation culture is 7.0±0.2-7.5±0.2, preferably 7.0±0.2;

[0023] After Oct4 protein induction: when the DO value is 10-20%, feed medium is added at an average flow rate of 0.3 ml / L / min; and / or, the fermentation time is 3-8 hours, preferably 5-7 hours; and / or, the pH of the fermentation is 7.0±0.1-7.5±0.1, preferably 7.0±0.1.

[0024] In this invention, the feed culture medium has an optimized carbon-to-nitrogen ratio, with an optimized C / N ratio of 10.37. This ratio is determined by dividing the amount of carbon in the carbon source of the feed culture medium by the amount of nitrogen in the amino nitrogen of the nitrogen source (yeast extract, peptone, ammonium sulfate, etc.).

[0025] In this invention, the host bacterium of the recombinant *E. coli* is BL21, and the recombinant expression vector is pET-30a(+); and / or,

[0026] The method for controlling the DO value is DO-stat intermittent automatic replenishment, that is, replenishment is automatically started when the DO value is higher than the threshold, and replenishment is automatically stopped when the DO value is lower than the threshold.

[0027] In this invention, the method includes one or more of the following conditions:

[0028] (1) The fermentation culture includes a shake flask seed culture step: after the working seed is thawed, it is inoculated into the expansion culture medium, the culture temperature is 33-37℃, and the culture time is 6-10h.

[0029] (2) The fermentation culture includes the step of inoculating the shake flask seed liquid into the fermentation medium as described, with an inoculation amount of 1-5%;

[0030] (3) The fermentation culture also includes cell lysis and inclusion body collection and lysis steps: the inclusion bodies and lysis solution are mixed at a mass-volume ratio of 1:(30-100), and stirred for 12-18 hours after thorough mixing; the supernatant of the lysis solution is collected by centrifugation; the stirring speed is, for example, 200 rpm, and the lysis solution is, for example, Na2HPO4.2H2O 1.78 g / L, KH2PO4 0.24 g / L, NaCl 8 g / L, KCl 0.2 g / L, Triton X-100 1 ml / L, urea 360 g / L, imidazole 5 mM, and pH is 7.8-8.2.

[0031] In this invention, the fermentation medium, the fed medium, or the combination of the media described herein are used in the fermentation production of Oct4 protein.

[0032] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0033] The reagents and raw materials used in this invention are all commercially available.

[0034] The positive and progressive effects of this invention are as follows: The culture medium described in this invention is conducive to the fermentation and growth of bacteria. Even after culturing bacteria for 16 hours, the OD value can still increase, and the OD value can reach more than 200 during subsequent fermentation. The method described in this invention can effectively control the concentration of carbon source in the fermentation broth, thereby reducing the impact of acetic acid accumulation on bacterial growth and target protein expression during fermentation, and significantly improving the purity of Oct4 protein.

[0035] The following are abbreviations:

[0036] VVM: Ventilation ratio, the ratio of the ventilation rate per minute to the actual liquid volume in the tank.

[0037] OD: Optical Density

[0038] DO: Dissolved oxygen content

[0039] rpm: revolutions per minute

[0040] h: hour. Attached Figure Description

[0041] Figure 1 This is a flowchart of the process for high-density fermentation expression of Oct4 protein in Escherichia coli.

[0042] Figure 2A The purity of Oct4 protein expressed in a 10L fermenter was determined using SDS-PAGE. Figure 2B To detect the expression of Oct4 protein in a 10L fermenter using Western blotting.

[0043] Figure 3 The effect of different induction times on the purity of Oct4 protein.

[0044] Figure 4A and Figure 4B To investigate the effect of different pH values ​​on the purity of Oct4 protein after induction.

[0045] Figure 5 To investigate the effect of different DO values ​​on the purity of Oct4 protein after induction.

[0046] Figure 6A This is a feeding monitoring chart for the experimental group (DO-stat). Figure 6B This is a monitoring chart of feeding for the control group (uniform feeding).

[0047] Figure 7A The growth of microorganisms under different feeding methods is shown. Figure 7B The purity of Oct4 protein when using different feeding methods.

[0048] Figure 8 The effect of using different fermentation media on cell growth during a 50L fermentation process was investigated.

[0049] Figure 9 The growth of bacteria was observed using different fed culture media.

[0050] Figure 10 The effects of different fermentation processes on cell growth during a 500L fermentation process were investigated.

[0051] Figure 11A The purity of Oct4 protein during a 500L fermentation process was compared to that using the control group process. Figure 11B The purity of Oct4 protein during a 500L fermentation process using the process of this invention. Detailed Implementation

[0052] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0053] Table 1. Instruments used in the embodiments

[0054] Shaking bed IS-RSH2 CRYSTAL liquid nitrogen tank YDS-65-216 Sichuan Jinfeng -80℃ refrigerator 906-ULTS Thermo Seed container (10L) T&J-AType10L Dibier Seed container (50L) BIO-DVT3000 Shanghai Diwoxin Seed container (500L) BR-F-500L Shanghai Dongfulong sterilization cabinet SGLB-A-350D Xinhua Biosensor Analyzer SBA-40C Shandong Academy of Sciences Institute of Biology

[0055] Table 2 shows the experimental reagents used in the examples.

[0056] yeast powder Angie FM802 Corn liquor dry powder Shandong Xiwang / Yeast peptone Angie FP101 trypsin OXID LP0042 Soy protein Kerry Hysoy4D Sodium chloride diligent CG01.01 Magnesium sulfate Sinopharm 20025118 Calcium chloride Biotechnology A501330-0500 ammonium sulfate Jiudian / α-Lactose monohydrate (IPTG) BBI A604280-0500 sucrose Shandong Xiwang / glycerin BBI A600232-0500 Defoamer FoamdOCTor F2875 Pennwhite Kana BBI A600286-0025 IPTG BBI A600168-0100 dipotassium hydrogen phosphate Biotechnology A501212-0500 Potassium dihydrogen phosphate Jiudian / <![CDATA[Na2HPO4]]> Jiudian / KCl diligent CG02.03 Triton X-100 BBI A600198-0500

[0057] Example 1: Construction of recombinant Escherichia coli expressing exogenous Oct4 protein

[0058] The process flow for purifying Oct4 protein in this invention is as follows: Figure 1 As shown.

[0059] 1. Amplification of the Oct4 gene

[0060] The plasmid (pEP4_E02S_CK2M_EN2L_20923) was used as a template for the human Oct4 gene. PCR amplification was performed using forward and reverse primers. The sequence is SEQ ID NO:1.

[0061] Forward primer (SEQ ID NO:3):

[0062] 5'cca att gcc atg g ct atc gaa ggt cgt atg gcg gga cac ctg gct tcg gat3'

[0063] Nco I Factor Xa

[0064] Reverse primer (SEQ ID NO:4): 3'ccg aga ggg tac gta agt ttg act ctt aag tttggc 5'

[0065] EcoR I

[0066] 2. Construction of recombinant expression vectors

[0067] The long amino acid sequence fragment (his-tag) in pET-30a(+) was replaced with an 8-amino acid sequence fragment (+) (pET30a_sHT). The amplified human Oct4 gene product was inserted into multiple cloning sites of the pET30a_sHT vector by ligating the Oct4 PCR product digested with restriction endonucleases NcoI / EcoRI and the pET-30a_sHT vector. The new bacterial expression plasmid, Oct4-pET30a_sHT, contains the human Oct4 sequence and a short his-tag at the n-terminus of the Oct4 protein. DNA sequencing was used to verify the sequence accuracy of the entire Oct4-pET30a_sHT expression plasmid, which was then stored at -80°C.

[0068] 3. Introduction of the target gene fragment into E. coli BL21

[0069] (1) Competent cells were prepared using a chemical method based on molecular cloning. The host cell was Escherichia coli BL21(DE3), and all competent cells were stored in a refrigerator at -80°C.

[0070] (2) Mix an appropriate amount of Oct4-pET30a_sHT plasmid with competent cells under ice, and then perform heat shock.

[0071] (3) Add amplification culture medium to the mixture of plasmid and competent cells to revive the bacteria.

[0072] Example 2: Production of Oct4 protein using recombinant Escherichia coli fermentation

[0073] 1. Construct the original seed bank

[0074] Culture medium for propagation: 5 g / L yeast powder, 10 g / L yeast peptone, 10 g / L NaCl, 2 g / L corn steep liquor powder, 2 g / L soybean protein powder, 1 g / L magnesium sulfate, 4.6 g / L ammonium sulfate. First, add water to 80% of the prepared volume. After all the raw materials are dissolved, bring the volume up to the required volume. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, wrap them well, and sterilize at 121℃ for 20 min.

[0075] (1) Preparation of solid culture medium: Add 2% agar powder and 30 mg / L kanamycin sulfate to the amplification culture medium to prepare solid culture medium in a plate.

[0076] (2) Aseptically dilute the resuscitation culture medium and spread it on the solid culture medium, and incubate it upside down at 37°C for 12-16 hours.

[0077] (3) Inoculate one single clone into a shaker tube containing expansion medium. Select six single clones and culture for 12-16 hours. Add an equal volume of 30-40% glycerol solution to the expansion medium, mix well, and aliquot into cryovials for storage in liquid nitrogen. Transfer the remaining expansion medium to a single-clone selection medium to induce recombinant protein expression. Analyze protein purity using SDS-PAGE, and select the highest expression level as the original seed bank. Use a 10L fermenter to test the purity of the target protein. The expressed Oct4 protein is shown below. Figure 2A and Figure 2B As shown, its amino acid sequence is SEQ ID NO:2.

[0078] 2. Construct the main seed library

[0079] (1) Take one original seed and thaw it. Inoculate it into the culture medium for 4-8 hours. The inoculation amount is 0.5-1 ml of seed liquid into an Erlenmeyer flask containing 200-400 ml of culture medium.

[0080] (2) Add 30-40% glycerol solution to the culture medium in equal volumes, mix well, and then dispense into cryovials and store in liquid nitrogen.

[0081] (3) Remove the cryopreservation tubes from the liquid nitrogen tank and test the following items: resistance, colony morphology, purity of target protein expression (using a 10L seed tank for fermentation test), plasmid copy number, mass loss rate, plasmid enzyme digestion, target gene sequencing, etc., to ensure that the main seed bank is compliant and qualified.

[0082] 3. Construct a working seed bank

[0083] (1) Take one main seed and thaw it. Inoculate it into the expansion culture medium and culture for 4-8 hours. The inoculation amount is 0.5-1 ml of seed liquid into an Erlenmeyer flask containing 200-400 ml of expansion culture medium.

[0084] (2) Add 30-40% glycerol solution to the culture medium in equal volumes, mix well, and then dispense into cryovials and store in a -80℃ freezer.

[0085] (3) Remove the cryovials from the -80℃ freezer and test the following items: resistance, colony morphology, purity of target protein expression (using a 10L seed tank for fermentation test), plasmid copy number, mass loss rate, plasmid enzyme digestion, target gene sequencing, etc., to ensure that the main seed bank is compliant and qualified.

[0086] 4. Seed culture in shake flasks

[0087] The inoculum volume is 0.5-1 ml of working seed culture to 200-400 ml of expansion culture medium. The incubation temperature is 33-37℃, and the incubation time is 6-10 hours.

[0088] 5. Seed tank fermentation

[0089] The optimal induction time, optimal pH after induction, optimal DO value after induction, and feeding method for recombinant E. coli fermentation were tested using a 10L fermenter. The optimal conditions for each were determined by testing the purity of the target protein.

[0090] The induction time was set to 1-9 hours, and the purity of the target protein was examined at different time points. The pH during fermentation was controlled at 6.5-7.0 and 7.0-7.5, respectively, and the cell OD was measured. 600To investigate the optimal pH range for cell growth, the pH was controlled at 7.0±0.1 in Experiment 1 and 7.5±0.1 in Experiment 2 after induction. The optimal pH range after induction was determined by SDS-page analysis of Oct4 protein purity. Before fermentation induction, DO (dissolved organic matter) was controlled at ≥30% in Experiments 1 and 2. After induction, DO was controlled at 30-50% in Experiment 1 and 10-20% in Experiment 2. The optimal DO range after induction was determined by SDS-page analysis of Oct4 protein purity. The experimental groups were fed intermittently using an automated feeding system (e.g., [missing information]). Figure 6A As shown), the average feed rate was controlled at 0.6 ml / L / min before induction and 0.3 ml / L / min after induction; the control group was fed continuously with a dynamic uniform feed rate (e.g., ...). Figure 6B As shown in the figure, the feeding rate was 0.6 ml / L / min before induction and 0.3 ml / L / min after induction. The purity of Oct4 protein was analyzed by SDS-page to determine the appropriate feeding method.

[0091] Note: Protein purity is calculated as the percentage of the target protein (Oct4 in this paper) in the total bands of the SDS-page gel image.

[0092] The results are shown in Table 3. The purity of the target protein reached its highest level at 7 hours of induction, and then gradually decreased (e.g., ...). Figure 3 As shown in the figure), the optimal induction time is 7 hours. Results showed that when the pH was controlled at 7.0 ± 0.1 after induction, the purity of the target protein was 19.48%, while when the pH was controlled at 7.5 ± 0.1 after induction, the purity was only 15.18% (as shown in the figure). Figure 4A (and Table 4); however, there was no significant difference in cell growth when the pH was controlled at 6.5-7.0 and 7.0-7.5 (e.g., ...). Figure 4B As shown in Table 5), the optimal pH after induction is 7.0. When the DO value is controlled at 10-20% after induction, the purity of the target protein is 25.43%, and when the DO value is controlled at 30-50%, the purity of the target protein is 20%. Therefore, the optimal DO value after induction is 10-20% (as shown in Table 6). Table 7 shows that there is no significant difference in cell growth between DO values ​​controlled at 30-50% (Experiment 1) and 10-20% (Experiment 2) (as shown in Table 7). Figure 5 (as shown in Table 7); Figure 7A and 7B The results showed that there was no significant difference in cell growth between the two feeding methods. The purity of Oct4 protein using the DO-stat feeding method increased from 17% in the control group to 22.5%, and the purity of the target protein increased by 32.35%.

[0093] Table 3. Effect of induction time on the purity of the target protein.

[0094] 1 9.59 2 9.22 3 13.27 4 12.51 5 18.09 6 18.81 7 21.89 8 13.98 9 14.05

[0095] Table 4 Effect of pH value on the purity of the target protein

[0096] Experiment 1 7.0±0.1 19.48％ Experiment 2 7.5±0.1 15.18％

[0097] Table 5. Determination of the optimal pH value after induction.

[0098]

[0099]

[0100] Table 6. Determination of the optimal DO value after induction.

[0101] Experiment 1 30-50％ 20％ Experiment 2 10-20％ 25.43％

[0102] Table 7. Effects of different DO values ​​on OD 600 Impact

[0103]

[0104]

[0105] In this invention, when the microorganisms grow and metabolize using carbon sources, they consume oxygen. When the aerobic rate is greater than the oxygen supply rate of the reactor, the dissolved oxygen in the fermentation broth will decrease, and vice versa. When the carbon source concentration decreases to a certain level (the lowest carbon source concentration detected offline is 0.04 g / L), the metabolic intensity of the cells decreases, and the amount of oxygen consumed decreases. When the oxygen supply in the reactor remains constant, the reaction is that dissolved oxygen increases (carbon source replenishment begins when dissolved oxygen rises to 15%. According to the equipment monitoring records, automatic feeding begins when dissolved oxygen is greater than 15%, and the maximum feeding time from the start of feeding to the stop is 2 minutes, with an average flow rate of 0.3 ml / L / min. The fermentation broth volume is 4.5 L, and the carbon source concentration in the feed medium is 200 g / L. It can be calculated that the maximum feed amount per fluctuation is 2.7 ml, and the added carbon source concentration is 2.7 × 200 ÷ 4.5 ÷ 1000 = 0.12 g / L. The lowest carbon source concentration when dissolved oxygen begins to rise is 0.04 g / L. Adding the added carbon source concentration, the total is 0.16 g / L. Considering cell consumption, the total carbon source concentration in the fermentation broth can be controlled at around 0.1 g / L). Therefore, supplementing the carbon source based on dissolved oxygen levels can keep the carbon source concentration at a low level. Offline sampling and monitoring of the carbon source concentration during fermentation were conducted. After the DO peak, DO-stat feeding was performed, which effectively controlled the carbon source concentration in the fermentation broth to 0.04-0.15 g / L (as shown in Table 8). The experiment showed good repeatability. In the later stages of fermentation, DO-stat feeding can be directly used to control the carbon source concentration in the fermentation broth at a low level.

[0106] Table 8. Relationship between OD value and residual sugar concentration during fermentation.

[0107]

[0108]

[0109] (1) Fermentation in a 50L seed tank

[0110] The formula for the trace element stock solution is as follows: FeSO4·7H2O 40g / L, CoCl2·6H2O 4g / L, H3BO3 0.5g / L, NaMoO4·2H2O 2g / L, ZnSO4·7H2O 2g / L, CuCl2·2H2O 1g / L, MnSO4·H2O 4.6g / L, CaCl2 1g / L, MgSO4 1g / L. Add concentrated hydrochloric acid until all trace elements are completely dissolved, then add water to make up to the prepared volume.

[0111] Use a 50L seed tank culture medium: 6g / L yeast extract, 12g / L yeast peptone, 2g / L corn steep liquor powder, 2g / L soybean protein powder, 8.2g / L dipotassium hydrogen phosphate, 1.15g / L potassium dihydrogen phosphate, 0.5g / L sodium bicarbonate, 0.196g / L magnesium sulfate, 0.5g / L calcium chloride, 3g / L ammonium sulfate, 5g / L sodium chloride, 32g / L glucose, 0.1ml / L antifoaming agent, and 0.4ml / L trace element stock solution. Adjust the volume to the required level. Sterilize at 121℃ for 20 minutes. Inoculate the seed culture in shake flasks at an inoculum size of 1-5%. Control the pH at 6.0-7.5 by adding ammonia, maintain an aeration rate of 0.5-2 vvm, and a stirring speed of 50-800 rpm. Control the dissolved oxygen (DO) to ≥30% by controlling the aeration rate and stirring speed. Incubate for 5-10 hours. In the culture medium, replacing yeast peptone with soybean peptone or tryptone, and glucose with glycerol, lactose, or sucrose, still maintains the same effect.

[0112] The fermentation medium of this invention has been significantly optimized, with the addition of corn steep liquor powder, soybean protein powder, sodium bicarbonate, trace elements, and other components (as shown in Table 9), and the optimal ratio of carbon and nitrogen sources in the medium has been established. The effectiveness of the fermentation medium was tested using a 10L fermenter, with a control group consisting of a standard culture medium.

[0113] Table 9 Comparison of ordinary culture medium and the fermentation culture medium of the present invention.

[0114]

[0115]

[0116] *Replacing yeast peptone with soy peptone or tryptone, and glucose with glycerol, lactose, or sucrose, will maintain the same effect.

[0117] Table 10 Comparison of production effects between the fermentation culture medium of the present invention and ordinary culture medium.

[0118]

[0119]

[0120] The experimental results are shown in Table 10. Using the control group fermentation medium, the OD value increased after 9.17 h of culture. 600 The OD increased to a maximum of 35, and then showed a downward trend. After culturing for 16 hours using the formula of this invention, the OD grew to 72.4 and still showed an upward trend (e.g., ...). Figure 8 (As shown). Therefore, it can be concluded that, all other things being equal, the culture medium composition of this invention is more conducive to the growth of Oct4 gene recombinant Escherichia coli.

[0121] (2) 500L fermentation

[0122] Fed culture medium: 70.5 g / L yeast extract, 146.4 g / L yeast peptone, 2 g / L corn steep liquor powder, 2 g / L soybean protein powder, 200 g / L glucose, 3 g / L (NH4)2SO4, 5.85 g / L MgSO4. First, add water to 80% of the prepared volume. After all ingredients are dissolved, bring the volume to the required level and sterilize at 121°C for 20 minutes. Replacing the yeast peptone with soybean peptone or trypsin, and replacing the glucose with glycerol, lactose, or sucrose, will maintain the same effect.

[0123] 500L seed tank culture medium: yeast powder 6g / L, yeast peptone 12g / L, corn steep liquor powder 2g / L, soybean protein 2g / L, dipotassium hydrogen phosphate 8.2g / L, potassium dihydrogen phosphate 1.15g / L, sodium bicarbonate 0.5g / L, magnesium sulfate 0.196g / L, calcium chloride 0.5g / L, ammonium sulfate 3g / L, sodium chloride 5g / L, glucose 32g / L, antifoaming agent 0.1ml / L, trace element stock solution 0.4ml / L. Adjust the volume to the required level and sterilize at 121℃ for 20 minutes. Inoculate 1-5% of the culture medium into a 50L seed tank. Replacing yeast peptone with soybean peptone or trypsin, and glucose with glycerol, lactose, or sucrose, will maintain the same effectiveness.

[0124] The pH was controlled at 6.0-7.5 by adding ammonia, with an aeration rate of 0.5-3 vvm and a stirring speed of 50-500 rpm. Before the DO curve peaked, DO was maintained at ≥30% by controlling the aeration rate and stirring speed. Once the DO curve peaked, fed-batch culture medium was started, maintaining the stirring speed and aeration rate unchanged from before the DO peak. DO-stat feedback was used for fed-batch culture, with an average flow rate of 0.6 ml / L / min before induction and 0.3 ml / L / min after induction, and a feed rate of 45-55 L. DO was controlled at 10-50% using fed-batch culture medium. When the fermentation OD... 600 Add IPTG when the pH value is 30-60, and induce fermentation for 3-8 hours. Collect the cells using a centrifuge after fermentation.

[0125] The residual sugar was detected using an SBA-40C biosensor analyzer, and the content of consumed glycerol was calculated using the periodic acid method. The residual sugar content in the fermentation broth was then detected, and carbon was added according to the residual sugar concentration. The carbon source was mixed with its components to prepare the fed culture medium, and the DO-stat feedback feeding was used for feeding.

[0126] Table 11 Composition of Feeding Culture Medium

[0127]

[0128]

[0129] * Yeast peptone can be replaced with soy peptone or tryptone, and glucose can be replaced with glycerol, lactose, or sucrose.

[0130] Comparative Example 1 of Feed Culture Medium

[0131] The fermentation medium used was the medium of this invention. The fed-batch media were as follows: Experiment 1: 6×TB (phosphate-free), Experiment 2: medium used during optimization, and Experiment 3: medium of this invention. OD 600 Once the culture medium reaches approximately 40%, supplemental feeding medium is added. The main indicators examined in the three groups of experiments were strain growth and OCT4 protein purity. Table 12 provides the formulation of the supplemental feeding medium.

[0132] Table 12 Comparison of Feeded Culture Media

[0133]

[0134] * Yeast peptone can be replaced with soy peptone or tryptone, and glucose can be replaced with glycerol, lactose, or sucrose.

[0135] Table 13 Comparison of Production Effects of Different Culture Media

[0136]

[0137] Table 13 shows that the growth trends of the three groups were basically the same before the addition of supplemental culture medium; there was no significant difference in the growth trends of experiments 2 and 3 after the addition of supplemental culture medium; after the addition of supplemental culture medium, the growth trend of experiment 1 was significantly slower than that of experiments 2 and 3 (e.g., Figure 9 (As shown); the purity of Oct protein in the three groups of experiments was 18.95%, 20.21%, and 23.68%, respectively. Experiment 3 showed that the supplemented culture medium was more conducive to Oct protein expression.

[0138] 6. Bacterial cell lysis and collection of crude inclusion bodies

[0139] Washing solution preparation: Na₂HPO₄ 1.42 g / L, KH₂PO₄ 0.24 g / L, NaCl 8 g / L, KCl 0.2 g / L, Triton X-100 1 ml / L, urea 30 g / L, imidazole 5 mM. First, add water to 80% of the prepared volume. After all the raw materials are dissolved, bring the volume to the required level. Adjust the pH to 6.0-7.5 using hydrochloric acid. Mix inclusion bodies and washing buffer at a mass-to-volume ratio of 1:(5-15) and mix thoroughly using a high-speed disperser. Collect inclusion bodies using a centrifuge. Repeat the above operation 2-4 times.

[0140] 7. Inclusion body lysis

[0141] (1) Preparation of lysis buffer: Na2HPO4·2H2O 1.78g / L, KH2PO4 0.24g / L, NaCl 8g / L, KCl 0.2g / L, Triton X-100 1ml / L, urea 360g / L, imidazole 5mM. First add water to 80% of the prepared volume. After all the raw materials are dissolved, make up the volume to the required volume. Adjust the pH to 7.8-8.2 with hydrochloric acid.

[0142] (2) The inclusion bodies and lysis solution are mixed at a mass-volume ratio of 1:(30-100), and then thoroughly mixed using a high-speed disperser before being stirred in a stirring system for 12-18 hours at a stirring speed of 200 rpm.

[0143] (3) Use a centrifuge to collect the lysate supernatant, i.e., the initial protein solution.

[0144] Table 14 Comparison of production effects of the culture medium of the present invention and TB culture medium.

[0145]

[0146]

[0147] When using a 500L industrial-scale fermenter for protein production, as shown in Table 14, compared with using TB medium, the fermentation time of the present invention is 15.65 hours, and the OD... 600In the control group, the OD growth trend consistently increased. The trend slowed down after 7.5 hours of fermentation, and then decreased after 8 hours, at which point fermentation was stopped (e.g., ...). Figure 10 As shown), OD during can placement 600 The purity of the target protein reached 27.5%, which was 82%, significantly higher than the expression purity of the Oct4 protein in the control group (e.g., 82). Figure 11A and 11B (As shown).

Claims

1. A method for producing Oct4 protein by fermentation, characterized in that, Recombinant Escherichia coli was cultured using a fermentation medium, with fed medium added according to the dissolved oxygen (DO) value during the culture process; the recombinant Escherichia coli expressed the Oct4 protein with the amino acid sequence shown in SEQ ID NO: 2; The fermentation medium comprises yeast powder, peptone, corn steep liquor powder, soybean protein powder, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, magnesium sulfate, calcium chloride, ammonium sulfate, sodium chloride, defoamer, trace element stock solution, and glucose; wherein the concentrations are as follows: yeast powder 6 g / L, peptone 12 g / L, corn steep liquor powder 2 g / L, soybean protein powder 2 g / L, dipotassium hydrogen phosphate 8.2 g / L, potassium dihydrogen phosphate 1.15 g / L, sodium bicarbonate 0.5 g / L, magnesium sulfate 0.196 g / L, calcium chloride 0.5 g / L, ammonium sulfate 3 g / L, sodium chloride 5 g / L, defoamer 0.1 ml / L, trace element stock solution 0.4 ml / L, and glucose 32 g / L; the pH is 6.5-7.

0. The fed culture medium comprises yeast extract, peptone, corn steep liquor powder, soybean protein powder, (NH4)2SO4, MgSO4, and glucose; wherein the concentration of yeast extract is 70.5 g / L, the concentration of peptone is 146.4 g / L, the concentration of corn steep liquor powder is 2 g / L, the concentration of soybean protein powder is 2 g / L, the concentration of (NH4)2SO4 is 3 g / L, the concentration of MgSO4 is 5.85 g / L, and the concentration of glucose is 20 g / L; the pH is the natural pH. The defoamer is FoamdOCTor F2875 Pennwhite; The peptone is yeast peptone; the trace element mother liquor includes FeSO4. 7H2O, CoCl2 6H2O, H3BO3, NaMoO4 2H2O, ZnSO4 7H2O, CuCl2 2H2O, MnSO4 H2O, CaCl2 and MgSO4; In the trace element mother liquor, FeSO4 The concentration of 7H2O is 40 g / L, and the concentration of CoCl2 is... The concentrations of 6H₂O and H₃BO₃ are 4 g / L, 0.5 g / L, and NaMoO₄. The concentration of 2H2O is 2 g / L, and the concentration of ZnSO4 is... The concentration of 7H2O is 2 g / L, and the concentration of CuCl2 is 2 g / L. The concentration of 2H2O is 1 g / L, and the concentration of MnSO4 is... The concentrations of H2O, CaCl2, and MgSO4 are 1 g / L. Before Oct4 protein induction: With the DO value controlled at 25-35%, fed culture medium was added at an average flow rate of 0.6 ml / L / min; when the OD value in the fermentation broth reached 25-35%, the culture medium was added continuously. 600 Add the inducing agent when the pH value is 30-60; the pH of the fermentation culture is 7.0±0.2-7.5±0.2; After Oct4 protein induction: when the DO value is 10-20%, feed medium is added at an average flow rate of 0.3 ml / L / min; the fermentation time is 3-8 hours; the pH of the fermentation culture is 7.0±0.

1.

2. The method as described in claim 1, characterized in that, The fermentation conditions are selected from one or more of the following: Aeration rate: 0.5-2 vvm; stirring speed: 50-800 rpm; The fermentation culture temperature was 37℃.

3. The method as described in claim 1, characterized in that, When the OD in the fermentation broth 600 Add an inducing agent when the value is 45-55.

4. The method as described in claim 1, characterized in that, Prior to the induction of the Oct4 protein, the pH of the fermentation culture was 7.0 ± 0.

2.

5. The method as described in claim 1, characterized in that, After the Oct4 protein is induced, the fermentation culture time is 5-7 hours.

6. The method as described in claim 1, characterized in that, The host bacterium of the recombinant Escherichia coli is BL21, and the recombinant expression vector is pET-30a(+); and / or, the method for controlling the DO value is DO-stat intermittent automatic feeding, that is, feeding is automatically started when the DO value is higher than the threshold, and feeding is automatically stopped when the DO value is lower than the threshold.

7. The method according to any one of claims 1-6, characterized in that, Includes one or more of the following conditions: (1) The fermentation culture includes a shake flask seed culture step: after the working seed is thawed, it is inoculated into the expansion culture medium, the culture temperature is 33-37℃, and the culture time is 6-10 h; (2) The fermentation culture includes the step of inoculating the seed culture from the shake flask into the fermentation medium, with an inoculation amount of 1-5%; (3) The fermentation culture also includes cell lysis and inclusion body collection and lysis steps: mix inclusion bodies and lysis broth at a mass-volume ratio of 1:(30-100), mix thoroughly and stir for 12-18 h; centrifuge and collect the lysis supernatant to obtain the product.

8. The method as described in claim 7, characterized in that, The pyrolysis solution is: Na2HPO4. The concentrations of the following ingredients were: 2H₂O 1.78 g / L, KH₂PO₄ 0.24 g / L, NaCl 8 g / L, KCl 0.2 g / L, Triton X-100 1 ml / L, urea 360 g / L, imidazole 5 mM, and pH 7.8-8.

2. The centrifugation stirring speed was 200 rpm.

Images