Improved self-induction culture medium and culture process

Abstract

The invention discloses an improved self-induction culture medium and a culture process, and belongs to the technical field of bioengineering. The improved self-induction culture medium is prepared from 10 g / L of glucose, 3-5 g / L of glycerin, 3 g / L of lactose, 24 g / L of yeast extract, 8 g / L of casein hydrolysate, 0.02-0.05 mM of malonic acid, 1 mM of methionine and 1-3 mM of MgSO4, a phosphate buffer solution is added to control the pH to be 7.2, and the improved self-induction culture medium is prepared by mixing after sterilization. Cells are inoculated to an improved self-induction culture medium for culture, automatic switching of growth-induction stages can be realized based on a pH and DO linkage dynamic feeding control method, the operation complexity is reduced, and the controllability is higher. The culture medium does not need a special inducer, cost is reduced, and protein expression quantity is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an improved autoinduction culture medium and a culture process. Background Art

[0002] Autoinduction media is a complex medium containing multiple growth factors and cell signaling molecules. These factors can induce autonomous cell differentiation and proliferation during cell culture, thereby achieving research goals such as cell differentiation and reprogramming. It is a very promising cell culture technology. Autoinduction media is typically composed of multiple basal media, growth factors, and cell signaling molecules, and is highly complex and specific.

[0003] However, the existing autoinduction culture medium has the following technical bottlenecks:

[0004] (1) Uncontrollable induction timing: The existing auto-induction medium for the efficient expression of foreign proteins in Escherichia coli (E. coli) utilizes the regulatory mechanism of E. coli on carbon sources and energy. Glucose is used as the repressor of the galactose operon. When glucose is exhausted, lactose is converted into allolactose, thereby activating the expression of the lac promoter and achieving automatic induction expression of foreign proteins. However, the traditional lactose / arabinose induction system is easily disturbed by carbon source metabolism, resulting in premature / delayed induction.

[0005] (2) Byproduct accumulation: When cultured at high density, acetic acid / lactic acid concentrations exceeding 5 g / L will inhibit cell growth;

[0006] (3) High formula cost: a large amount of growth factors and cell signaling molecules need to be added;

[0007] (4) Poor stability: Various growth factors and cell signaling molecules in the autoinduction culture medium are easily affected by environmental changes and have relatively poor stability.

[0008] Although autoinduction culture technology has the advantages of high efficiency, versatility and easy operation, its defects such as high cost, poor stability and uncontrollable induction still need further improvement and optimization. Summary of the Invention

[0009] The present invention provides an improved auto-induction culture medium and a culture process, which can solve the problem of uncontrollable induction system of the auto-induction culture medium in the prior art.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] A modified autoinduction medium, wherein the composition of the modified autoinduction medium is as follows:

[0012] Glucose 10g / L, glycerol 3-5g / L, lactose 3g / L, yeast extract 24g / L, casein hydrolysate 8g / L, malonic acid 0.02-0.05mM, methionine 1mM, MgSO41-3mM, phosphate buffer was added to control the pH to 7.2.

[0013] The improved autoinduction medium is prepared by sterilizing each component separately and then mixing them together. Glucose and lactose are sterilized at 115° C. for 15 minutes, and the other components are sterilized at 121° C. for 20 minutes to avoid Maillard reaction.

[0014] Malonic acid is a succinate dehydrogenase inhibitor that can reduce the formation of by-products.

[0015] When used in E. coli cell culture, glucose is preferentially consumed to maintain cell growth. Dynamic feeding occurs when DO exceeds a threshold during metabolism. Lactose metabolism triggers induction, inducing cells to automatically initiate T7 RNA polymerase expression.

[0016] The present invention also provides a culture process, which uses the improved autoinduction medium as described above for culture, and the steps of the culture process are as follows:

[0017] Single clones were picked from the plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 12-16 hours. The cells were inoculated into the modified autoinduction medium at a volume ratio of 1%-5%, and cultured at 37°C for 18-24 hours. The lactose induction was automatically started after glucose was exhausted, and the bacteria were collected to detect the expression of the target protein.

[0018] For cells cultured in the modified autoinduction medium, dissolved oxygen-linked feeding was set, and a DO threshold was set. When DO>30%, glycerol was added, and the stirring rate was intermittently increased. The glycerol feeding rate was 0.5-1 mL / min.

[0019] The pH threshold is set for culturing cells in the modified autoinduction medium. When the pH exceeds 7.2, the mixed carbon source solution is automatically added at a rate of 2 mL / min until the pH drops. The pH is stabilized at 6.8-7.2 by feeding to avoid drastic fluctuations that affect bacterial metabolism. The feed flow rate is adjusted according to the rate of pH change.

[0020] The proportions of the mixed carbon source solution are as follows:

[0021] Glucose 200g / L, lactose 60g / L and MgSO4 5g / L.

[0022] Furthermore, when the cells cultured in the culture process have the characteristic of expressing proteins at low temperature, the temperature of the improved autoinduction culture medium is lowered to 20-30° C. during the culture process to improve the expression of soluble proteins.

[0023] Furthermore, the specific growth rate of the cells in the culture process is controlled at 0.15-0.25h-1.

[0024] Furthermore, the cells in the culture process have an acetate production coefficient of less than 0.2 g / g DCW. The acetate production coefficient is the number of grams of acetate produced per gram of dry cell weight.

[0025] Furthermore, in the culture process, OUR (oxygen uptake rate) is maintained at 20-25 mmol / L / h during the induction stage.

[0026] Furthermore, the culture process regulates the carbon source supply rate through dissolved oxygen feedback when used to culture Escherichia coli.

[0027] Furthermore, when used to culture CHO cells, the culture process induces gene expression through temperature control, and the glucose content feedback regulates the carbon source supply rate to maintain the glucose content at 0.5-1 g / L.

[0028] Furthermore, when used to culture Pichia pastoris, the culture process induces gene expression through methanol, and the dissolved oxygen (DO) feedback regulates the carbon source supply rate to maintain the dissolved oxygen (DO) at 30%-60%.

[0029] Furthermore, the protein expression level of the culture process reaches 3.8 g / L-10 g / L.

[0030] Beneficial effects of the present invention:

[0031] (1) The culture process provided by the present invention has the characteristics of intelligent regulation, which can realize automatic switching between the "growth-induction" stage, without the need for manual addition of inducers, reducing the complexity of operation. The dynamic feeding control method based on the linkage of pH and DO can achieve optimal growth and product synthesis conditions through feeding control, and has stronger controllability. Through intelligent carbon and nitrogen source ratio and induction timing control, high-density and high-expression auto-induction culture is achieved, which is suitable for recombinant protein production in mammalian cells, Escherichia coli and yeast systems.

[0032] (2) The culture medium provided by the present invention, combined with the culture process, can achieve a breakthrough in production efficiency, increase protein expression, and enhance production capacity per unit volume.

[0033] (3) The improved autoinduction culture medium is low-cost, reducing the cost by 40% compared to existing culture media (eliminating inducers and special additives), and increasing the downstream purification yield by 25% (reducing host protein contamination by 60%).

[0034] (4) The materials of the culture medium are environmentally friendly, which can shorten the culture cycle, reduce the carbon footprint, and reduce the discharge of organic waste liquid. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0036] Example 1

[0037] This example establishes a culture system based on the expression of green fluorescent protein in Escherichia coli.

[0038] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.04 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0039] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0040] Cultivation process:

[0041] A single colony of the BL21(DE3) strain carrying the pET-28a-GFP plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were inoculated into the modified autoinduction medium described above at a volume ratio of 3% and cultured at 37°C for 4 hours. The culture temperature was then lowered to 20°C to enhance soluble protein expression. The total culture time was 24 hours. Feeding was linked to the culture process, and a pH threshold was set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO was >30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) at 20%-30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0042] The results showed that the expression level of green fluorescent protein (GFP) reached 4.2 g / L and the acetic acid concentration was 1.2 g / L.

[0043] Example 2

[0044] The only difference from Example 1 is that the amount of malonate added to the culture medium was adjusted from 0.04 mM to 0.02 mM.

[0045] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.02 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0046] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0047] Cultivation process:

[0048] A single colony of the BL21(DE3) strain carrying the pET-28a-GFP plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were inoculated into the modified autoinduction medium described above at a volume ratio of 3% and cultured at 37°C for 4 hours. The culture temperature was then lowered to 20°C to enhance soluble protein expression. The total culture time was 24 hours. Feeding was linked to the culture process, and a pH threshold was set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO was >30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) at 20%-30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0049] The results showed that the expression level of green fluorescent protein (GFP) reached 3.9 g / L and the acetic acid concentration was 1.0 g / L.

[0050] Example 3

[0051] The only difference from Example 1 is that the amount of malonate added to the culture medium was adjusted from 0.04 mM to 0.05 mM.

[0052] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.05 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0053] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0054] Cultivation process:

[0055] A single colony of the BL21(DE3) strain carrying the pET-28a-GFP plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were inoculated into the modified autoinduction medium described above at a volume ratio of 3% and cultured at 37°C for 4 hours. The culture temperature was then lowered to 20°C to enhance soluble protein expression. The total culture time was 24 hours. Feeding was linked to the culture process, and a pH threshold was set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO was >30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) at 20%-30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0056] The results showed that the expression level of green fluorescent protein (GFP) reached 4.4 g / L and the acetic acid concentration was 1.6 g / L.

[0057] Example 4

[0058] The only difference from Example 1 is that this example establishes a culture system based on the expression of recombinant human serum albumin by culturing E. coli.

[0059] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.04 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0060] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0061] Cultivation process:

[0062] A single colony of the BL21(DE3) strain carrying the pET-28a-HSA plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were inoculated into the modified autoinduction medium described above at a 3% volume ratio and incubated at 37°C for 4 hours. The culture temperature was then lowered to 20°C to enhance soluble protein expression. The total incubation time was 24 hours. Feeding was performed during the culture process. A pH threshold was set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO was >30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) between 20% and 30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0063] The results showed that the expression level of recombinant human serum albumin (HSA) reached 3.8 g / L and the acetic acid concentration was 0.8 g / L.

[0064] Example 5

[0065] The only difference from Example 1 is that this example establishes a culture system based on the expression of heat-stable β-galactosidase protein by culturing Escherichia coli.

[0066] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.04 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0067] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0068] Cultivation process:

[0069] A single colony of the BL21(DE3) strain carrying the pET-28a-β-Gal plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were inoculated at a 3% volume ratio into the modified autoinduction medium described above and cultured at 37°C for 4 hours. The culture temperature was then lowered to 20°C to enhance soluble protein expression. The total culture time was 24 hours. Feeding was performed during the culture process. A pH threshold was set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO > 30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) between 20% and 30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0070] The results showed that the expression level of β-galactosidase protein (β-Gal) was 5 g / L and the acetic acid concentration was 1.2 g / L.

[0071] Example 6

[0072] The only difference from Example 1 is that this example establishes a culture system based on the expression of recombinant human interferon α protein by culturing E. coli.

[0073] Prepare a modified autoinduction medium with the following composition: glucose 10 g / L, glycerol 4 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.04 mM, methionine 1 mM, MgSO4 2 mM, and add phosphate buffer to control the pH to 7.2.

[0074] Glucose and lactose were sterilized at 115°C for 15 min, respectively, and the other components were sterilized at 121°C for 20 min. After sterilization, the components were mixed to obtain a modified autoinduction medium.

[0075] Cultivation process:

[0076] A single colony of the BL21(DE3) strain carrying the pET-28a-IFNα plasmid was picked from a plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 15 hours. The cells were then inoculated into the modified autoinduction medium described above at a 3% volume ratio and incubated at 37°C for 4 hours. The culture was then cooled to 20°C to enhance soluble protein expression for a total of 24 hours. Feeding was performed during the culture process, with a pH threshold set. When the pH exceeded 7.2, a mixed carbon source solution (200 g / L glucose, 60 g / L lactose, and 5 g / L MgSO4) was automatically added at a rate of 2 mL / min to stabilize the pH within the range of 6.8-7.2. A DO threshold was set. When the DO exceeded 30%, glycerol was added at a rate of 0.5 mL / min to maintain the dissolved oxygen (DO) between 20% and 30%. During the culture process, the specific growth rate of the cells was controlled at 0.15-0.25 h⁻¹, the acetic acid production coefficient of the cells was <0.2 g / gDCW, and the OUR (oxygen uptake rate) during the induction phase was maintained at 20-25 mmol / L / h. After the culture was completed, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The cells were weighed and the weight was recorded. Subsequent lysis and purification steps and electrophoresis detection were performed according to conventional procedures.

[0077] The results showed that the expression level of IFN-α protein was 4.3 g / L and the acetic acid concentration was 0.9 g / L.

[0078] Comparative Example 1

[0079] The only difference from Example 1 is that this comparative example adopts IPTG induction method for culture.

[0080] Prepare LB medium (per L):

[0081] Tryptone 10g, yeast extract 5g, sodium chloride 10g.

[0082] Cultivation process:

[0083] A single colony of the BL21(DE3) strain carrying the pET-28a-GFP plasmid was picked from the plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 12 hours. The bacterial suspension was then transferred to 1 L of LB medium containing the corresponding resistance and cultured at 37°C and 220 rpm until OD600 = 0.8. IPTG was added to a final concentration of 0.1 mmol / L and expression was induced at 20°C for 12 hours. The bacterial suspension was collected, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The bacterial cells were weighed and the weight was recorded. Subsequent lysis and purification steps were performed according to routine procedures, and electrophoresis was performed for detection.

[0084] The results showed that the expression level of green fluorescent protein (GFP) was 2.1 g / L and the acetic acid concentration was 4.5 g / L.

[0085] Comparative Example 2

[0086] The only difference from Example 4 is that this comparative example adopts IPTG induction method for culture.

[0087] Prepare LB medium (per L):

[0088] Tryptone 10g, yeast extract 5g, sodium chloride 10g.

[0089] Cultivation process:

[0090] A single colony of the BL21(DE3) strain carrying the pET-28a-HSA plasmid was picked from the plate and inoculated into LB medium containing antibiotics. The culture was shaken at 37°C and 220 rpm for 12 hours. The bacterial suspension was then transferred to 1 L of LB medium containing the corresponding resistance and cultured at 37°C and 220 rpm until OD600 = 0.8. IPTG was added to a final concentration of 0.1 mmol / L and expression was induced at 20°C for 12 hours. The bacterial suspension was collected, the culture medium was centrifuged, the supernatant was removed, and the precipitated bacterial sludge was collected. The bacterial cells were weighed and the weight was recorded. Subsequent lysis and purification steps were carried out according to routine procedures, and electrophoresis was performed for detection.

[0091] The results showed that the expression level of recombinant human serum albumin (HSA) was 1.5 g / L and the acetic acid concentration was 4.2 g / L.

[0092] Comparative Example 3

[0093] The only difference from Example 5 is that this comparative example adopts IPTG induction method for culture.

[0094] Prepare LB medium (per L):

[0095] Tryptone 10g, yeast extract 5g, sodium chloride 10g.

[0096] Cultivation process:

[0097] The BL21 (DE3) strain carrying the pET-28a-β-Gal plasmid was picked from the plate and inoculated into LB medium containing antibiotics, and cultured at 37°C and 220 rpm for 12 hours. The bacterial suspension was then transferred to LB medium containing the corresponding resistance and cultured at 37°C and 220 rpm until the OD 600 =0.8, add IPTG to a final concentration of 0.1 mmol / L, and induce expression at 30°C for 12 h. Collect the bacterial suspension, centrifuge the culture medium, remove the supernatant, collect the precipitated bacterial sludge, and weigh the cells. Proceed with subsequent lysis and purification steps and electrophoresis analysis as per standard procedures.

[0098] The results showed that the expression level of β-Gal protein was 2.2 g / L and the acetic acid concentration was 4.3 g / L.

[0099] Comparative Example 4

[0100] The only difference from Example 6 is that this comparative example adopts IPTG induction method for culture.

[0101] Prepare LB medium (per L):

[0102] Tryptone 10g, yeast extract 5g, sodium chloride 10g.

[0103] Cultivation process:

[0104] The BL21 (DE3) strain carrying the pET-28a-IFNα plasmid was picked from the plate and inoculated into LB medium containing antibiotics, and cultured at 37°C and 220 rpm for 12 hours. The bacterial suspension was then transferred to LB medium containing the corresponding resistance and cultured at 37°C and 220 rpm until the OD 600 =0.8, add IPTG to a final concentration of 0.1 mmol / L, and induce expression at 28°C for 12 hours. Collect the bacterial suspension, centrifuge the culture medium, remove the supernatant, collect the precipitated bacterial sludge, and weigh the cells. Proceed with subsequent lysis and purification steps and electrophoresis analysis as per standard procedures.

[0105] The results showed that the expression level of IFN-α protein was 1.8 g / L and the acetic acid concentration was 3.8 g / L.

[0106] The improved auto-induction culture medium and culture process provided by the present invention can achieve low-cost and efficient cell culture, increase protein expression, eliminate the need for artificial addition of inducers, reduce operational complexity, and achieve optimal growth and product synthesis conditions through feed control based on the dynamic feeding control method linked to pH and DO. The controllability is strong, and the culture medium material has environmentally friendly properties, which can reduce the discharge of organic waste liquid.

[0107] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0108] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A modified autoinduction medium, characterized in that The formulation of the improved autoinduction medium is as follows: Glucose 10 g / L, glycerol 3-5 g / L, lactose 3 g / L, yeast extract 24 g / L, casein hydrolysate 8 g / L, malonic acid 0.02-0.05 mM, methionine 1 mM, MgSO4 1-3 mM, phosphate buffer added to control pH 7.2; The improved autoinduction culture medium is prepared by mixing and sterilizing the components separately.

2. A culture process, characterized in that: The improved autoinduction medium according to claim 1 is used for culturing, and the steps of the culturing process are as follows: Single clones were picked from the plate and inoculated into LB medium containing antibiotics. The cells were shaken and cultured at 37°C and 220 rpm for 12-16 hours. 1%-5% of the cells were inoculated into the modified autoinduction medium at a volume ratio and cultured at 37°C for 18-24 hours. The cells were collected and tested for the expression of the target protein. The cells were cultured in the modified autoinduction medium and fed with dissolved oxygen linkage. The DO threshold was set. When DO>30%, glycerol was added at a rate of 0.5-1 mL / min. The improved auto-induction medium sets a pH threshold for culturing cells. When the pH exceeds 7.2, a mixed carbon source solution is automatically added to stabilize the pH at 6.8-7.2 through feeding. The proportions of the mixed carbon source solution are as follows: Glucose 200g / L, lactose 60g / L and MgSO4 5g / L.

3. A culture process according to claim 2, characterized in that, When the cells cultured in the culture process have the characteristic of expressing proteins at low temperature, the temperature of the culture in the improved auto-induction culture medium is lowered to 20-30°C.

4. A culture process according to claim 2, characterized in that, The specific growth rate of cells in the culture process is controlled at 0.15-0.25h-1.

5. A culture process according to claim 2, characterized in that, The acetate production coefficient of the cells in the culture process is <0.2 g / g DCW.

6. A culture process according to claim 2, characterized in that, During the induction phase of the culture process, the oxygen uptake rate is maintained at 20-25 mmol / L / h.

7. A culture process according to claim 2, characterized in that, When the culture process is used for culturing Escherichia coli, the carbon source supply rate is regulated by dissolved oxygen feedback.

8. A culture process according to claim 2, characterized in that, When used to culture CHO cells, the culture process induces gene expression through temperature control, and the glucose content feedback regulates the carbon source supply rate to maintain the glucose content at 0.5-1 g / L.

9. A culture process according to claim 2, characterized in that, The culture process, when used for culturing Pichia pastoris, induces gene expression through methanol, and regulates the carbon source supply rate through dissolved oxygen feedback to maintain the dissolved oxygen at 20%-30%.

10. A culture process according to claim 2, characterized in that: The protein expression amount of the culture process reaches 3.8g / L-10g / L.