CN114672531A - Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control - Google Patents
Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control Download PDFInfo
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Abstract
The invention discloses a method for improving the expression quantity of escherichia coli protein through stage dissolved oxygen control; the method comprises the following steps: the method comprises the following steps of strain activation, seed culture and fermentation tank fermentation, wherein the fermentation tank fermentation is divided into an initial culture stage, a feed supplement growth stage and an induction stage; wherein the induction phase is divided into a first phase induction and a second phase induction; the first stage of induction controls the dissolved oxygen to be more than 25 percent, and the second stage of induction controls the dissolved oxygen to be 0 to 10 percent. The invention divides the induction into two stages and controls the culture conditions of each stage, so that the escherichia coli genetic engineering bacteria can realize high-density fermentation. In the first stage of induction, the fermentation tank is in the maximum oxygen supply state, so that the escherichia coli is in a high metabolism state, and the rapid expression of the protein is realized; during the second stage of induction, the oxygen supply state of the fermentation tank is reduced, and then the supplement amounts of a carbon source and a nitrogen source are reduced, so that the thallus is in a lower metabolic state, the aging and autolysis of the thallus can be slowed down, the vitality of the thallus is maintained, and the further expression of the protein is realized.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving the expression quantity of escherichia coli protein through stage dissolved oxygen control.
Background
In recent years, due to the wide application of recombinant DNA technology and the improvement of the market demands for drugs, health care products and the like produced by the recombinant DNA technology, the combination process of the recombinant DNA technology and the large-scale culture technology is accelerated, so that natural proteins which cannot be obtained in large quantities originally, particularly genetic engineering drugs, can be produced in large quantities, and the recombinant DNA technology can be applied to clinic. The high-density fermentation technology is adopted to improve the fermentation density of the thalli and finally improve the protein expression, so that the culture volume can be reduced, the downstream separation and extraction can be enhanced, the production period can be shortened, the equipment investment can be reduced, the production cost can be reduced, and the competitiveness of the product in the market can be greatly improved. Therefore, it is an important direction for the development of fermentation industry in recent years.
The dissolved oxygen concentration is an important indicator of the growth of the bacteria. Escherichia coli needs a large amount of oxygen to metabolize in the growth and reproduction process, the demand for oxygen is increased along with the increase of the density of the thalli, the oxygen consumption of the thalli is extremely high in the later period of high-density fermentation, and the growth and expression of the thalli are seriously influenced if insufficient oxygen supply is realized at the moment. Therefore, the supply of oxygen is very important. However, different species of microorganisms have different oxygen demands. The oxygen demand of the same microorganism also varies with the age of the microorganism and the culture conditions. Oxygen consumption during cell growth and product synthesis are also different.
The control measures of the dissolved oxygen concentration mainly include stirring speed, tank pressure change and ventilation. However, the ventilation amount cannot be increased infinitely, and when the ventilation amount is too large, the foam in the fermentation tank is increased, and the utilization rate of the fermentation tank is reduced. The oxygen partial pressure can be increased or decreased by changing the tank pressure, thereby achieving the purpose of changing the dissolved oxygen concentration.
At present, the high-density fermentation process is generally in a state of relatively sufficient oxygen supply in an induction stage, and the dissolved oxygen is generally maintained at more than 20 percent, so that escherichia coli is in a relatively high metabolic state, and the rapid expression of an inclusion body can be realized. However, in the later stage of induction, bacterial aging and autolysis are easy to occur, the activity of the bacterial is reduced, and protein degradation occurs, so that the further improvement of the protein expression level is limited.
Disclosure of Invention
The invention provides a method for improving the expression quantity of escherichia coli protein through stage dissolved oxygen control, and the fermentation process can obviously improve the protein expression quantity of genetically engineered bacteria through the stage dissolved oxygen control method.
The specific technical scheme is as follows:
a method for improving the expression level of Escherichia coli protein by means of staged dissolved oxygen control, which comprises the following steps: the method comprises the following steps of strain activation, seed culture and fermentation of a fermentation tank, and is characterized in that the fermentation of the fermentation tank is divided into an initial culture stage, a feed supplement growth stage and an induction stage, wherein the induction stage is divided into a first stage induction and a second stage induction; controlling the dissolved oxygen at more than 25% by the first stage induction, and controlling the dissolved oxygen at more than 0-10% by the second stage induction.
Preferably, the initial culture phase, the feed growth phase and the first phase induce dissolved oxygen to be controlled to be more than 25%, and the second phase induces dissolved oxygen to be controlled to be 0-10%.
The method for improving the expression quantity of the escherichia coli protein through the stage dissolved oxygen control specifically comprises the following steps:
(1) an initial culture stage: inoculating the strain to a fermentation culture medium, wherein the pH is 6.6-6.8, the dissolved oxygen is 25% -100%, the dissolved oxygen naturally decreases along with the growth of the strain, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation, pressure and the like when the dissolved oxygen decreases to 25%;
(2) and (3) a feed supplement growth stage: feeding a carbon source in a flowing manner when the pH naturally rises to 6.9, and feeding a nitrogen source in a flowing manner when the dissolved oxygen naturally rises to 60%; then, the pH is 6.8-7.0, and the dissolved oxygen is 25% -50%;
(3) the first stage of induction: when the concentration of the thalli reaches the maximum value, the induction enters a first stage, the inducer is IPTG, the pH value is 7.0-7.2, and the dissolved oxygen is 25% -50%;
(4) and (3) induction in the second stage: the pH is 7.0-7.2, the dissolved oxygen is 0% -10%, and the feeding is reduced to be near the initial feeding.
And in the second stage, the oxygen supply state of the fermentation tank is reduced by reducing parameters such as rotating speed, ventilation, pressure and the like, so that the supplement amount of the carbon source and the nitrogen source is reduced, and the dissolved oxygen is kept at 0-10%.
The initial culture stage is an adaptation period of inoculating strains into a fermentation tank, and the primary purpose of the initial culture stage is to enable the strains to adapt to the environment of the fermentation tank; the feeding growth phase is the logarithmic growth phase of the thalli, and the feeding culture medium is fed according to the dissolved oxygen flow in the phase, so that the genetic engineering bacteria are rapidly proliferated at a certain specific growth rate, and high-density fermentation is realized; in the first stage of induction, the fermentation tank is in the maximum oxygen supply state, so that the escherichia coli is in a higher metabolic state, and the rapid expression of the inclusion body can be realized; during the second stage of induction, the oxygen supply state of the fermentation tank is reduced by reducing parameters such as rotating speed, ventilation, pressure and the like, and then the supplement amounts of a carbon source and a nitrogen source are reduced, so that the thallus is in a lower metabolic state, the aging and autolysis of the thallus can be slowed down, the vitality of the thallus is maintained, and the further expression of the inclusion body is realized.
Wherein the fermentation medium is: 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1.0g/L of magnesium sulfate.
Preferably, the carbon source is fed with glucose; the preferred concentration of glucose is 300 g/L.
Preferably, the nitrogen source feed is yeast extract powder; the preferred concentration of yeast extract powder is 200 g/L.
Preferably, the fermentation temperature is 30-37 ℃.
Preferably, in the step (1), the inoculation amount of the strain is 2-10% by volume of the fermentation medium.
Preferably, in the step (1), the fermentation culture time is 3-7 h.
Preferably, in the step (2), the fermentation culture time is 7-15 h.
Preferably, in the step (3), the fermentation culture time is 5-13 h.
Preferably, in the step (4), the fermentation culture time is 1-7 h.
Preferably, the seed culture process comprises a primary seed culture process and a secondary seed culture process; the primary seed culture temperature is 30 ℃, and the time is 12-16 h; the culture temperature of the secondary seeds is 37 ℃, and the culture time is 3-6 h.
Preferably, the culture medium of the primary seeds and the secondary seeds is 10g/L of sodium chloride, 5g/L of yeast extract powder and 10g/L of peptone.
Specifically, the invention takes two strains as an example, namely a genetically engineered strain A and a genetically engineered strain B.
Compared with the prior art, the invention has the following advantages:
(1) the process of the invention divides the fermentation into four stages, and controls the culture conditions of each stage, thus realizing the high-density fermentation of the genetic engineering bacteria;
(2) the process can realize the rapid proliferation of the thalli through the feed supplement growth stage;
(3) the process can realize the rapid expression of the inclusion body through the first stage induction;
(4) the process can greatly improve the expression quantity of inclusion bodies in the genetic engineering bacteria through the second stage induction;
(5) the second stage of the process of the invention induces and reduces ventilation, rotating speed, pressure, feeding and the like, so that the thalli can be in a lower metabolic state, the aging and autolysis of the thalli are slowed down, and the vitality of the thalli is maintained;
(6) the second stage of the process of the invention induces and reduces ventilation, rotating speed, pressure, feeding and the like, can reduce the consumption of water, electricity, gas and materials, and is beneficial to energy conservation and emission reduction.
Drawings
FIG. 1 is a change curve of dissolved oxygen in the fermentation process of example 1;
FIG. 2 is a change curve of dissolved oxygen in the fermentation process of example 2;
FIG. 3 is a change curve of dissolved oxygen in the fermentation process of example 3;
FIG. 4 is a change curve of dissolved oxygen in the fermentation process of example 4;
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.
The genetically engineered bacterium A is E.coli BL21(DE3), and the recombinant protein is liraglutide;
coli BL21(DE3), and the recombinant protein is human proinsulin.
Example 1 fermentation of 30L of genetically engineered bacteria A, comprising only the first stage induction;
1) first-order seed culture: inoculating the strain into a primary seed culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 12-16 h to obtain a primary seed solution; the primary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
2) Secondary seed culture: inoculating the primary seeds into a secondary seed culture medium, and performing shake culture at 37 ℃ and 250rpm for 3-6 hours to obtain a secondary seed solution; the secondary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
3)30L fermentation
a. An initial culture stage: preparing 12L fermentation culture medium, sterilizing at 121 deg.C for 20min, and cooling to 30 deg.C; adjusting the pH value to 6.7, inoculating 1.2L of secondary seeds into a fermentation tank, wherein the dissolved oxygen is 25-100%, the dissolved oxygen naturally decreases along with the growth of thalli, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation, pressure and the like when the dissolved oxygen decreases to 25%;
the fermentation medium comprises 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1.0g/L of magnesium sulfate.
b. And (3) a feed supplement growth stage: feeding glucose by using a carbon source when the pH naturally rises to 6.9, and feeding yeast extract by using a nitrogen source when the dissolved oxygen naturally rises to 60%; then, the temperature is 30 ℃, the pH is 6.8-7.0, and the dissolved oxygen is 25% -50%.
The whole feeding process is mainly based on dissolved oxygen regulation, if the dissolved oxygen is low, parameters such as rotating speed, ventilation, pressure and the like are improved, and the dissolved oxygen is maintained at 25-50%; if the dissolved oxygen is higher, the material supplement amount is increased, and the dissolved oxygen is maintained at 25-50%; and paying attention to pH change in the whole feeding growth stage, maintaining the pH to be stable as much as possible, and adjusting the pH by feeding a carbon-nitrogen ratio as much as possible if the pH fluctuates.
The feed medium is 200g/L of yeast extract powder and 300g/L of glucose.
c. The first stage induction: after the materials are supplemented for 11 hours, the induction is carried out in the first stage, the induction is carried out by adding an inducer IPTG with the final concentration of 0.5M, the temperature is 30 ℃, the pH value is 7.0-7.2, and the dissolved oxygen is 25-50%. The maximum oxygen supply capacity of the device is maintained at this stage, the vigorous metabolism of the thalli is ensured, and the inclusion bodies are quickly expressed. Course sampling induced 7h, 8h, 9h samples.
4) Crushing of thallus
And (3) resuspending the samples for 7h, 8h and 9h by using a buffer solution for 1h, crushing the samples for 2 times by using a homogenizer at 900bar and 40Hz after uniform resuspension, centrifugally collecting the pre-washed inclusion bodies, then washing the inclusion bodies for 1h by using the buffer solution through resuspension, and collecting the washed inclusion bodies.
5) Denaturation of inclusion bodies
And (4) washing the inclusion bodies after the induction for 7h, 8h and 9h in the first stage, performing overnight denaturation, and determining the protein amount. The protein amount of the fermentation liquor is 10.92g/L after 7h of induction, the protein amount of the fermentation liquor is 13.18g/L after 8h of induction, and the protein amount of the fermentation liquor is 12.10g/L after 9h of induction. It is known that the amount of the protein in the first stage induction time of 9h is less than 8h, i.e. the protein is degraded in the later stage of the first stage induction.
Example 2 fermentation of genetically engineered bacterium a for 30L, comprising a first stage induction and a second stage induction;
1) first-order seed culture: inoculating the strain into a primary seed culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 12-16 h to obtain a primary seed solution; the primary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
2) Secondary seed culture: inoculating the primary seeds into a secondary seed culture medium, and performing shake culture at 37 ℃ and 250rpm for 3-6 h to obtain a secondary seed solution; the secondary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
3)30L fermentation
a. An initial culture stage: preparing 12L fermentation culture medium, sterilizing at 121 deg.C for 20min, and cooling to 30 deg.C; adjusting the pH value to 6.7, inoculating 1.2L of secondary seeds into a fermentation tank, wherein the dissolved oxygen is 25-100%, the dissolved oxygen naturally decreases along with the growth of thalli, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation, pressure and the like when the dissolved oxygen decreases to 25%;
the fermentation medium comprises 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1g/L of magnesium sulfate.
b. And (3) a feed supplement growth stage: feeding glucose by using a carbon source when the pH naturally rises to 6.9, and feeding yeast extract by using a nitrogen source when the dissolved oxygen naturally rises to 60%; then, the temperature is 30 ℃, the pH value is 6.8-7.0, and the dissolved oxygen is 25% -50%.
The whole feeding process is mainly based on dissolved oxygen regulation, if the dissolved oxygen is low, parameters such as rotating speed, ventilation, pressure and the like are improved, and the dissolved oxygen is maintained at 25-50%; if the dissolved oxygen is higher, the material supplementing amount is increased, and the dissolved oxygen is maintained at 25-50%; and paying attention to pH change in the whole feeding growth stage, maintaining the pH to be stable as much as possible, and adjusting the pH by feeding a carbon-nitrogen ratio as much as possible if the pH fluctuates.
The feed medium is 200g/L of yeast extract powder and 300g/L of glucose.
c. The first stage of induction: after the materials are supplemented for 11 hours, the induction is carried out in the first stage, the induction is carried out by adding an inducer IPTG with the final concentration of 0.5M, the temperature is 30 ℃, the pH value is 7.0-7.2, and the dissolved oxygen is 25-50%. The maximum oxygen supply capacity of the device is maintained at this stage, the vigorous metabolism of the thalli is ensured, and the inclusion bodies are quickly expressed.
d. And (3) induction in the second stage: the first stage is induced for 9 hours and then the second stage is induced, the temperature is 30 ℃, the pH is 7.0-7.2, and the dissolved oxygen is 0-10%. The oxygen supply state of the fermentation tank is reduced by reducing parameters such as rotating speed, ventilation, pressure and the like, so that the supplement amount of a carbon source and a nitrogen source is reduced, and the supplement amount is reduced to be close to the initial supplement value in the supplement growth stage, so that the thallus is in a lower metabolic state, the aging and autolysis of the thallus can be slowed down, the vitality of the thallus is maintained, and the further expression of the inclusion body is realized. The process samples were taken and the second stage induced samples for 0h, 2h, 4 h.
4) Crushing of thallus
And (3) resuspending the sample for 0h, 2h and 4h in the second stage by using a buffer solution for 1h, crushing the sample for 2 times by using a homogenizer at 900bar and 40Hz after uniform resuspension, centrifugally collecting the inclusion bodies before washing, then washing the inclusion bodies for 1h by using the buffer solution for resuspension, and collecting the washed inclusion bodies.
5) Denaturation of inclusion bodies
And (3) washing the samples of 0h, 2h and 4h after induction in the second stage, carrying out overnight denaturation on the inclusion bodies, and determining the protein amount. The protein amount of the second stage induction 0h is 12.70g/L fermentation liquor, the protein amount of the second stage induction 2h is 16.69g/L fermentation liquor, and the protein amount of the second stage induction 4h is 15.62g/L fermentation liquor. It can be seen that the amount of 2h protein induced in the second stage is higher than 0h, i.e. the protein will continue to be expressed after the second stage induction, which exceeds 0h by about 31.42%.
Example 3 fermentation of genetically engineered bacterium B, containing only the first stage induction;
1) first-order seed culture: inoculating the strain into a primary seed culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 12-16 h to obtain a primary seed solution; the primary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
2) Secondary seed culture: inoculating the primary seeds into a secondary seed culture medium, and performing shake culture at 37 ℃ and 250rpm for 3-6 hours to obtain a secondary seed solution; the secondary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
3)30L fermentation
a. An initial culture stage: preparing 12L fermentation culture medium, sterilizing at 121 deg.C for 20min, and cooling to 30 deg.C; adjusting the pH value to 6.7, inoculating 1.2L of secondary seeds into a fermentation tank, wherein the dissolved oxygen is 25-100%, the dissolved oxygen naturally decreases along with the growth of thalli, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation, pressure and the like when the dissolved oxygen decreases to 25%;
the fermentation medium comprises 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1g/L of magnesium sulfate.
b. And (3) a feed supplement growth stage: feeding glucose by using a carbon source when the pH naturally rises to 6.9, and feeding yeast extract by using a nitrogen source when the dissolved oxygen naturally rises to 60%; then, the temperature is 30 ℃, the pH is 6.8-7.0, and the dissolved oxygen is 25% -50%.
The whole feeding process is mainly based on dissolved oxygen regulation, if the dissolved oxygen is low, parameters such as rotating speed, ventilation, pressure and the like are improved, and the dissolved oxygen is maintained at 25-50%; if the dissolved oxygen is higher, the material supplementing amount is increased, and the dissolved oxygen is maintained at 25-50%; and paying attention to pH change in the whole feeding growth stage, maintaining the pH stable as much as possible, and adjusting the pH by feeding the carbon-nitrogen ratio as much as possible if the pH fluctuates.
The feed medium is 200g/L of yeast extract powder and 300g/L of glucose.
c. The first stage of induction: feeding the materials for 8.5h, then performing first-stage induction, adding an inducer IPTG with the final concentration of 0.5M, and inducing at the temperature of 30 ℃, the pH value of 7.0-7.2 and the dissolved oxygen content of 25-50%. The maximum oxygen supply capacity of the device is maintained at this stage, the vigorous metabolism of the thalli is ensured, and the inclusion bodies are quickly expressed. The process sampling induced 5h, 6h samples.
4) Crushing of thallus
And (3) inducing the sample bacteria for 5h and 6h in the first stage to be resuspended for 1h by using a buffer solution, crushing for 2 times by using a homogenizer at 850bar and 40Hz after the resuspension is uniform, centrifugally collecting the inclusion bodies before washing, then resuspending and washing the inclusion bodies for 1h by using the buffer solution, and collecting the washed inclusion bodies.
5) Denaturation of inclusion bodies
After the samples of 5h and 6h of induction in the first stage are washed, the inclusion bodies are denatured overnight, and the amount of protein is measured. The first stage induces 6.76g/L fermentation liquor of 5h protein, and induces 5.55g/L fermentation liquor of 6h protein, which shows that the induced 5h protein is higher, and the protein can be degraded along with the extension of the induction time.
Example 4 fermentation of genetically engineered bacterium B for 30L, comprising a first stage induction and a second stage induction;
1) first-order seed culture: inoculating the strain into a primary seed culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 12-16 h to obtain a primary seed solution; the primary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 deg.C for 20 min.
2) Secondary seed culture: inoculating the primary seeds into a secondary seed culture medium, and performing shake culture at 37 ℃ and 250rpm for 3-6 h to obtain a secondary seed solution; the secondary seed culture medium is LB culture medium, sodium chloride 10g/L, yeast extract powder 5g/L, peptone 10g/L, sterilizing at 121 ℃ for 20 min.
3)30L fermentation
a. An initial culture stage: preparing 12L fermentation culture medium, sterilizing at 121 deg.C for 20min, and cooling to 30 deg.C; adjusting the pH value to 6.7, inoculating 1.2L of secondary seeds into a fermentation tank, wherein the dissolved oxygen is 25-100%, the dissolved oxygen naturally decreases along with the growth of thalli, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation, pressure and the like when the dissolved oxygen decreases to 25%;
the fermentation medium comprises 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1g/L of magnesium sulfate.
b. And (3) a feed supplement growth stage: feeding glucose by using a carbon source when the pH naturally rises to 6.9, and feeding yeast extract by using a nitrogen source when the dissolved oxygen naturally rises to 60%; then, the temperature is 30 ℃, the pH is 6.8-7.0, and the dissolved oxygen is 25% -50%.
The whole feeding process is mainly based on dissolved oxygen regulation, if the dissolved oxygen is low, parameters such as rotating speed, ventilation, pressure and the like are improved, and the dissolved oxygen is maintained at 25-50%; if the dissolved oxygen is higher, the material supplementing amount is increased, and the dissolved oxygen is maintained at 25-50%; and paying attention to pH change in the whole feeding growth stage, maintaining the pH to be stable as much as possible, and adjusting the pH by feeding a carbon-nitrogen ratio as much as possible if the pH fluctuates.
The feed medium is 200g/L of yeast extract powder and 300g/L of glucose.
c. The first stage of induction: feeding the materials for 9h, then performing first-stage induction, adding an inducer IPTG with the final concentration of 0.5M for induction, wherein the temperature is 30 ℃, the pH value is 7.0-7.2, and the dissolved oxygen is 25-50%. The maximum oxygen supply capacity of the device is maintained at this stage, the vigorous metabolism of the thalli is ensured, and the inclusion bodies are quickly expressed.
d. And (3) induction in the second stage: the first stage is induced for 5 hours and then the second stage is induced, the temperature is 30 ℃, the pH is 7.0-7.2, and the dissolved oxygen is 0-10%. The oxygen supply state of the fermentation tank is reduced by reducing parameters such as rotating speed, ventilation, pressure and the like, so that the supplement amount of a carbon source and a nitrogen source is reduced, and the supplement amount is reduced to be close to the initial supplement value in the supplement growth stage, so that the thallus is in a lower metabolic state, the aging and autolysis of the thallus can be slowed down, the vitality of the thallus is maintained, and the further expression of the inclusion body is realized. Process sampling the second stage induced samples for 0h, 2h, 4h, 7 h.
4) Crushing of thallus
And (3) resuspending the sample for 0h, 2h, 4h and 7h in buffer solution for 1h, crushing the sample for 2 times at 850bar and 40Hz by a homogenizer after uniform resuspension, centrifugally collecting the inclusion bodies before washing, then washing the inclusion bodies for 1h by the buffer solution through resuspension, and collecting the washed inclusion bodies.
5) Denaturation of inclusion bodies
And (3) washing the samples of 0h, 2h, 4h and 7h of induction in the second stage, carrying out overnight denaturation on the inclusion bodies, and determining the protein amount. In the second stage, the protein content of 0h is induced to be 6.32g/L of fermentation liquor, the protein content of 2h is induced to be 7.03g/L of fermentation liquor, the protein content of 4h is induced to be 7.95g/L of fermentation liquor, and the protein content of 7h is induced to be 7.10g/L of fermentation liquor. It can be seen that the amount of the protein induced for 4h in the second stage is higher than 0h, i.e. the protein will continue to be expressed after the second stage induction, which exceeds 0h by about 25.79%.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and all equivalent substitutions or substitutions made on the above-mentioned embodiments are included in the scope of the present invention.
Claims (10)
1. A method for improving the expression level of Escherichia coli protein by means of staged dissolved oxygen control, which comprises the following steps: the method comprises the following steps of strain activation, seed culture and fermentation tank fermentation, and is characterized in that the fermentation tank fermentation is divided into an initial culture stage, a feed supplement growth stage and an induction stage, wherein the induction stage is divided into a first stage induction and a second stage induction; controlling the dissolved oxygen to be more than 25% in the first stage of induction, and controlling the dissolved oxygen to be 0-10% in the second stage of induction.
2. The method for increasing the expression level of E.coli protein through staged dissolved oxygen control as claimed in claim 1, wherein the initial culture stage, the feeding growth stage and the first stage induce dissolved oxygen to be controlled at 25% or more, and the second stage induce dissolved oxygen to be controlled at 0-10%.
3. The method for controlling and improving the expression level of the Escherichia coli protein through the staged dissolved oxygen according to claim 2, comprising the following steps:
(1) an initial culture stage: inoculating the strain to a fermentation culture medium, wherein the pH is 6.6-6.8, the dissolved oxygen is 25% -100%, the dissolved oxygen naturally decreases along with the growth of the strain, and the dissolved oxygen is maintained to be more than 25% by adjusting parameters such as rotating speed, ventilation and pressure when the dissolved oxygen decreases to 25%;
(2) and (3) a feed supplement growth stage: feeding a carbon source in a flowing mode when the pH value naturally rises to 6.9, and feeding a nitrogen source in a flowing mode when the dissolved oxygen naturally rises to 60%; then, the pH is 6.8-7.0, and the dissolved oxygen is 25% -50%;
(3) the first stage of induction: when the concentration of the bacteria reaches the maximum value, the induction enters a first stage, an inducer is IPTG, the pH value is 7.0-7.2, and the dissolved oxygen is 25% -50%;
(4) and a second stage of induction: the pH is 7.0-7.2, the dissolved oxygen is 0-10%, and the feeding is reduced to the initial feeding.
4. The method for increasing the expression level of Escherichia coli protein by means of staged dissolved oxygen control according to claim 3, wherein the fermentation medium is: 10g/L of glucose, 8g/L of peptone, 12g/L of yeast extract powder, 17g/L of disodium hydrogen phosphate dodecahydrate, 3g/L of potassium dihydrogen phosphate, 0.5g/L of sodium chloride, 1.0g/L of ammonium chloride and 1.0g/L of magnesium sulfate.
5. The method for controlling and improving the expression level of Escherichia coli protein according to claim 3, wherein the carbon source is fed with glucose; the preferred concentration of glucose is 300 g/L; the nitrogen source supplement is yeast extract powder; the preferred concentration of yeast extract powder is 200 g/L.
6. The method for controlling and increasing the expression level of Escherichia coli protein through staged dissolved oxygen according to claim 3, wherein the fermentation temperature is 30-37 ℃.
7. The method for increasing the expression level of Escherichia coli protein through staged dissolved oxygen control as claimed in claim 3 or 6, wherein in step (1), the inoculum size of the strain is 2-10%.
8. The method for improving the expression level of the Escherichia coli protein through the staged dissolved oxygen control according to claim 3 or 7, wherein in the step (1), the fermentation culture time is 3-7 h; preferably, in the step (2), the fermentation culture time is 7-15 h; preferably, in the step (3), the fermentation culture time is 5-13 h; preferably, in the step (4), the fermentation culture time is 1-7 h.
9. The method for improving the expression level of Escherichia coli protein by means of staged dissolved oxygen control according to any one of claims 1 to 3, wherein the seed culture process comprises a primary seed culture process and a secondary seed culture process; the culture temperature of the first-stage seeds is 30 ℃ and the culture time is 12-16 h, and the culture temperature of the second-stage seeds is 37 ℃ and the culture time is 3-6 h.
10. The method for controlling and improving the expression level of Escherichia coli protein through staged dissolved oxygen as claimed in claim 9, wherein the culture medium of the primary seed and the secondary seed is 10g/L sodium chloride, 5g/L yeast extract powder and 10g/L peptone.
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