CN115125224B - Method for producing DNA polymerase with activity in large scale - Google Patents

Method for producing DNA polymerase with activity in large scale Download PDF

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CN115125224B
CN115125224B CN202210959921.5A CN202210959921A CN115125224B CN 115125224 B CN115125224 B CN 115125224B CN 202210959921 A CN202210959921 A CN 202210959921A CN 115125224 B CN115125224 B CN 115125224B
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fermentation
culture
medium
dissolved oxygen
dna polymerase
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CN115125224A (en
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陈鲤群
高上
袁智涛
王涛
姜文倩
林峻
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Fuzhou University
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Fuzhou University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a method for producing DNA polymerase with activity in large scale. The method comprises the following steps: 1) Streaking a strain carrying a target gene on an LB plate with ampicillin resistance, and selecting a single colony for culture to be used as seed liquid; 2) Preparing a high-density fermentation medium, and adding the high-density fermentation medium into a fermentation tank; 3) Transferring the seed liquid into the fermentation tank in the step 2) for culturing; 4) When the dissolved oxygen is reduced to below 30%, starting to add a feed medium, and controlling the dissolved oxygen to be above 30%; 5) When culturing to 4g/L of the maximum bacterial wet weight distance, adding IPTG at one time to induce to make the final concentration of IPTG be 1+ -0.1 mM; 6) Stopping fermentation after the optimal induction time is reached, and placing the fermentation tank to obtain thalli. The invention improves the fermentation stability of the target product, reduces the generation of inclusion bodies, and can realize the large-scale production of DNA polymerase.

Description

Method for producing DNA polymerase with activity in large scale
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing DNA polymerase with activity in a large scale.
Technical Field
DNA polymerase is a core enzyme for nucleic acid amplification, and has strong thermal stability and polymerase activity. DNA polymerase is widely used in research and diagnosis, public health, food safety, agricultural production and other fields requiring rapid detection.
The DNA polymerase is a recombinase expressed by E.coli. The expression of the target molecule can be realized by using a triangular flask and a common LB culture medium, but the expression quantity of the target protein is unstable after large-scale fermentation production or inclusion bodies are formed to deactivate the target protein due to the complexity of an expression system and the dissimilarity of culture conditions after process amplification.
Disclosure of Invention
The invention aims to provide a method for producing active DNA polymerase in a large scale, which can realize that a target product of large-scale fermentation of the DNA polymerase is more stable and active target protein is produced.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for mass-producing an active DNA polymerase comprising the steps of:
1) Seed culture: streaking a strain which is preserved at-80 ℃ and carries a target gene on an LB plate with ampicillin resistance, picking a single colony, inoculating the single colony into a small shake flask for overnight culture, inoculating the single colony into a 2L triangular flask, and performing expanded culture to obtain seed liquid;
2) Preparing a culture medium: preparing a high-density fermentation medium, adding the high-density fermentation medium into a fermentation tank, sterilizing for 30min at 121 ℃, reducing the temperature of the medium to a required temperature, connecting an oxygen dissolving electrode, and calibrating the oxygen dissolving to be 100%;
3) Fermentation: transferring the seed liquid obtained in the step 1) into the culture medium of the step 2) for culturing at an inoculum size of 5+/-0.2%, wherein the liquid loading amount of a fermentation tank is 45% -55%, the dissolved oxygen is kept at 30% -50%, the tank pressure is 0-0.035Mpa, the fermentation temperature is 36-37 ℃, and the stirring rotating speed is 300-800rpm;
4) And (3) material supplementing: when the dissolved oxygen is reduced to below 30%, the feeding culture medium is started to be added, the dissolved oxygen is controlled to be above 30%, and the feeding speed meets the following conditions: the feed rate in mL/h was equal to 4 times the volume in L (e.g., 14L broth, 56 mL/h);
5) Induction: when the culture is carried out until the wet weight of the bacterial cells is 4g/L from the maximum wet weight of the bacterial cells, isopropyl-beta-D-thiopyran galactoside (IPTG) is added for induction at one time to ensure that the final concentration of the IPTG is 1+/-0.1 mM, and the feeding speed of a feeding culture medium is controlled to meet the following conditions: the feeding speed in mL/h is 10 times of the volume value of the fermentation liquor in L (for example, 14L of fermentation liquor, the feeding speed is 140 mL/h), and the dissolved oxygen is controlled to be more than 30%;
6) Stopping fermentation after reaching the optimal induction time (6 hours), and placing the fermentation tank to obtain thalli.
The strain in the step 1) takes escherichia coli ER2566 as a host and pET15b as a vector, and can be used for high-density fermentation of DNA polymerase.
The seed medium used in step 1) for the shake flask is as follows: 10g of tryptone, 5g of yeast extract and 5g of NaCl, fixing the volume to 1L, adjusting the pH to 7.0, and sterilizing for 30min at 121 ℃.
The fermentation medium is as follows: 177g of peptone, 354g of yeast powder, 59g of glycerol, 100g of glucose and KH 2 PO 4 39g、K 2 HPO 4 225g、Mg 2 SO 4 67g, to 14L.
The feed medium was as follows: 10g/L tryptone, 10g/L yeast powder and 5g/L glycerol
In the fermentation process, 30% ammonia water and 10% sulfuric acid are used for regulating the pH.
The beneficial effects of the invention are as follows: inclusion body formation is relatively complex, and is related to the rate of protein production within the cytoplasm, with higher concentrations of newly produced polypeptide and insufficient time to fold, thereby forming amorphous, amorphous protein aggregates; in addition, inclusion body formation is considered to be related to culture conditions of the host bacteria, such as medium composition, temperature, pH, ionic strength, and the like. Aiming at the fermentation production of the DNA polymerase, the invention reduces the nutrition components of the culture medium, and after changing some culture conditions, the purified DNA polymerase is found to have biological activity, probably because the culture medium and the culture conditions or the induction conditions are not the most suitable for the production of thalli, so that the thalli cannot be rapidly propagated, the proteins in cytoplasm cannot be produced in large quantity, and the conditions for forming inclusion bodies cannot be reached.
The invention improves the fermentation stability of the target product, reduces the generation of inclusion bodies, and can realize the large-scale production of DNA polymerase.
Drawings
FIG. 1 is a chart showing coomassie brilliant blue staining of bacterial cells expressed by the methods of the prior art and the present invention. Wherein, the marker is Spectra Multicolor High Range Protein Ladder of thermo, the goods number: #26625; the original method (prior art) refers to the fermentation method described in chinese patent application CN 110777100A.
FIG. 2 is an electrophoretogram of a protein purified by the prior art method and the method of the present invention. Wherein, the Marker is broad-spectrum rainbow pre-dyeing protein Marker of century, the goods number: CW2841M; the original method (prior art) refers to the fermentation method described in chinese patent application CN 110777100A.
FIG. 3 is an electrophoresis chart of enzyme activity detection. Wherein, the Marker is a DL500 DNA Marker of takara, the goods number: 3590Q, standard was purchased commercial DNA polymerase.
Detailed Description
The following media were prepared:
seed culture medium: 10g of tryptone, 5g of yeast extract and 5g of NaCl, fixing the volume to 1L, adjusting the pH to 7.0, and sterilizing for 30min at 121 ℃.
Fermentation medium: 177g of peptone, 354g of yeast powder, 59g of glycerol, 100g of glucose and KH 2 PO 4 39g、K 2 HPO 4 225g、Mg 2 SO 4 67g, to 14L.
Feed medium: 10g/L tryptone, 10g/L yeast powder and 5g/L glycerol
30% ammonia water and 10% sulfuric acid are prepared for regulating pH in the fermentation process.
Example 1
A method for mass-producing an active DNA polymerase comprising the steps of:
1) Seed culture: scribing strains (taking escherichia coli ER2566 as a host and pET15b as a carrier) which are preserved at the temperature of minus 80 ℃ and carry target genes on an LB plate with ampicillin resistance, picking single bacterial colonies, inoculating the single bacterial colonies into a small shake flask for overnight culture, inoculating the single bacterial colonies into a 2L triangular flask, and performing amplification culture to obtain seed liquid;
2) Preparing a culture medium: preparing a high-density fermentation medium, adding the high-density fermentation medium into a fermentation tank, sterilizing for 30min at 121 ℃, reducing the temperature of the medium to a required temperature, connecting an oxygen dissolving electrode, and calibrating the oxygen dissolving to be 100%;
3) Fermentation: transferring the seed liquid obtained in the step 1) into the culture medium of the step 2) for culture with the inoculum size of 5%, wherein the liquid loading amount of a fermentation tank is 50%, the dissolved oxygen is kept at 40%, the tank pressure is 0.02Mpa, the fermentation temperature is 36.5 ℃, and the stirring rotation speed is 500 rpm;
4) And (3) material supplementing: when the dissolved oxygen is reduced to below 30%, the feeding culture medium is started to be added, the dissolved oxygen is controlled to be above 30%, and the feeding speed meets the following conditions: the feed rate in mL/h was equal to 4 times the volume of the broth in L, in this example, 14L, so the feed rate was controlled to be 56 mL/h;
5) Induction: when the bacterial cells are cultured until the wet weight distance between the bacterial cells and the maximum wet weight of the bacterial cells is 4g/L, IPTG is added for induction at one time to ensure that the final concentration of the IPTG is 1 mM, and the feeding speed of the feeding culture medium is controlled to meet the following conditions: the value of the feeding speed in mL/h is equal to 10 times of the value of the volume of the fermentation broth in L, and in the embodiment, the volume of the fermentation broth is 14L, so that the feeding speed is controlled to be 140 mL/h, and the dissolved oxygen is controlled to be more than 30%;
6) Stopping fermentation after 6 hours of fermentation induction, and placing the fermentation tank to obtain thalli.
And (3) obtaining a bacterial precipitate after fermentation, crushing the bacterial precipitate by a cell crusher, centrifuging, taking a supernatant, and performing coomassie staining. Meanwhile, experiments were carried out by the method described in chinese patent application CN 110777100A for comparison.
FIG. 1 is a chart showing coomassie brilliant blue staining of bacterial cells expressed by the methods of the prior art and example 1. As can be seen from FIG. 1, the destination stripe size is 64kDa. The method of the invention has no obvious difference in the expression of the target protein from the prior art method.
FIG. 2 is an electrophoretogram of a protein purified by the prior art method and the method of the present invention. After protein purification, enzyme activity detection was performed. The electrophoresis pattern of the enzyme activity detection is shown in FIG. 3, and the result shows that the band is provided, which indicates that the amplification enzyme of the method of the invention has activity.
Example 2
A method for mass-producing an active DNA polymerase comprising the steps of:
1) Seed culture: scribing strains (taking escherichia coli ER2566 as a host and pET15b as a carrier) which are preserved at the temperature of minus 80 ℃ and carry target genes on an LB plate with ampicillin resistance, picking single bacterial colonies, inoculating the single bacterial colonies into a small shake flask for overnight culture, inoculating the single bacterial colonies into a 2L triangular flask, and performing amplification culture to obtain seed liquid;
2) Preparing a culture medium: preparing a high-density fermentation medium, adding the high-density fermentation medium into a fermentation tank, sterilizing for 30min at 121 ℃, reducing the temperature of the medium to a required temperature, connecting an oxygen dissolving electrode, and calibrating the oxygen dissolving to be 100%;
3) Fermentation: transferring the seed liquid obtained in the step 1) into the culture medium of the step 2) for culturing at an inoculation amount of 4.8%, wherein the liquid loading amount of a fermentation tank is 45%, the dissolved oxygen is kept to be 30%, the tank pressure is 0Mpa, the fermentation temperature is 36 ℃, and the stirring rotation speed is 800rpm;
4) And (3) material supplementing: when the dissolved oxygen is reduced to below 30%, the feeding culture medium is started to be added, the dissolved oxygen is controlled to be above 30%, and the feeding speed meets the following conditions: the feed rate in mL/h was equal to 4 times the volume of the broth in L, in this example, 14L, so the feed rate was controlled to be 56 mL/h;
5) Induction: when the bacterial cells are cultured until the wet weight distance between the bacterial cells and the maximum wet weight of the bacterial cells is 4g/L, IPTG is added for induction at one time to ensure that the final concentration of the IPTG is 1 mM, and the feeding speed of the feeding culture medium is controlled to meet the following conditions: the feed rate in mL/h was 10 times the volume of the broth in L, in this example, 14L, so the feed rate was controlled to 140 mL/h; simultaneously controlling the dissolved oxygen to be more than 30 percent;
6) Stopping fermentation after induction for 6 hours, and placing the fermentation tank to obtain thalli.
Example 3
A method for mass-producing an active DNA polymerase comprising the steps of:
1) Seed culture: scribing strains (taking escherichia coli ER2566 as a host and pET15b as a carrier) which are preserved at the temperature of minus 80 ℃ and carry target genes on an LB plate with ampicillin resistance, picking single bacterial colonies, inoculating the single bacterial colonies into a small shake flask for overnight culture, inoculating the single bacterial colonies into a 2L triangular flask, and performing amplification culture to obtain seed liquid;
2) Preparing a culture medium: preparing a high-density fermentation medium, adding the high-density fermentation medium into a fermentation tank, sterilizing for 30min at 121 ℃, reducing the temperature of the medium to a required temperature, connecting an oxygen dissolving electrode, and calibrating the oxygen dissolving to be 100%;
3) Fermentation: transferring the seed liquid obtained in the step 1) into the culture medium of the step 2) for culturing at an inoculation amount of 5+/-0.2%, wherein the liquid loading amount of a fermentation tank is 55%, the dissolved oxygen is kept to be 50%, the tank pressure is 0.035Mpa, the fermentation temperature is 37 ℃, and the stirring rotation speed is 300 rpm;
4) And (3) material supplementing: when the dissolved oxygen is reduced to below 30%, the feeding culture medium is started to be added, the dissolved oxygen is controlled to be above 30%, and the feeding speed meets the following conditions: the feed rate in mL/h was equal to 4 times the volume of the broth in L, in this example, 14L, so the feed rate was controlled to be 56 mL/h;
5) Induction: when the bacterial cells are cultured until the wet weight distance between the bacterial cells and the maximum wet weight of the bacterial cells is 4g/L, IPTG is added for induction at one time to ensure that the final concentration of the IPTG is 1+/-0.1 and mM, and the feeding speed of the feeding culture medium is controlled to meet the following conditions: the feed rate in mL/h was 10 times the volume of the broth in L, in this example, 14L, so the feed rate was controlled to be 56 mL/h; simultaneously controlling the dissolved oxygen to be more than 30 percent;
6) Stopping fermentation after induction for 6 hours, and placing the fermentation tank to obtain thalli.

Claims (2)

1. The method for improving the content of active DNA polymerase in the genetically engineered bacteria is characterized by comprising the following steps:
1) Seed culture: streaking a strain carrying a target gene on an LB plate with ampicillin resistance, picking a single colony, inoculating the single colony into a shake flask for overnight culture, inoculating the single colony into a triangular flask, and performing expansion culture to obtain seed liquid;
the strain takes escherichia coli ER2566 as a host and pET15b as a vector;
2) Preparing a culture medium: preparing a high-density fermentation medium, adding the high-density fermentation medium into a fermentation tank, sterilizing, reducing the temperature of the medium to a required temperature, connecting an oxygen dissolving electrode, and calibrating the oxygen dissolving to be 100%;
the high densityThe preparation method of the fermentation medium comprises the following steps: taking 177g of peptone, 354g of yeast powder, 59g of glycerol, 100g of glucose and KH 2 PO 4 39g、K 2 HPO 4 225g、Mg 2 SO 4 67g, constant volume to 14L, sterilizing at 121 ℃ for 30 min;
3) Fermentation: transferring the seed liquid obtained in the step 1) into the culture medium of the step 2) for culture at an inoculum size of 5+/-0.2%, wherein the liquid loading amount of a fermentation tank is 45% -55%, and the culture conditions are as follows: maintaining dissolved oxygen at 30% -50%, tank pressure at 0-0.035Mpa, fermentation temperature at 36-37deg.C, stirring speed at 300-800rpm;
4) And (3) material supplementing: when the dissolved oxygen is reduced to below 30%, the feeding culture medium is started to be added, the dissolved oxygen is controlled to be above 30%, and the feeding speed of the feeding culture medium meets the following conditions: the value of the feeding speed in mL/h is equal to 4 times of the value of the volume of the fermentation liquor in L;
the feed medium comprises the following components in percentage by weight: 10g/L of tryptone, 10g/L of yeast powder and 5g/L of glycerol;
5) Induction: when the bacterial cells are cultured until the wet weight distance between the bacterial cells and the maximum wet weight of the bacterial cells is 4g/L, IPTG is added for induction at one time to ensure that the final concentration of the IPTG is 1+/-0.1 and mM, and the feeding speed of the feeding culture medium is controlled to meet the following conditions: the value of the feeding speed in ml/h is equal to 10 times of the value of the volume of the fermentation liquor in L, and the dissolved oxygen is controlled to be more than 30 percent;
6) Stopping fermentation after induction for 6 hours, and placing the fermentation tank to obtain thalli.
2. The method for increasing the content of active DNA polymerase in genetically engineered bacteria according to claim 1, wherein the seed culture medium used in the shake flask in step 1) is prepared by the following method: taking 10g of tryptone, 5g of yeast extract and 5g of NaCl, fixing the volume to 1L, adjusting the pH to 7.0, and sterilizing for 30min at 121 ℃.
CN202210959921.5A 2022-08-11 2022-08-11 Method for producing DNA polymerase with activity in large scale Active CN115125224B (en)

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UA80809C2 (en) * 2001-11-05 2007-11-12 Process for preparation of protective antigen protein of anthracis with e.coli
CN102002517B (en) * 2010-09-29 2012-11-28 北京正旦国际科技有限责任公司 Fermentation medium for preparing recombination IL-1ra (Interleukin-1 Receptor Antagonist) and fermentation method thereof
EP2708598A1 (en) * 2012-09-14 2014-03-19 Basf Se Serinol production in glycerol catabolism deficient escherichia coli strains
CN104560845A (en) * 2013-10-29 2015-04-29 天津强微特生物科技有限公司 Method for establishing recombinant bacillus subtilis for expressing thermophilic xylanase
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