CN112852703A - Culture medium for improving expression quantity of recombinant protein in bacillus subtilis, preparation method and application - Google Patents
Culture medium for improving expression quantity of recombinant protein in bacillus subtilis, preparation method and application Download PDFInfo
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Abstract
The invention provides a culture medium for improving the expression quantity of recombinant protein in bacillus subtilis, a preparation method and application. In each 1000mL system of the culture medium, the composition of the culture medium comprises: 25-35g of corn soaking powder, 36-48g of bean pulp extract, 40-60g of maltose, 4-6g of potassium chloride, 3-5g of ammonium sulfate, 0.075-0.125g of manganese sulfate, 0.075-0.125g of calcium chloride and the balance of deionized water. Inoculating the bacillus subtilis for expressing the recombinant protein into the culture medium, culturing for 30-42h at 28-36 ℃, then centrifuging the bacterial liquid, collecting the supernatant, and purifying to obtain the bacterial liquid with the expression level of the recombinant protein. When the culture medium is used for expressing the recombinant protein by using the bacillus subtilis, the expression quantity of the recombinant protein is improved by 28-65% compared with that of an LB culture medium, and the components of the culture medium are cheap and easily obtained, are safe and nontoxic and can be fermented on a large scale.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium for improving the expression quantity of recombinant protein in bacillus subtilis and a preparation method thereof.
Background
Currently, Escherichia coli (Escherichia coli) is the most widely used in prokaryotic expression systems, and has numerous advantages, such as low culture condition requirements, simple operation, short expression period, high gene product expression level, and the like. However, the disadvantages of the E.coli expression system are not negligible. The expression of recombinant protein by using an escherichia coli expression system can cause inactive protein to form inclusion bodies or polymers, so that subsequent steps are required to be added, non-glycosylated, misfolded or non-functional protein can be expressed, meanwhile, escherichia coli lacks a secretion expression mechanism, so that thallus can be broken when the recombinant protein is obtained, and the cell wall of the escherichia coli has endotoxin, so that the purification difficulty is increased. Moreover, acetate can be accumulated in the fermentation process of the escherichia coli, and the acetate can influence the growth of the escherichia coli and the protein expression.
Compared with Escherichia coli, Bacillus subtilis also has low requirement on culture conditions, short expression period and endotoxin-free cell wall, and can secrete recombinant protein outside cells, thereby simplifying purification steps. Although Bacillus subtilis secretes more protease and affects the synthesized recombinant protein, protease-deficient strains have been constructed, and by using such strains, the recombinant protein can be prevented from being decomposed. At present, a large number of expression systems and expression methods of bacillus subtilis are constructed, for example, the expression quantity of beta-galactosidase of a bacillus subtilis maltose operator promoter expression system constructed by Yangming and the like reaches the expression quantity close to that of a commercial efficient expression system pET28a of escherichia coli; the invention provides a method for expressing foreign protein by using bacillus subtilis, and the like, and reduces the introduction of foreign harmful substances or impurities.
However, in the current bacillus subtilis expression method, LB culture medium is mostly adopted for the expression of exogenous protein, but tryptone and yeast extract adopted by LB culture medium are expensive, the yield of recombinant protein is low, and the method is not suitable for large-scale production of fermentation exogenous protein. Based on the above problems in the process of expressing recombinant proteins by Bacillus subtilis, in order to reduce fermentation costs and increase the expression yield of recombinant proteins, the culture medium for expressing recombinant proteins by Bacillus subtilis needs to be improved.
After search of the prior art, the invention patent with application number 201610130056.8 discloses a culture medium for escherichia coli to secrete and express recombinant protein, wherein every 1000ml of the culture medium contains: tryptone 12-16 g, yeast extract 6-10 g, D-sorbitol 60-100 g, sodium chloride 8-16 g, D-glucose 1-2 g, 1mol/LTris-HCl buffer (pH7.2)20-100 ml, adding deionized water to 1000 ml. However, in the culture medium, the price of the tryptone and the yeast extract is expensive, and the yield of the recombinant protein is low and is not more than 7mg/L at most.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a culture medium for improving the expression quantity of recombinant protein in bacillus subtilis, a preparation method and application.
The purpose of the invention is realized by the following scheme:
the first aspect of the present invention provides a culture medium for increasing the expression amount of a recombinant protein in bacillus subtilis, wherein the culture medium comprises the following components per 1000mL of the culture medium: 25-35g of corn soaking powder, 36-48g of bean pulp extract, 40-60g of maltose, 4-6g of potassium chloride, 3-5g of ammonium sulfate, 0.075-0.125g of manganese sulfate, 0.075-0.125g of calcium chloride and the balance of deionized water. Wherein the corn steep powder is used as carbon source, the soybean meal extract is used as nitrogen source, maltose is used as inducer, and inorganic salt such as potassium chloride, ammonium sulfate, manganese sulfate, calcium chloride, etc. is added, pH is 6-8, and can be used for improving expression level of recombinant protein such as earthworm antibacterial peptide.
Preferably, the medium composition comprises, per 1000mL system of the medium: 30g of corn soaking powder, 42g of soybean meal extract, 50g of maltose, 5g of potassium chloride, 4g of ammonium sulfate, 0.1g of manganese sulfate, 0.1g of calcium chloride and the balance of deionized water.
Preferably, the maltose is firstly prepared into a maltose solution with the concentration of 50% (W/V) by using deionized water, the manganese sulfate is firstly prepared into a manganese sulfate solution with the concentration of 10g/L by using deionized water, and the calcium chloride is firstly prepared into a calcium chloride solution with the concentration of 10g/L by using deionized water.
The second aspect of the present invention provides a method for preparing the above-mentioned culture medium for increasing the expression level of recombinant protein in bacillus subtilis, comprising the following steps:
s1, weighing 25-35g of corn extract powder, 36-48g of soybean meal extract, 4-6g of potassium chloride and 3-5g of ammonium sulfate, adding 800ml of deionized water for dissolving, adjusting the pH value to 6-8 by using a sodium hydroxide solution, and carrying out autoclaving to obtain a basic culture solution YD;
s2, weighing 125g of maltose, adding 200mL of deionized water to dissolve the maltose, fixing the volume to 250mL, and carrying out autoclaving to obtain a maltose solution with the concentration of 50% (W/V);
s3, weighing 1g of manganese sulfate, adding 60mL of deionized water to dissolve the manganese sulfate, fixing the volume to 100mL, and performing high-pressure sterilization to obtain 10g/L manganese sulfate solution;
s4, weighing 1g of calcium chloride, adding 60mL of deionized water to dissolve the calcium chloride, fixing the volume to 100mL, and carrying out autoclaving to obtain 10g/L calcium chloride solution;
s5, slowly adding 80-120mL of 50% (W/V) maltose solution, 7.5-1.25mL of 10g/L manganese sulfate solution and 7.5-1.25mL of 10g/L calcium chloride solution into the basic culture solution YD prepared in the step S1, and using sterilized deionized water to make the volume reach 1000mL to obtain the culture medium for improving the expression amount of the recombinant protein in the bacillus subtilis.
Further, in step S5, antibiotics may be added for screening recombinants or providing selection pressure.
The third aspect of the invention provides an application of the culture medium for improving the expression level of the recombinant protein in the bacillus subtilis, wherein the bacillus subtilis for expressing the recombinant protein is inoculated into the culture medium of the first aspect, the culture medium is cultured for 30-42h at 28-36 ℃, then the bacterial liquid is centrifuged to extract the supernatant, and the bacterial liquid with the expression level of the recombinant protein is obtained after purification.
Further, in the bacillus subtilis for expressing the recombinant protein, a promoter used for expression is a modified maltose operator promoter Pglv-M1, and the sequence of the promoter is shown as SEQ ID NO: 1, and the following components: gtgttacgggacgagctatctcatggtataaatggaattgtaaaatttatcaaggaggtcgtcat are provided.
Further, before inoculation of Bacillus subtilis for expression of recombinant proteins, it was activated overnight in LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7, autoclaved at 121 ℃ for 20 min). Then inoculating the bacillus subtilis into the culture medium for improving the expression quantity of the recombinant protein in the bacillus subtilis according to the proportion of 1-3%, and culturing for 30-42h at 28-36 ℃.
Further, the expressed recombinant protein is a soluble recombinant protein.
Further, the strain used for expressing the recombinant protein is bacillus subtilis WB800N or other bacillus subtilis used for expression.
The culture medium can improve the expression quantity of the recombinant protein, and adopts corn soaking powder as a carbon source and bean pulp extract as a nitrogen source: (1) during the culture process, the bacillus subtilis can decompose and utilize corn steep powder to produce various metabolites, such as fructose-1, 6-diphosphate, glucose-6-phosphate and the like, the metabolites can activate protein kinases of bacillus subtilis, the activated protein kinases can acylate serine residue at position 46 in a phosphate carrier protein Hpr molecule, and the acylated phosphate carrier protein Hpr can regulate and control the synthesis of protein; (2) the soybean meal extract is a high-quality protein source, can provide a large amount of amino acids for the bacillus subtilis, and promotes the growth of the bacillus subtilis and synthesizes protein; (3) the manganese element and the calcium element are trace elements required by the growth of the bacillus subtilis, and the proper amount of the manganese element and the calcium element can promote the growth of the bacillus subtilis and synthesize protein.
Compared with the prior art, the invention has the following beneficial effects:
1) corn steep powder is used as a carbon source, a soybean meal extract is used as a nitrogen source, maltose is used as an inducer, and inorganic salts such as potassium chloride, ammonium sulfate, manganese sulfate, calcium chloride and the like are added, so that the raw materials of the culture medium are wide in source and low in price compared with LB (lysogeny broth) culture medium, and the method is suitable for large-scale fermentation;
2) when the culture medium is used for expressing the recombinant protein by using the bacillus subtilis, the expression quantity of the recombinant protein is improved by 28-65 percent compared with that of an LB culture medium.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
The invention provides a culture medium for improving the expression quantity of recombinant protein in bacillus subtilis, and lays a foundation for large-scale fermentation expression of the recombinant protein by the bacillus subtilis.
According to the invention, (1) the corn steep powder is used as a carbon source, the soybean meal extract is used as a nitrogen source, the maltose is used as an inducer, and inorganic salts such as potassium chloride, ammonium sulfate, manganese sulfate, calcium chloride and the like are added, so that the raw materials of the culture medium are wide in source and low in price compared with LB culture medium, and the method is suitable for large-scale fermentation. (2) When the recombinant protein is expressed by using the bacillus subtilis, the expression quantity of the recombinant protein is improved by 28-65 percent compared with that of an LB culture medium.
Example 1 optimization of the content of Medium Components
The content optimization test was performed on corn steep powder, soybean meal extract, maltose, potassium chloride, ammonium sulfate, manganese sulfate, and calcium chloride, and the experimental procedure was as in example 3. Specific experimental data are shown in table 1:
TABLE 1 Plackett-Burman assay for screening of the content of important constituents of the culture Medium
From the above test results, it was found that the influence of the corn steep powder concentration, the soybean meal extract concentration and the maltose concentration on the expression level was the greatest, and the remaining components were inoculated at 2% and cultured at 32 ℃ for 36 hours while keeping the potassium chloride concentration at 5g/L, ammonium sulfate at 4g/L, manganese sulfate and calcium chloride at 0.1g/L and pH at 7.
TABLE 2 steepest climb test for screening of the content of essential components of the culture
The steepest climbing test result screened by the content of the important components of the culture medium can be known that the optimal range is 25-35mg/L of corn steep powder, 36-48mg/L of soybean meal extract and 40-60mg/L of maltose.
Example 2
1. Reagents and materials
The corn extract powder and the bean pulp extract are purchased from Beijing Hongrunbao Shunjin Co Ltd;
potassium chloride was purchased from Shanghai Merlin Biotech, Inc.;
ammonium sulfate is available from qiaoyi biotechnology (shanghai) ltd;
maltose purchased from bio-engineering (shanghai) gmbh;
manganese sulfate and calcium chloride are purchased from chemical reagents of national drug group, ltd.
2. Preparation of culture Medium
(1) Weighing 27g of corn extract powder, 42g of soybean meal extract, 6g of potassium chloride and 3g of ammonium sulfate, adding 800ml of deionized water for dissolving, adjusting the pH value to 6 by using a sodium hydroxide solution, and carrying out autoclaving at 121 ℃ for 20 min;
(2) weighing 125g of maltose, adding 200ml of deionized water for dissolving, fixing the volume to 250ml, and carrying out autoclaving at 115 ℃ for 30min to obtain a maltose solution with the concentration of 50% (W/V);
(3) weighing 1g of manganese sulfate, adding 60ml of deionized water to dissolve the manganese sulfate, fixing the volume to 100ml, and carrying out high-pressure sterilization at 121 ℃ for 20min to obtain 10g/L of manganese sulfate solution;
(4) weighing 1g of calcium chloride, adding 60ml of deionized water for dissolving, fixing the volume to 100ml, and carrying out autoclaving at 121 ℃ for 20min to obtain a 10g/L calcium chloride solution;
(5) to the solution prepared in step (1), 120ml of a 50% (W/V) maltose solution, 7.5ml of a 10g/L manganese sulfate solution, 7.5ml of a 10g/L calcium chloride solution, and 100. mu.L of 5mg/ml chloramphenicol were slowly added, and a volume of 1L was made up with sterilized deionized water.
3. Expression and purification of earthworm antibacterial peptide SUMO-Lumbricin I (6-34)
A method for preparing Bacillus subtilis WB800N containing SUMO-Lumbricin I (6-34) expression plasmid is disclosed in patent publication No. CN 111304233A.
(1) Selecting a single bacillus subtilis colony containing an SUMO-Lumbricin I (6-34) expression plasmid, inoculating the single bacillus subtilis colony in 50mL of LB culture medium, and culturing at 37 ℃ and 180rpm overnight;
(2) the overnight inoculum was inoculated at 3% ratio to 500mL of the medium prepared in the previous step and incubated at 36 ℃ and 225rpm for 30 h.
(3) Centrifuging the bacterial liquid to obtain supernatant, adding ammonium sulfate to precipitate, centrifuging to obtain precipitate, redissolving, dialyzing, purifying with nickel affinity chromatography column and molecular sieve chromatography column, detecting protein concentration with non-interference protein concentration determination kit (Producer Biopsis, China), and converting to obtain culture medium supernatant containing SUMO-Lumbricin I (6-34)33.37 mg/L.
Example 3
1. Reagents and materials
The corn extract powder and the bean pulp extract are purchased from Beijing Hongrunbao Shunjin Co Ltd;
potassium chloride was purchased from Shanghai Merlin Biotech, Inc.;
ammonium sulfate is available from qiaoyi biotechnology (shanghai) ltd;
maltose purchased from bio-engineering (shanghai) gmbh;
manganese sulfate and calcium chloride are purchased from chemical reagents of national drug group, ltd;
2. preparation of culture Medium
(1) Weighing 30g of corn extract powder, 42g of soybean meal extract, 5g of potassium chloride and 4g of ammonium sulfate, adding 800ml of deionized water for dissolving, adjusting the pH value to 7 by using a sodium hydroxide solution, and carrying out autoclaving at 121 ℃ for 20 min;
(2) weighing 125g of maltose, adding 200ml of deionized water for dissolving, fixing the volume to 250ml, and carrying out autoclaving at 115 ℃ for 30min to obtain a maltose solution with the concentration of 50% (W/V);
(3) weighing 1g of manganese sulfate, adding 60ml of deionized water to dissolve the manganese sulfate, fixing the volume to 100ml, and carrying out high-pressure sterilization at 121 ℃ for 20min to obtain 10g/L of manganese sulfate solution;
(4) weighing 1g of calcium chloride, adding 60ml of deionized water for dissolving, fixing the volume to 100ml, and carrying out autoclaving at 121 ℃ for 20min to obtain a 10g/L calcium chloride solution;
(5) to the solution prepared in step (1), 100ml of 50% (W/V) maltose solution, 10ml of 10g/L manganese sulfate solution, 10ml of 10g/L calcium chloride solution, and 100. mu.L of 5mg/ml chloramphenicol were slowly added, and a volume of 1L was made up with sterilized deionized water.
3. Expression and purification of earthworm antibacterial peptide SUMO-Lumbricin I (6-34)
A method for preparing Bacillus subtilis WB800N containing SUMO-Lumbricin I (6-34) expression plasmid is disclosed in patent publication No. CN 111304233A.
(1) Selecting a single bacillus subtilis colony containing an SUMO-Lumbricin I (6-34) expression plasmid, inoculating the single bacillus subtilis colony in 50mL of LB culture medium, and culturing at 37 ℃ and 180rpm overnight;
(2) the overnight inoculum was inoculated at 2% ratio to 500mL of the medium prepared in the previous step and incubated at 32 ℃ for 36h at 500 rpm.
(3) Centrifuging the bacterial solution to obtain supernatant, adding ammonium sulfate to precipitate, centrifuging to obtain precipitate, dissolving again, dialyzing, purifying with nickel affinity chromatography column and molecular sieve chromatography column, detecting protein concentration with non-interference protein concentration determination kit (Producer Biopsis, China), and converting to obtain culture medium supernatant containing SUMO-Lumbricin I (6-34)40.65mg/L,
example 4
In this example, the media composition included: 27g of corn soaking powder, 42g of bean pulp extract, 60g of maltose, 6g of potassium chloride, 3g of ammonium sulfate, 0.075g of manganese sulfate, 0.075g of calcium chloride and the balance of deionized water, wherein the pH value is 6, the inoculation amount is 1%, and the corn soaking powder is cultured at 36 ℃ for 30 h. The other steps were the same as in example 3, and after purification, the protein concentration was measured using a non-interfering protein concentration measuring kit (Bio-Ltd., China), and the concentration was converted to 33.05mg/L SUMO-Lumbricin I (6-34) in the culture supernatant.
Example 5
In this example, the media composition included: 34.9g of corn soaking powder, 45.3g of soybean meal extract, 52.3g of maltose, 5g of potassium chloride, 4g of ammonium sulfate, 0.1g of manganese sulfate, 0.1g of calcium chloride and the balance of deionized water, wherein the pH value is 7. The inoculation amount is 2%, and the culture is carried out for 36h at 32 ℃. The other steps were the same as in example 3, and after purification, the protein concentration was measured using a non-interfering protein concentration measuring kit (Bio-Ltd., China), and the concentration was converted to 42.61mg/L SUMO-Lumbricin I (6-34) in the culture supernatant.
Comparative example 1
1. Reagents and materials
Tryptone, yeast extract and maltose were purchased from bio-engineering (shanghai) gmbh;
sodium chloride was purchased from the national pharmaceutical group chemical agents limited.
2. Preparation of culture Medium
(1) Weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding 800ml of deionized water for dissolving, adjusting the pH to 7 by using a sodium hydroxide solution, and carrying out autoclaving at 121 ℃ for 20 min;
(2) weighing 125g of maltose, adding 200ml of deionized water for dissolving, fixing the volume to 250ml, and carrying out autoclaving at 115 ℃ for 30min to obtain a maltose solution with the concentration of 50% (W/V);
(3) to the solution prepared in step (1), 100ml of a 50% (W/V) maltose solution, 100. mu.L of 5mg/ml chloramphenicol, was slowly added, and a volume of 1L was made with sterilized deionized water.
3. Expression and purification of earthworm antibacterial peptide SUMO-Lumbricin I (6-34)
(1) Selecting a single bacillus subtilis colony containing an SUMO-Lumbricin I (6-34) expression plasmid, inoculating the single bacillus subtilis colony in 50mL of LB culture medium, and culturing at 37 ℃ and 180rpm overnight;
(2) the overnight inoculum was inoculated at 2% ratio to 500mL LB medium prepared in the previous step and incubated at 37 ℃ and 225rpm for 36 h.
(3) Centrifuging the bacterial liquid to obtain supernatant, adding ammonium sulfate to precipitate, centrifuging to obtain precipitate, redissolving, dialyzing, purifying with nickel affinity chromatography column and molecular sieve chromatography column, detecting protein concentration with non-interference protein concentration determination kit (Producer Biopsis, China), and converting to obtain culture medium supernatant containing SUMO-Lumbricin I (6-34)25.82 mg/L.
As is clear from the above examples, the culture medium of the present invention increased the yield of example 2 by 29.24% compared to 25.82mg/L of SUMO-Lumbricin I (6-34) obtained in LB medium (5% maltose, cultured at 37 ℃ for 36 hours). The improvement is 57.44% in example 3, 28% in example 4 and 65% in example 5.
The reason why the expression amount of the culture medium of the present invention is higher than that of the LB culture medium is as follows: (1) the price of the corn soaking powder and the soybean meal extract is lower than that of the main components of tryptone and yeast extract of the LB culture medium, so that the concentration of the corn soaking powder and the soybean meal extract can be increased under the condition of lower cost, and a large amount of carbon sources and nitrogen sources are provided for the bacillus subtilis; (2) the additionally added manganese element and calcium element can promote the growth of the bacillus subtilis and synthesize protein.
The culture medium can improve the yield of the expression quantity of the recombinant protein secreted and expressed by the bacillus subtilis, adopts corn steep powder as a carbon source, bean pulp extract as a nitrogen source, maltose as an inducer, and adds inorganic salts such as potassium chloride, ammonium sulfate, manganese sulfate, calcium chloride and the like, has the pH value of 6-8, is cheap and easily available in components, is safe and nontoxic, and can be used for large-scale fermentation. The specific culture method is that the bacillus subtilis containing the expression plasmid is inoculated in the culture medium according to 1-3 percent after being cultured overnight, and is cultured for 30-42h at the temperature of 28-36 ℃, and the culture method has simple flow and easy operation. The culture medium and the culture method are used for expressing recombinant protein, in particular to expressing recombinant protein by utilizing bacillus subtilis. The culture medium and the culture method can utilize Bacillus subtilis WB800N or other strains for protein expression.
In the above examples of the present invention, SUMO-Lumbricin I (6-34) was used as the recombinant protein, but those skilled in the art can understand that other recombinant proteins in the prior art are also suitable for the method of the present invention based on the technical scheme of the present invention. Therefore, the method has multiple purposes and has good application prospect.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Sequence listing
<110> Shanghai university of transportation
<120> culture medium for improving expression quantity of recombinant protein in bacillus subtilis, preparation method and application
<130> KAG45921
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtgttacggg acgagctatc tcatggtata aatggaattg taaaatttat caaggaggtc 60
gtcat 65
Claims (10)
1. A culture medium for increasing the expression amount of recombinant protein in Bacillus subtilis, wherein the culture medium comprises the following components in every 1000mL system: 25-35g of corn soaking powder, 36-48g of bean pulp extract, 40-60g of maltose, 4-6g of potassium chloride, 3-5g of ammonium sulfate, 0.075-0.125g of manganese sulfate, 0.075-0.125g of calcium chloride and the balance of deionized water.
2. The culture medium for increasing the expression amount of the recombinant protein in the bacillus subtilis according to claim 1, wherein the culture medium comprises the following components in every 1000mL system: 30g of corn soaking powder, 42g of soybean meal extract, 50g of maltose, 5g of potassium chloride, 4g of ammonium sulfate, 0.1g of manganese sulfate, 0.1g of calcium chloride and the balance of deionized water.
3. The culture medium for improving the expression level of the recombinant protein in the bacillus subtilis as claimed in claim 1, wherein the maltose is firstly prepared into a 50% (W/V) maltose solution by using deionized water, the manganese sulfate is firstly prepared into a 10g/L manganese sulfate solution by using deionized water, and the calcium chloride is firstly prepared into a 10g/L calcium chloride solution by using deionized water.
4. A method for preparing the culture medium for increasing the expression level of the recombinant protein in the Bacillus subtilis according to claim 1, comprising the steps of:
s1, weighing 25-35g of corn extract powder, 36-48g of soybean meal extract, 4-6g of potassium chloride and 3-5g of ammonium sulfate, adding 800ml of deionized water for dissolving, adjusting the pH value to 6-8 by using a sodium hydroxide solution, and carrying out autoclaving to obtain a basic culture solution YD;
s2, weighing 125g of maltose, adding 200mL of deionized water to dissolve the maltose, fixing the volume to 250mL, and carrying out autoclaving to obtain a maltose solution with the concentration of 50% (W/V);
s3, weighing 1g of manganese sulfate, adding 60mL of deionized water to dissolve the manganese sulfate, fixing the volume to 100mL, and performing high-pressure sterilization to obtain 10g/L manganese sulfate solution;
s4, weighing 1g of calcium chloride, adding 60mL of deionized water to dissolve the calcium chloride, fixing the volume to 100mL, and carrying out autoclaving to obtain 10g/L calcium chloride solution;
s5, slowly adding 80-120mL of 50% (W/V) maltose solution, 7.5-1.25mL of 10g/L manganese sulfate solution and 7.5-1.25mL of 10g/L calcium chloride solution into the basic culture solution YD prepared in the step S1, and using sterilized deionized water to make the volume reach 1000mL to obtain the culture medium for improving the expression amount of the recombinant protein in the bacillus subtilis.
5. The method of claim 4, wherein in step S5, antibiotics are added to screen for recombinants or provide selection pressure.
6. The use of the culture medium according to claim 1 for increasing the expression level of a recombinant protein in Bacillus subtilis, wherein Bacillus subtilis for expressing a recombinant protein is inoculated into the culture medium according to claim 1, cultured at 28-36 ℃ for 30-42h, and then centrifuged to obtain a supernatant, which is purified to obtain a bacterial solution with an increased expression level of a recombinant protein.
7. The use of the medium according to claim 6 for increasing the expression level of a recombinant protein in Bacillus subtilis, wherein the promoter used for expressing the recombinant protein in Bacillus subtilis is modified maltose-operator promoter Pglv-M1, whose sequence is shown in SEQ ID NO: 1 is shown.
8. The use of the medium according to claim 6 for increasing the expression level of a recombinant protein in Bacillus subtilis, wherein the Bacillus subtilis is activated overnight in LB medium before inoculation.
9. The use of the medium according to claim 6 for increasing the expression level of a recombinant protein in Bacillus subtilis, wherein the recombinant protein is a soluble recombinant protein.
10. The use of the culture medium for increasing the expression amount of a recombinant protein in Bacillus subtilis according to claim 6, wherein the strain used for expressing the recombinant protein is Bacillus subtilis WB 800N.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686550A (en) * | 2022-04-29 | 2022-07-01 | 成都搏锋智宇生物科技有限公司 | Fermentation preparation method for producing tenebrio molitor antibacterial peptide |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243117A (en) * | 2013-05-24 | 2013-08-14 | 江南大学 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
| CN103396969A (en) * | 2013-08-19 | 2013-11-20 | 北京航天恒丰科技发展有限公司 | Fermentation culture medium capable of mixedly culturing brevibacillus laterosporus and bacillus subtilis and fermentation method thereof |
| CN103911396A (en) * | 2014-04-25 | 2014-07-09 | 山东仙普爱瑞科技股份有限公司 | Bacillus subtilis fermentation medium |
| CN108473484A (en) * | 2015-10-01 | 2018-08-31 | 赛诺米克斯公司 | It can be used as the compound of TRPM8 conditioning agents |
| CN111304233A (en) * | 2020-02-27 | 2020-06-19 | 上海交通大学 | Expression method of earthworm antibacterial peptide |
-
2021
- 2021-03-17 CN CN202110286083.5A patent/CN112852703A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243117A (en) * | 2013-05-24 | 2013-08-14 | 江南大学 | Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis |
| CN103396969A (en) * | 2013-08-19 | 2013-11-20 | 北京航天恒丰科技发展有限公司 | Fermentation culture medium capable of mixedly culturing brevibacillus laterosporus and bacillus subtilis and fermentation method thereof |
| CN103911396A (en) * | 2014-04-25 | 2014-07-09 | 山东仙普爱瑞科技股份有限公司 | Bacillus subtilis fermentation medium |
| CN108473484A (en) * | 2015-10-01 | 2018-08-31 | 赛诺米克斯公司 | It can be used as the compound of TRPM8 conditioning agents |
| CN111304233A (en) * | 2020-02-27 | 2020-06-19 | 上海交通大学 | Expression method of earthworm antibacterial peptide |
Non-Patent Citations (5)
| Title |
|---|
| 冒鑫哲等: "枯草芽孢杆菌高产角蛋白酶发酵条件优化", 《食品与发酵工业》 * |
| 张志焱等: "一株枯草芽孢杆菌产抗菌肽培养基筛选及发酵工艺优化的研究", 《中国畜牧兽医》 * |
| 张慧等: "枯草芽孢杆菌(Bacillus subtilis)CS27液体发酵条件优化", 《热带作物学报》 * |
| 李达等: "饲用枯草芽孢杆菌高密度发酵培养基的优化", 《东北农业科学》 * |
| 管国强: "枯草芽孢杆菌ZC1高密度培养的条件优化", 《中国调味品》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686550A (en) * | 2022-04-29 | 2022-07-01 | 成都搏锋智宇生物科技有限公司 | Fermentation preparation method for producing tenebrio molitor antibacterial peptide |
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