CN107760644A - A kind of culture medium of feeding Escherichia coli high density fermentation and its application - Google Patents
A kind of culture medium of feeding Escherichia coli high density fermentation and its application Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 87
- 230000004151 fermentation Effects 0.000 title claims abstract description 87
- 239000001963 growth medium Substances 0.000 title claims abstract description 61
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 31
- 239000008103 glucose Substances 0.000 claims abstract description 31
- 150000003839 salts Chemical class 0.000 claims abstract description 28
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- 239000012137 tryptone Substances 0.000 claims abstract description 20
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 14
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims abstract description 14
- 239000007836 KH2PO4 Substances 0.000 claims abstract description 13
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 13
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 13
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 54
- 102100029655 Tripartite motif-containing protein 72 Human genes 0.000 claims description 36
- 101150088302 trim72 gene Proteins 0.000 claims description 36
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical class Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 235000011167 hydrochloric acid Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
- 108010021466 Mutant Proteins Proteins 0.000 claims description 4
- 102000008300 Mutant Proteins Human genes 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004473 Threonine Substances 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000006052 feed supplement Substances 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 210000002027 skeletal muscle Anatomy 0.000 claims description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 2
- 241000305071 Enterobacterales Species 0.000 claims 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims 1
- 238000009938 salting Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 239000002054 inoculum Substances 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 239000013595 supernatant sample Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 241001597008 Nomeidae Species 0.000 description 5
- 229910052927 chalcanthite Inorganic materials 0.000 description 5
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 238000010899 nucleation Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000000843 powder Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241001052560 Thallis Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
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- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The present invention provides a kind of culture medium of Escherichia coli high density fermentation, belongs to biological fermentation field.Batch every liter of the culture medium of the present invention includes following proportioning component：Tryptone 10g/L, dusty yeast 20g/L, sodium chloride 0.5g/L, glucose 8.5g/L, KH2PO4·H2O5g/L, K2HPO4·3H2O15g/L, 2mMMgCl2·6H2O solution 1ml/L, Trace salts solution 1ml/L, pH 7.0.The culture medium of the present invention can significantly improve the fermentation density of Recombinant organism, increase destination protein expression quantity, so as to improve specific production rate, save production cost.
Description
Technical field
The invention belongs to technical field of biological fermentation, be related to a kind of feeding Escherichia coli high density fermentation culture medium and its
Using.
Background technology
High density fermentation be lifted protein expression important channel, and culture medium be high density fermentation material impact because
Element.In the case where thalline expressing protein efficiency is suitable, the fermentation density of thalline directly influences the production cost of bio-pharmaceutical,
In order to improve the expression total amount of bacterial fermentation thalli growth density and target product, the specific production rate of tunning is improved, enters one
Step reduces production cost, and we developed a kind of culture suitable for Recombinant organism high density fermentation culture
Base.
The invention provides a kind of culture medium of feeding Escherichia coli high density fermentation, the culture medium is remarkably improved thalline
Density and expressing quantity.It is particularly applicable to the expression and fermentation of MG53 albumen and its mutant.
The content of the invention
The technical problems to be solved by the invention are the provision of a kind of culture medium of feeding Escherichia coli high density fermentation,
Using the culture medium, higher density biomass can be effectively obtained, improves destination protein expression quantity.
In order to solve the above technical problems, the technical scheme is that：
The present invention provides a kind of culture medium of feeding Escherichia coli high density fermentation, and described Escherichia coli are Escherichia coli
Engineered strain BL21 (DE3)/pET22b (+), the main component and its content of high density fermentation culture medium are as follows：Tryptone 8
~15g/L, 15~25g/L of dusty yeast, 0~1g/L of sodium chloride, 6~10g/L of glucose, KH2PO4·H22~6g/L of O,
K2HPO4·3H210~20g/L of O, 2mM MgCl2·6H20.5~2ml/L of O, Trace salts solution 0.5~2ml/L, pH 7.0；
The main component of supplemented medium is as follows：Tryptone, dusty yeast, glucose.
Wherein, the main component of the high density fermentation culture medium and its content are preferably as follows：8~12g/ of tryptone
L, 15~20g/L of dusty yeast, 0.3~0.6g/L of sodium chloride, 8~10g/L of glucose, KH2PO4·H24~6g/L of O, K2HPO4·
3H210~20g/L of O, 2mM MgCl2·6H2O 0.5-1.0ml/L, Trace salts solution 0.5~1.0ml/L, pH 7.0.
Wherein, the main component of the high density fermentation culture medium and its content are preferably as follows：Tryptone 10g/L,
Dusty yeast 20g/L, sodium chloride 0.5g/L, glucose 8.5g/L, KH2PO4·H2O 5g/L, K2HPO4·3H2O 15g/L,
2mMMgCl2·6H2O solution 1ml/L, Trace salts solution 1ml/L, pH 7.0.
Wherein, the Trace salts solution is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O
0.71g、CuSO4·5H2O 0.4g、H3BO4 0.06g、MnSO4·H2O 0.8g、CaCl2 0.07g、Na2MoSO4·2H2O
1g、CoCl2·6H2O 0.01g, are settled to 100ml, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
The present invention provides a kind of culture medium of feeding Escherichia coli high density fermentation, the compound method of the fermentation medium
It is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, in fermentation tank, 121 DEG C of sterilizings 20
Minute；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, is cooled to 37
DEG C produce.
The present invention provides a kind of culture medium of feeding Escherichia coli high density fermentation, the feed-batch culture of the fermentation medium
The main component and content of base are as follows：Tryptone 5-10g/L, dusty yeast 5-10g/L, glucose 200-500g/L.
The present invention provides a kind of culture medium of feeding Escherichia coli high density fermentation, and the culture medium can be used for large intestine bar
Bacterium BL21 (DE3) is host, and pET22b (+) is carrier, available for expressing and ferment Human Skeletal Muscle MG53 albumen and its mutant.
Wherein, described MG53 mutant is sported for the serine sites in MG53 Coiled-coil domains and removed
Amino acid beyond threonine and tyrosine.
The present invention a kind of feeding Escherichia coli high density fermentation culture medium can with high density fermentation recombination bacillus coli,
Higher density biomass can be effectively obtained, improves destination protein expression quantity, substantially reduces production cost, improves production effect
Rate.
Brief description of the drawings
Fig. 1 is SDS-PAGE gel electrophoresis of protein figures, wherein：
1st, standard items
2、MARKER
3rd, Supernatant samples before inducing
4th, Supernatant samples after former culture medium fermentation MG53 inductions
5th, Supernatant samples after the fermentation MG53 inductions of high density fermentation culture medium formula 1
6th, Supernatant samples after the fermentation MG53 inductions of high density fermentation culture medium formula 2
7th, Supernatant samples after the fermentation MG53 inductions of high density fermentation culture medium formula 3
8th, Supernatant samples after the fermentation MG53 mutation inductions of high density fermentation culture medium formula 3
Embodiment
Technical scheme of the present invention is described further with reference to specific embodiment.
INSTRUMENT MODEL, reagent and its source used in the following example of the present invention is as follows：
Fermentation tank：The logical sequence bio tech ltd of BLBIO-10SJA-50SJA-VIP Shanghai hundred
Protein purification instrument：AKTA pure GE
Gel imager：Universal Hood III BIO-RAD
Ultraviolet-uisible spectrophotometer：752 Shanghai essence tech equipment Co., Ltds
Tryptone：Cat.No：LP0042OXOID
Dusty yeast：Cat.No：LP0021OXOID
Former culture medium prescription：Tryptone 12g/L, dusty yeast 24g/L, glycerine 4g/L, KH2PO4 2.3g/L、K2HPO4·
3H2O 16.4g/L, pH 7.0；Supplemented medium：Glycerine；
Culture medium compound method is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned each component；
(2) tryptone, dusty yeast, glycerine are dissolved with distilled water in fermentation tank；Under the conditions of 121 DEG C, sterilize 20 minutes；
(3)KH2PO4、K2HPO4·3H2O solution individually sterilizes.
(4) when fermentation tank is cooled to 50 DEG C or so, KH is added2PO4、K2HPO4·3H2O solution, it is cooled to 37 DEG C and produces.
MG53 protein fermentations：
Colibacillus engineering strain BL21 (DE3)/pET22b (+)/MG53 is activated by test tube slant, Shaking culture,
Seeding tank is inoculated into 5% inoculum concentration and expands culture preparation seed liquor, is sent out seed liquor in above-mentioned culture medium with 5% inoculum concentration
Ferment, dissolved oxygen are controlled more than 20%, 37 DEG C of cultivation temperature, when dissolved oxygen starts bounce-back, are started to be slowly added to supplemented medium, are trained
Support to exponential phase, add derivant and induced, obtain MG53 albumen.
The high density fermentation culture medium formula 1 of embodiment 1 is used for the MG53 albumen that ferments
1.1st, the main component of Escherichia coli high density fermentation culture medium and its content are as follows：Tryptone 8g/L, yeast
Powder 15g/L, sodium chloride 1g/L, glucose 6g/L, KH2PO4·H2O 2g/L、K2HPO4·3H2O 15g/L、2mM MgCl2·
6H2O 1ml/L, Trace salts solution 1ml/L, pH 7.0；The main component and content of supplemented medium are as follows：Tryptone 5g/
L, dusty yeast 10g/L, glucose 200g/L；
Wherein, the Trace salts solution is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O
0.71g、CuSO4·5H2O 0.4g、H3BO4 0.06g、MnSO4·H2O 0.8g、CaCl2 0.07g、Na2MoSO4·2H2O
1g、CoCl2·6H2O 0.01g, are settled to 100ml, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
1.2nd, culture medium compound method is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, in fermentation tank, 121 DEG C of sterilizings 20
Minute；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, is cooled to 37
DEG C produce
1.3rd, high density fermentation MG53 albumen：
Colibacillus engineering strain BL21 (DE3)/pET22b (+)/MG53 is activated by test tube slant, Shaking culture,
Seeding tank is inoculated into 5% inoculum concentration and expands culture preparation seed liquor, is sent out seed liquor in above-mentioned culture medium with 5% inoculum concentration
Ferment, dissolved oxygen are controlled more than 20%, 37 DEG C of cultivation temperature, when dissolved oxygen starts bounce-back, are started to be slowly added to supplemented medium, are trained
Support to exponential phase, add derivant and induced, obtain MG53 albumen.
The high density fermentation culture medium formula 2 of embodiment 2 is used for the MG53 albumen that ferments
2.1st, the main component of Escherichia coli high density fermentation culture medium and its content are as follows：Tryptone 15g/L, yeast
Powder 20g/L, sodium chloride 1g/L, glucose 8g/L, KH2PO4·H2O 5g/L、K2HPO4·3H2O 20g/L、2mM MgCl2·
6H2O 1ml/L, Trace salts solution 1ml/L, pH 7.0；The main component and content of supplemented medium are as follows：Tryptone
10g/L, dusty yeast 5g/L, glucose 300g/L；
Wherein, the Trace salts solution is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O
0.71g、CuSO4·5H2O 0.4g、H3BO4 0.06g、MnSO4·H2O 0.8g、CaCl2 0.07g、Na2MoSO4·2H2O
1g、CoCl2·6H2O 0.01g, are settled to 100ml, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
2.2nd, culture medium compound method is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, in fermentation tank, 121 DEG C of sterilizings 20
Minute；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, is cooled to 37
DEG C produce
2.3rd, high density fermentation MG53 albumen：
Colibacillus engineering strain BL21 (DE3)/pET22b (+)/MG53 is activated by test tube slant, Shaking culture,
Seeding tank is inoculated into 5% inoculum concentration and expands culture preparation seed liquor, is sent out seed liquor in above-mentioned culture medium with 5% inoculum concentration
Ferment, dissolved oxygen are controlled more than 20%, 37 DEG C of cultivation temperature, when dissolved oxygen starts bounce-back, are started to be slowly added to supplemented medium, are trained
Support to exponential phase, add derivant and induced, obtain MG53 albumen.
The high density fermentation culture medium formula 3 of embodiment 3 is used for the MG53 albumen that ferments
3.1st, the main component of Escherichia coli high density fermentation culture medium and its content are as follows：Tryptone 10g/L, yeast
Powder 20g/L, sodium chloride 0.5g/L, glucose 8.5g/L, KH2PO4·H2O 5g/L、K2HPO4·3H2O 15g/L、2mM
MgCl2·6H2O 1ml/L, Trace salts solution 1ml/L, pH 7.0；The main component and content of supplemented medium are as follows：Pancreas egg
White peptone 8g/L, dusty yeast 8g/L, glucose 250g/L；
Wherein, the Trace salts solution is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O
0.71g、CuSO4·5H2O 0.4g、H3BO4 0.06g、MnSO4·H2O 0.8g、CaCl2 0.07g、Na2MoSO4·2H2O
1g、CoCl2·6H2O 0.01g, are settled to 100ml, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
3.2, culture medium compound method it is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, in fermentation tank, 121 DEG C of sterilizings 20
Minute；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, is cooled to 37
DEG C produce
3.3rd, high density fermentation MG53 albumen：
Colibacillus engineering strain BL21 (DE3)/pET22b (+)/MG53 is activated by test tube slant, Shaking culture,
Seeding tank is inoculated into 5% inoculum concentration and expands culture preparation seed liquor, is sent out seed liquor in above-mentioned culture medium with 5% inoculum concentration
Ferment, dissolved oxygen are controlled more than 20%, 37 DEG C of cultivation temperature, when dissolved oxygen starts bounce-back, are started to be slowly added to supplemented medium, are trained
Support to exponential phase, add derivant and induced, obtain MG53 albumen.
The high density fermentation culture medium formula 3 of embodiment 4 is used for the MG53 mutant proteins that ferment
4.1st, the main component of Escherichia coli high density fermentation culture medium and its content are as follows：Tryptone 10g/L, yeast
Powder 20g/L, sodium chloride 0.5g/L, glucose 8.5g/L, KH2PO4·H2O 5g/L、K2HPO4·3H2O 15g/L、2mM
MgCl2·6H2O 1ml/L, Trace salts solution 1ml/L, pH 7.0；The main component and content of supplemented medium are as follows：Pancreas egg
White peptone 8g/L, dusty yeast 8g/L, glucose 250g/L；
Wherein, the Trace salts solution is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O
0.71g、CuSO4·5H2O 0.4g、H3BO4 0.06g、MnSO4·H2O 0.8g、CaCl2 0.07g、Na2MoSO4·2H2O
1g、CoCl2·6H2O 0.01g, are settled to 100ml, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
4.2nd, culture medium compound method is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, in fermentation tank, 121 DEG C of sterilizings 20
Minute；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, is cooled to 37
DEG C produce
4.3rd, high density fermentation MG53 mutant proteins：
By colibacillus engineering strain BL21 (DE3)/pET22b (+)/MG53 mutant, activate, shake by test tube slant
Bottle culture, seeding tank expansion culture preparation seed liquor is inoculated into 5% inoculum concentration, with 5% inoculum concentration by seed liquor in above-mentioned training
Support and fermented in base, dissolved oxygen is controlled more than 20%, 37 DEG C of cultivation temperature, when dissolved oxygen starts bounce-back, starts to be slowly added to feed supplement
Culture medium, cultivate to exponential phase, add derivant and induced, obtain MG53 mutant proteins.
Interpretation of result：
The different culture media fermentation protein expression quantity of table 1 compares
By MG53 albumen by the testing result phase after former culture medium prescription and three groups of high density fermentation culture medium formula, fermentings
Compare, the results are shown in Table 1 and Fig. 1：The all more former culture medium prescription of MG53 expressing quantities increases；Three groups of high density fermentation cultures
Based formulas no matter the wet bacterium amount of unit volume or protein content after purification, be obviously improved compared with original formulation, its middle-high density
The effect of fermentative medium formula 3 is optimal.
High density fermentation culture medium fermentation optimal set is applied to the fermentation of MG53 mutant, found optimal with MG53 fermentations
Group obtains the wet bacterium amount of unit system of product, protein content, expressing quantity result are suitable after purification.
It the above is only highly preferred embodiment of the present invention, culture medium of the invention includes but is not limited to above-described embodiment, this hair
Bright unaccomplished matter, belong to the common knowledge of those skilled in the art.Therefore the change in the case where not departing from present general inventive concept
And modification, it should belong within protection scope of the present invention.
Claims (8)
1. a kind of culture medium of feeding Escherichia coli high density fermentation, it is characterised in that described Escherichia coli are Escherichia coli
Engineered strain BL21 (DE3)/pET22b (+), the main component and its content of high density fermentation culture medium are as follows：Tryptone 8
~15g/L, 15~25g/L of dusty yeast, 0~1g/L of sodium chloride, 6~10g/L of glucose, KH2PO4·H2O2~6g/L,
K2HPO4·3H2O10~20g/L, 2mM MgCl2·6H2O0.5~2ml/L, Trace salts solution 0.5~2ml/L, pH 7.0；Mend
Expect that the main component of culture medium is as follows：Tryptone, dusty yeast, glucose.
A kind of 2. culture medium of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that the height
The main component and its content of density fermentation culture medium are as follows：8~12g/L of tryptone, 15~20g/L of dusty yeast, sodium chloride
0.3~0.6g/L, 8~10g/L of glucose, KH2PO4·H2O4~6g/L, K2HPO4·3H2O10~20g/L, 2mM MgCl2·
6H2O0.5-1.0ml/L, Trace salts solution 0.5~1.0ml/L, pH 7.0.
A kind of 3. culture medium of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that the height
The main component and its content of density fermentation culture medium are as follows：Tryptone 10g/L, dusty yeast 20g/L, sodium chloride 0.5g/L,
Glucose 8.5g/L, KH2PO4·H2O5g/L, K2HPO4·3H2O15g/L, 2mMMgCl2·6H2O solution 1ml/L, trace salt are molten
Liquid 1ml/L, pH 7.0.
4. the culture medium of a kind of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that described micro-
Measuring salting liquid is：15ml concentrated hydrochloric acids add appropriate water dilution, sequentially add ZnSO4·7H2O0.71g、CuSO4·
5H2O0.4g、H3BO40.06g、MnSO4·H2O0.8g、CaCl20.07g、Na2MoSO4·2H2O1g、CoCl2·6H2O0.01g,
100ml is settled to, after thorough dissolving, aseptically with 0.22 μm of membrane filtration.
A kind of 5. culture medium of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that fermentation training
The collocation method for supporting base is as follows：
(1) by the accurate component for weighing predetermined weight of proportioning of above-mentioned fermentation medium each component；
(2) 115 DEG C of glucose solution, sterilize 20 minutes.MgCl2·6H2O and Trace salts solution filtration sterilization；
(3) other whole components of fermentation medium dissolve in distilled water solution, and in fermentation tank, 121 DEG C sterilize 20 minutes；
(4) when being cooled to 50 DEG C or so, MgCl is added2·6H2O solution, Trace salts solution, glucose solution, it is cooled to 37 DEG C i.e.
.
6. the culture medium of a kind of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that feed supplement is trained
Main component and the content for supporting base are as follows：5~10g/L of tryptone, 5~10g/L of dusty yeast, 200~500g/L of glucose.
7. the culture medium of a kind of feeding Escherichia coli high density fermentation according to claim 1, it is characterised in that described big
Enterobacteria is for host with e. coli bl21 (DE3), and pET22b (+) is carrier, available for expressing and the Human Skeletal Muscle that ferments
MG53 albumen and its mutant protein.
8. MG53 mutant according to claim 7, it is characterised in that the recombination human source MG53 protein mutants are
Serine sites in MG53 Coiled-coil domains sport the amino acid in addition to threonine and tyrosine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090029410A1 (en) * | 2004-10-08 | 2009-01-29 | Novozymes A/S | Lipases from Thermophilic Anaerobes |
| CN101914603A (en) * | 2010-07-08 | 2010-12-15 | 暨南大学 | Fermentation method for production of recombination protein by lactose-induced pMFH carrier |
| CN102643888A (en) * | 2012-04-27 | 2012-08-22 | 天津生机集团股份有限公司 | High density fermentation method for prokaryotic expression of chicken interferon alpha |
| CN107266551A (en) * | 2016-04-06 | 2017-10-20 | 慎东 | A kind of MG53 mutant and its preparation method and application |
-
2017
- 2017-12-06 CN CN201711273899.4A patent/CN107760644A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090029410A1 (en) * | 2004-10-08 | 2009-01-29 | Novozymes A/S | Lipases from Thermophilic Anaerobes |
| CN101914603A (en) * | 2010-07-08 | 2010-12-15 | 暨南大学 | Fermentation method for production of recombination protein by lactose-induced pMFH carrier |
| CN102643888A (en) * | 2012-04-27 | 2012-08-22 | 天津生机集团股份有限公司 | High density fermentation method for prokaryotic expression of chicken interferon alpha |
| CN107266551A (en) * | 2016-04-06 | 2017-10-20 | 慎东 | A kind of MG53 mutant and its preparation method and application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536423A (en) * | 2019-01-11 | 2019-03-29 | 宁夏医科大学 | A kind of cultural method and its special culture media of bacillus cereus |
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