A kind of culture medium of feeding Escherichia coli high density fermentation and its application
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.