CN106434426A - High-efficiency expression culture medium for escherichia coli - Google Patents
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Abstract
The invention provides a high-efficiency expression culture medium for escherichia coli. The high-efficiency expression culture medium for escherichia coli comprises an escherichia coli culture medium and additives, the escherichia coli culture medium comprises peptone, yeast powder and sodium chloride, and the additives include peptone, yeast extracts, a carbon source substance, an ionic element, surfactant and protein stabilizer. As vitamin, multivalence peptone, casein peptone, the ionic element, surfactant and other nutrient substances are added into the high-efficiency expression culture medium for escherichia coli, the culture medium can support high-density growth of escherichia coli under the fermentation tank culture condition, and soluble protein of high proportion can be obtained.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of escherichia coli high-level expression culture medium.
Background technology
Escherichia coli (Escherichia Coli) are as the host of exogenous gene expression, and genetic background understands, technology is grasped
Making simple, condition of culture is simple, and contamination resistance is strong, large scale fermentation economy, the extremely attention of genetic engineering expert.Big at present
Enterobacteria is most widely used general, most successful expression system, often does the first-selected system of high efficient expression, moves with yeast, insect, lactation
Thing referred to as 4 big heterologous expression systems.The foreign gene (albumen) that each expression system is suitably expressed is different, and wherein Escherichia coli are more
It is suitable for the expression of prokaryotic gene.Escherichia coli are as the important system of expression alien gene, by everybody extensive concern.Highly dense
It is that bio-pharmaceuticals mass produces mode used that degree is cultivated, and its product yield is influenced by factors, as：Bacterial strain, password
Sub-Preference, expression vector, temperature, induced concentration and opportunity, dissolved oxygen, pH value etc..As genetic engineering especially molecule is raw
What thing learned a skill develops rapidly, and bacterial strain, codon preference, vector modification have had qualitative leap, biological culture engineering such as fermentation tank
Research deeply, the culture environment research such as dissolved oxygen, pH is also more careful, drastically increases target product especially solubility product
The yield of thing, improves productivity effect.
Basis as cell growth---culture medium is not kept pace with the times progress, and research is stagnated.Producer is used mostly to use
More classical formula or oneself configuration formula, used medium cannot form extensive batch and produce, thus affect and produce into
This and product are stable.
Content of the invention
Extensive batch cannot be formed produce to solve above-mentioned culture medium, thus affect production cost and product is stable
Technical problem, the present invention provides a kind of culture medium can form extensive batch and produces, and reduces production cost and raising product is steady
Fixed escherichia coli high-level expression culture medium.
The escherichia coli high-level expression culture medium that the present invention provides includes Escherichia coli culture medium and additive, described large intestine
Baccilus medium is the Escherichia coli culture medium of peptone, dusty yeast, sodium chloride, the component of described additive include peptone,
Yeast extract, carbon source material, ion elements, surfactant and protein stabiliser.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described peptone is multiple
Peptone, including peptone, casein peptone, tryptone, soy peptone and wheat bran hydrolysate, described peptone content is 1-
6g/L, described casein peptone content is 1-6g/L, and described tryptone content is 1-3g/L, and described soy peptone content is 1-
3g/L, described wheat bran hydrolysate content is 0.1-0.3g/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described yeast extract
Addition is 3-6g/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described carbon source material includes
Glucose, sucrose, maltose and glycerine, described glucose content is 1-10g/L, and described cane sugar content is 1-10g/L, described wheat
Bud sugar content is 1-5g/L, and described glycerine is 1-3g/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described ion elements includes
Sodium chloride, ferrous sulfate heptahydrate, MnSO4·nH2O, white vitriol, seven water aluminium chloride, copper chloride, cobalt chloride and HBO4, described
Sodium chloride content is 3-8g/L, and described ferrous sulfate heptahydrate content is 1-20mg/L, described MnSO4·nH2O content is 1-10mg/
L, described white vitriol content is 0.1-2mg/L, and described seven water aluminium chloride content are 0.1-10mg/L, described copper chloride content
For 0.10-1mg/L, described cobalt chloride content is 0.1-4mg/L, described HBO4Content is 0.01-1mg/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described surfactant is
Pluronic, its content is 1-5g/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described protein stabiliser bag
Including Aprotinin, PMSF, TPCK and sorbierite, described Aprotinin content is 0.02-6 μ g/ml, and described PMSF content is 17-170 μ
G/ml, described TPCK content is 70-100 μ g/ml, and described sorbierite is 6-60g/L.
In a kind of preferred embodiment of the escherichia coli high-level expression culture medium that the present invention provides, described additive also includes
Putrescine, described putrescine content is 0.1-1g/L.
The described escherichia coli high-level expression culture medium that utilizes providing in the present invention cultivates Escherichia Coli bacterium
The method planted, comprises the steps：
Step one, Escherichia Coli bacterial screening and preservation：Select E.coli B and derivative strain thereof as table
Reach Host Strains, select the alternately bacterial strain such as BL21 (DE3)/BL21 (Rosetta)/BL21 (PLysS/E)；
Step 2, the selection of culture medium and optimization：Make synthetic media；
Step 3, fermentation tank are cultivated bacterial classification：It is dissolved oxygen 30-50% in parameter, rotating speed 150-300 rev/min, pH6.9-
The fermentation tank of 7.6 adds culture medium abduction delivering.
The points for attention of the escherichia coli high-level expression culture medium of the present invention and beneficial effect：
First, Escherichia Coli bacterial screening and preservation
Different coli strains also exists very big difference, Host Strains on condition of culture and exogenous gene expression ability
Selection to the accumulation of expression product and isolated and purified most important.Some bacterial classifications can reach higher bacterium in incubation
Volume density, the then product acid having is more.If E.coli K and E.coli B and derivative strain thereof are the Host Strains more commonly used, these are two years old
Class bacterial strain has different growth characteristics, and wherein related to a High Density Cultivation significant difference is exactly that E.coli B bacterial strain exists
The acetic acid producing in high-density culture process is far less than E.coli K.Therefore, when High Density Cultivation, choose most suitable
Expressive host bacterium.Our experiment also demonstrates this point, unless considered other factors such as gene expression, plasmid stabilisation, as far as possible
B strain is selected to carry out next step experiment.The bacterial strain being beneficial to soluble protein expression may be different because expressing gene is different, institute
To generally select the alternately bacterial strain such as BL21 (DE3)/BL21 (Rosetta)/BL21 (PLysS/E).
2nd, the selection of culture medium and optimization
In order to reach High Density Cultivation, generally select synthetic media, its main component include carbon source, nitrogen source, vitamin,
Trace element etc..The biomass of high-cell-density cultivation can reach 200g/L, needs to put into the matrix ability times over biomass
Meet cell fast-growth and the needs of great expression gene outcome.But the concentration of matrix Middle nutrition material can not be too high, exceedes
Certain limit can make growth inhibitory substance accumulate in a large number, makes the expression of colibacillary growth and breeding and genes of interest be pressed down
System.
3rd, the escherichia coli high-level expression culture medium of the present invention can form the production of extensive batch, it is possible to decrease produces into
This, improve product stability.
Brief description
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, required in embodiment being described below make
Accompanying drawing be briefly described, it should be apparent that, below describe in accompanying drawing be only some embodiments of the present invention, for
From the point of view of those of ordinary skill in the art, on the premise of not paying creative work, can also be obtained other according to these accompanying drawings
Accompanying drawing, wherein：
Fig. 1 is the technological process of a kind of embodiment of preparation method of the escherichia coli high-level expression culture medium that the present invention provides
Figure；
Fig. 2 is Escherichia coli (Rosetta) obtainable thalline in the escherichia coli high-level expression culture medium that the present invention provides
Weight in wet base；
Fig. 3 is expressions in the escherichia coli high-level expression culture medium that the present invention provides for the Escherichia coli (Rosetta).
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments wholely.Based on
Embodiment in the present invention, it is all other that those of ordinary skill in the art are obtained under the premise of not making creative work
Embodiment, broadly falls into the scope of protection of the invention.
Described escherichia coli high-level expression culture medium 1 includes Escherichia coli culture medium and additive, and described Escherichia coli are trained
Foster base is the Escherichia coli culture medium of peptone, dusty yeast, sodium chloride, and the component of described additive includes that peptone, yeast carry
Take thing, carbon source material, ion elements, surfactant and protein stabiliser.
Described peptone is multiple protein peptone, including peptone, casein peptone, tryptone, soy peptone and wheat bran
Hydrolysate, described peptone content is 1-6g/L, and described casein peptone content is 1-6g/L, and described tryptone content is 1-
3g/L, described soy peptone content is 1-3g/L, and described wheat bran hydrolysate content is 0.1-0.3g/L.
The addition of described yeast extract is 3-6g/L.
Described carbon source material includes glucose, sucrose, maltose and glycerine, and described glucose content is 1-10g/L, described
Cane sugar content is 1-10g/L, and described maltose content is 1-5g/L, and described glycerine is 1-3g/L.
Described ion elements includes sodium chloride, ferrous sulfate heptahydrate, MnSO4·nH2O, white vitriol, seven water aluminium chloride,
Copper chloride, cobalt chloride and HBO4, described sodium chloride content is 3-8g/L, and described ferrous sulfate heptahydrate content is 1-20mg/L, institute
State MnSO4·nH2O content is 1-10mg/L, and described white vitriol content is 0.1-2mg/L, and described seven water aluminium chloride content are
0.1-10mg/L, described copper chloride content is 0.10-1mg/L, and described cobalt chloride content is 0.1-4mg/L, described HBO4Content
For 0.01-1mg/L.
Described surfactant is pluronic, and its content is 1-5g/L.
Described protein stabiliser includes Aprotinin, PMSF, TPCK and sorbierite, and described Aprotinin content is 0.02-6 μ g/
Ml, described PMSF content is 17-170 μ g/ml, and described TPCK content is 70-100 μ g/ml, and described sorbierite is 6-60g/L.
Described additive also includes putrescine, and described putrescine content is 0.1-1g/L.
Refer to Fig. 1, be a kind of embodiment of preparation method of the escherichia coli high-level expression culture medium that the present invention provides
Process chart.
Utilize method S1 that described escherichia coli high-level expression culture medium cultivates Escherichia Coli bacterial classification, including
Following steps：
S11, Escherichia Coli bacterial screening and preservation：Select E.coli B and derivative strain thereof as expression place
Main bacterium, selects the alternately bacterial strain such as BL21 (DE3)/BL21 (Rosetta)/BL21 (PLysS/E)；
S12, the selection of culture medium and optimization：Make synthetic media；
S13, fermentation tank are cultivated bacterial classification：It is dissolved oxygen 30-50% in parameter, rotating speed 150-300 rev/min, pH6.9-7.6's
Fermentation tank adds culture medium abduction delivering.
Escherichia coli high-level expression culture medium 1 of the present invention and preparation method thereof has the advantages that：
The escherichia coli high-level expression culture medium of the present invention can form extensive batch and produce, it is possible to decrease production cost,
Improve product stability.
In order to reach High Density Cultivation, generally select synthetic media, its main component include carbon source, nitrogen source, vitamin,
Trace element etc..The biomass of high-cell-density cultivation can reach 200g/L, needs to put into the matrix ability times over biomass
Meet cell fast-growth and the needs of great expression gene outcome.But the concentration of matrix Middle nutrition material can not be too high, exceedes
Certain limit can make growth inhibitory substance accumulate in a large number, makes the expression of colibacillary growth and breeding and genes of interest be pressed down
System.
1) peptone：Peptone contains abundant amino acid especially sulfur-containing amino acid, enriches for bacterial growth offer
Amino acid, is also the important sources of vitamin and other growth factors.
2) yeast extract：Yeast extract is that yeast will wherein protein, nucleic acid, vitamin etc. extract after broken wall,
Again through active skull cap components such as the amino acid rich in little molecule of biological enzymolysis, peptide, nucleotides, vitamins.
3) carbon source material：Carbohydrate is good sources of carbon and the energy substance that Escherichia coli are easier to utilize.First it is considered
Content, mainly affects the carbon-nitrogen ratio in culture medium.Carbon source is too much, then the relatively low pH in easy execution ground, suppresses thalli growth, and carbon source is not
Foot, then easily cause aging and the self-dissolving of thalline.In addition, carbon-nitrogen ratio is improper also can cause thalline pro rata absorption nutrients
Matter, thus directly affect the growth of thalline and the synthesis of product.Secondly the constituent of carbohydrate is considered, the high fermentation of glucose content
During can produce the accumulation of more acetic acid, impact growth.
4) ion elements：Trace element is the confactor of multiple enzymes in Metabolism of E. coli approach, and its content affects
Colibacillary metabolism.
5) surfactant：The gas that the interpolation of surfactant or defoamer can produce because of stirring with less sweat
Bubble, less shearing force.Reduce and pollute and the impact on protein stability.
6) protein stabiliser：Protease inhibitors can suppress the activity of intracellular multiple enzyme, thus protects cell to produce
The integrality of albumen, improves yield.Some reagent such as sorbierite can play stable albumen natural structure.
The escherichia coli high-level expression culture medium 1 of the present invention is tested as follows, to test its effect：
E. coli bl21 (Rosetta) bacterial strain is cultivated by this research at autonomous invention culture medium JYK-EBM, and
And using conventional medium LB, M9 and certain brand X product culture medium domestic as comparison, through in 50L fermentation tank in container,
(parameter is set to dissolved oxygen 30-50%, rotating speed 150-300 rev/min, pH6.9-7.6) adds lactose-induced expression to obtain for 10 hours
Bacterial strain thalline weight in wet base up to 200g/L, in control group, thalline weight in wet base is then for being 70g/L in LB culture medium, in M9 culture medium is
82g/L, X culture medium is 190g/L (as shown in Figure 2).
Refer to Fig. 2, be that Escherichia coli (Rosetta) can in the escherichia coli high-level expression culture medium that the present invention provides
The thalline weight in wet base obtaining.
Thalline, through resuspended after PBS 3 times, washes away original fluid.After ultrasonication, 12000rpm is centrifugal obtains supernatant.
The albumen of solubility expression is i.e. in supernatant, and total leukorrhea content in Kjeldahl nitrogen determination supernatant, SDS-PAGE method analyzes target
Albumen accounts for the ratio (target protein ratio in data) of total protein content, thus calculates target protein content in supernatant.
Equally, the description of expression：Certain soluble protein (T4-DNA Ligase in PET30a in BL-21
(DE3)) 36% is risen to by 20% containing accounting for total protein content.I.e. target protein ratio exceeds 16 percentages than chief competitor
Point (as shown in Figure 2).
Refer to Fig. 3, be that Escherichia coli (Rosetta) are in the escherichia coli high-level expression culture medium that the present invention provides
Expression.
Inner Mongol Jin Yuankang bioengineering Co., Ltd Escherichia coli culture medium JYK-EBM product is wet at thalline in sum
While heavily increasing, the ratio of soluble protein also increases, thus substantially increases target protein content.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
Equivalent structure or equivalence flow process that bright specification and accompanying drawing content are made convert, or are directly or indirectly used in other related skills
Art field, all in like manner includes in the scope of patent protection of the present invention.
Claims (9)
1. an escherichia coli high-level expression culture medium, it is characterised in that include Escherichia coli culture medium and additive, described greatly
Enterobacteria culture medium is the Escherichia coli culture medium of peptone, dusty yeast, sodium chloride, and the component of described additive includes albumen
Peptone, yeast extract, carbon source material, ion elements, surfactant and protein stabiliser.
2. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described peptone is multiple eggs
White peptone, including peptone, casein peptone, tryptone, soy peptone and wheat bran hydrolysate, described peptone content is 1-
6g/L, described casein peptone content is 1-6g/L, and described tryptone content is 1-3g/L, and described soy peptone content is 1-
3g/L, described wheat bran hydrolysate content is 0.1-0.3g/L.
3. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that adding of described yeast extract
Dosage is 3-6g/L.
4. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described carbon source material includes Portugal
Grape sugar, sucrose, maltose and glycerine, described glucose content is 1-10g/L, and described cane sugar content is 1-10g/L, described Fructus Hordei Germinatus
Sugar content is 1-5g/L, and described glycerine is 1-3g/L.
5. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described ion elements includes chlorine
Change sodium, ferrous sulfate heptahydrate, MnSO4·nH2O, white vitriol, seven water aluminium chloride, copper chloride, cobalt chloride and HBO4, described chlorine
Changing sodium content is 3-8g/L, and described ferrous sulfate heptahydrate content is 1-20mg/L, described MnSO4·nH2O content is 1-10mg/L,
Described white vitriol content is 0.1-2mg/L, and described seven water aluminium chloride content are 0.1-10mg/L, and described copper chloride content is
0.10-1mg/L, described cobalt chloride content is 0.1-4mg/L, described HBO4Content is 0.01-1mg/L.
6. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described surfactant is general
Lang Nike, its content is 1-5g/L.
7. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described protein stabiliser includes
Aprotinin, PMSF, TPCK and sorbierite, described Aprotinin content is 0.02-6 μ g/ml, and described PMSF content is 17-170 μ g/
Ml, described TPCK content is 70-100 μ g/ml, and described sorbierite is 6-60g/L.
8. escherichia coli high-level expression culture medium according to claim 1, it is characterised in that described additive also includes corruption
Amine, described putrescine content is 0.1-1g/L.
9. one kind utilizes the escherichia coli high-level expression culture medium that in claim 1 to 8, any one is described to cultivate
The method of Escherichia Coli bacterial classification, it is characterised in that comprise the steps：
Step one, Escherichia Coli bacterial screening and preservation：Select E.coli B and derivative strain thereof as expression place
Main bacterium, selects the alternately bacterial strain such as BL21 (DE3)/BL21 (Rosetta)/BL21 (PLysS/E)；
Step 2, the selection of culture medium and optimization：Make synthetic media；
Step 3, fermentation tank are cultivated bacterial classification：It is dissolved oxygen 30-50% in parameter, rotating speed 150-300 rev/min, pH6.9-7.6's
Fermentation tank adds culture medium abduction delivering.
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