CN104561200A - Method for effectively improving the soluble expression of recombinant protein in escherichia coli - Google Patents
Method for effectively improving the soluble expression of recombinant protein in escherichia coli Download PDFInfo
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- CN104561200A CN104561200A CN201310517277.7A CN201310517277A CN104561200A CN 104561200 A CN104561200 A CN 104561200A CN 201310517277 A CN201310517277 A CN 201310517277A CN 104561200 A CN104561200 A CN 104561200A
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Abstract
The invention discloses a method for effectively improving the soluble expression of recombinant protein in escherichia coli. The method is mainly characterized in that 1) the synthesis speed of protein is controlled by controlling the supply speed of supplementary C, N and P nutrient sources later, 2) an obvious supplementation gap exists between a thallus reproduction stage and a protein inducible expression stage, and 3) the method can be used in combination with protein low-temperature expression, molecular chaperone co-expression, mark fusion expression and other well-known methods for improving the soluble expression of recombinant protein. The method is applicable to studying the expression of related foreign protein in escherichia coli in bioengineering, molecular biology, genetics, gene engineering and the like, and can remarkably improve the soluble expression of recombinant protein.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of effective method improving foreign protein solubility expression in escherichia coli host.
Background technology
Escherichia expression system is expression of recombinant proteins system the most frequently used in current genetically engineered. have that growth cycle is short, method is easy, structure is quick, expression level advantages of higher, but due to the defect that escherichia expression system exists, make expressed albumen usually easily form inclusion body that is insoluble, inactive.Although can be obtained the solubility of albumen by later stage refolding strategy operation, process is complicated and yield is low, significantly increases the cost of protein purification.In order to obtain bioactive expression of recombinant proteins product, existing relevant scholar or researchist have groped many methods to improve the solubility expression of recombinant protein in intestinal bacteria, provide molecular chaperone protein matter, disulfide bond isomerase, coexpression methods such as proline isomerase or by means of staphylococcus aureus protein A, glutathione-S-transferase, the sulphur reduction fusion tag solubility expression such as albumen or change as reduced the methods such as inducing temperature by culture condition, and at biotechnology, genetically engineered or molecular biology, extensive application is obtained in the transgene expressions such as genetics, but for some albumen, still there is process complexity, complex operation, later-period purification high in cost of production problem.
Summary of the invention
The object of the present invention is to provide a kind of simple method that effectively, can prevent inclusion body products from being formed, can enrich and strengthen the means that researchist improves foreign gene solubility expression, have easy and simple to handle, production cost is low and can scale amplify feature.
The present invention is by the following technical solutions:
1) seed activation: by-80 DEG C of frozen recombinant protein colibacillus engineerings in an aseptic environment according to 0.1% inoculum size and add the microbiotic of respective amount according to concrete host and carrier situation, overnight incubation or 12 ~ 14h in 150 ~ 220rpm shaking table in 36 ~ 38 DEG C of LB or other substratum.
2) fermentor tank inoculation: under flame protection, the inoculum size access seed liquor according to 1 ~ 10% and the microbiotic adding respective amount carry out thalline breeding and cultivate, and temperature 32 ~ 38 DEG C, pH controls 6.5 ~ 7.5, and saturated oxyty controls 10 ~ 50%.Fermentation period and raising thalli growth amount is extended by flow feeding mode in the later stage.
3) abduction delivering: when soon reaching induction period, be interrupted feed supplement and continue 10 ~ 30 minutes, by one or several the limiting nutrient source approach exhaustions in C, N, P in assay determination substratum or lower than under level of control time, start to carry out the abduction delivering that the mode such as temperature variation or inductor interpolation carries out recombinant protein.Separate the mode that stream adds carry out feed supplement subsequently through controlling nutrition source and other feed component.This stage maintains 2 ~ 36 hours.Notice that other nutrition sources should add according to thalline nutritional needs normal stream, formula is improved by process assay in centre.
4) gather in the crops thalline: centrifugal 5 ~ 20 min of 5000 rpm, remove supernatant, collect thalline, be placed in-20 DEG C of preservations.
Present method controlled mainly through the later stage and albumen synthesizes the speed that mode that relevant nutrition source feed supplement stream adds synthesizes to the albumen that slows down, thus improved the solubility expression of recombinant protein in intestinal bacteria.
The feed supplement in described abduction delivering stage can be synthetic medium, semisynthetic medium, complex medium, be preferably full-synthetic culture medium, trace element can be added if desired supplement, as being semisynthetic medium, complex medium, limiting nutrient source contained in its natural organic matter should be noted interior.
The feed supplement in described abduction delivering stage should should be interrupted for some time with the feed supplement of thalli growth stage, the limiting nutrient source in substratum is impelled to exhaust or lower than below later stage level of control, be preferably 10 ~ 30 minutes interval time, be preferably and adopt content detection to control.
The described method improving recombinant protein solubility expression in coli expression system, method such as fusion tag, molecular chaperones and the culture condition change etc. of improvement solubility expression that can be known with other combinationally use.
Embodiment
Be described in conjunction with specific embodiments.
Embodiment 1
Bacterial strain: E.coli IL10 BL21 (DE3) PET15b engineering bacteria (independently purchase and build);
Substratum: LB liquid nutrient medium
Shaking flask is prepared: according to LB formula (sodium-chlor l0g/L, peptone l0g/L, yeast leaching powder 5g/L), after all ingredients is weighed up, add the tertiary effluent of respective amount, make it dissolve and regulate PH to 7.0, packing shaking flask 50ml/bottle (250ml), 121 ° of C 20min sterilizing in high-pressure sterilizing pot.
10L fermentation tank culture medium prepare: according to 6L amount according to LB formula weigh up all ingredients, add the tertiary effluent of respective amount, add in fermentor tank, and take the mode of online sterilizing be cooled to after 121 ° of C 20min sterilizing 37 DEG C for subsequent use.
Thalli growth stage feed supplement preparation: according to Tryptones 50 g/L, yeast leaching powder 25 g/L prepares 1 liter, for subsequent use after 121 ° of C 20min sterilizing in high-pressure sterilizing pot after mixing.
The stage feed supplement of thalline abduction delivering is prepared: according to glucose 100 g/L, preparation 500ml, ammonium nitrate 50 g/L, 3 water dipotassium hydrogen phosphate 14 g/L, and potassium primary phosphate 5.2 g/L, mixed preparing 500ml are for subsequent use after 121 ° of C 20min sterilizing in high-pressure sterilizing pot.
Prepared by first order seed: by recombinant protein colibacillus engineering frozen for-80 ° of C in an aseptic environment according to the inoculum size of 0.1% and the kantlex of final concentration 50ug/ml, in LB substratum 37 DEG C, 200rpm overnight incubation.
Fermentor tank inoculation and thalli growth: under flame protection; amount by 5% adds seed prepared by 300ml and final concentration is the kantlex of 50ug/ml; the ammoniacal liquor adjust ph to 7.0 of online employing mass percent 20%; then at 37 DEG C; pH is 7.0; dissolved oxygen controls the saturated oxyty about 30%, carries out thalli growth cultivation.After dissolved oxygen rises to 50%, then need flow feeding.
Control sample abduction delivering: when cell density OD600 reaches 22 time, directly adding final concentration is that 0.2mM IPTG carries out abduction delivering, feed supplement formula and stream add control as growth phase, induce after 10 hours and sample centrifugal 10 min of 5000 rpm sample in contrast.
The inventive method abduction delivering: when cell density OD600 reaches 22 time, stop flow feeding, after continuing to cultivate 30min, reducing sugar content in sampling and measuring fermented liquid, the front blank sample of induction made by remaining sample, and when reducing sugar is low to moderate 0.05%, adding final concentration is 0.2mM inductor IPTG, stream adds glucose, controls concentration of reduced sugar about 0.1%.Period adds 30ml/ the non-C source feed supplement of mixing at interval of 30min stream.Induce after 10 hours and terminate.
Results thalline: centrifugal 10 min of 5000 rpm, collects thalline.
Sample preparation and detection: take each sample thalline of 2.5g and add 100ml 20 mM Tris-HCl, pH8.0 breaks bacterium damping fluid, stir 10 ~ 20 min and make it to mix, ultrasonication is under 400W power, take 10 minutes ultrasound condition total times, open 2 seconds, close 2 seconds broken thalline.After in broken bacterium liquid, take out 20ul as whole protein sample, the broken bacterium liquid of residue is in 4 DEG C, centrifugal 10min under 12000rpm, as soluble proteins sample, carry out SDS-PAGE inspection after treatment, by bandscan software scans, from result, recombinant protein solubility expression improves 50% than control sample.
Embodiment 2
Bacterial strain: E.coli IL10 BL21 (DE3) PET15b engineering bacteria (our company independently purchases and builds);
Substratum: 2YT liquid nutrient medium (sodium-chlor 5g/L, peptone l6g/L, yeast leaching powder 10g/L)
Shaking flask is prepared: according to 2YT formula, after being weighed up by all ingredients, add the tertiary effluent of respective amount, make it dissolve and regulate PH to 7.0, packing shaking flask 50ml/bottle (250ml), 121 ° of C 20min sterilizing in high-pressure sterilizing pot.
10L fermentation tank culture medium prepare: according to 6L amount according to 2YT formula weigh up all ingredients, adopt tertiary effluent be settled to 6L, add in fermentor tank, and take the mode of online sterilizing be cooled to after 121 ° of C 20min sterilizing 37 DEG C for subsequent use.
Thalli growth stage feed supplement preparation: according to Tryptones 50 g/L, yeast leaching powder 25 g/L prepares 1 liter, for subsequent use after 121 ° of C 20min sterilizing in high-pressure sterilizing pot after mixing.
The stage feed supplement of thalline abduction delivering is prepared: according to ammonium nitrate 100 g/L, preparation 500ml, glucose 100 g/L, 3 water dipotassium hydrogen phosphate 14 g/L, potassium primary phosphate 5.2 g/L, mixed preparing 500ml, for subsequent use after 121 ° of C 20min sterilizing in high-pressure sterilizing pot.
Prepared by first order seed: by recombinant protein colibacillus engineering frozen for-80 ° of C in an aseptic environment according to the inoculum size of 0.1% and the kantlex of final concentration 50ug/ml, in LB substratum 37 DEG C, 200rpm overnight incubation.
Fermentor tank inoculation and thalli growth: under flame protection; amount by 5% adds seed prepared by 300ml and final concentration is the kantlex of 50ug/ml; the ammoniacal liquor adjust ph to 7.0 of online employing mass percent 20%; then at 37 DEG C; pH is 7.0; dissolved oxygen controls the saturated oxyty about 30%, carries out thalli growth cultivation.After dissolved oxygen rises to 50%, then need flow feeding.
Control sample abduction delivering: when cell density OD600 reaches 22 time, directly adding final concentration is that 0.2mM IPTG carries out abduction delivering, feed supplement formula and stream add control as growth phase, induce after 6 hours and sample centrifugal 10 min of 5000 rpm sample in contrast.
The inventive method abduction delivering: when cell density OD600 reaches 22 time, stop flow feeding, after continuing to cultivate 20min, amino nitrogen content in sampling and measuring fermented liquid, the front blank sample of induction made by remaining sample, and when amino nitrogen content is low to moderate 1%, adding final concentration is that 0.2mM IPTG induces, stream adds ammonium nitrate, controls amino nitrogen about 1%.Period adds 30ml/ the non-N source feed supplement of mixing at interval of 30min stream.Induce after 6 hours and terminate.
Results thalline: centrifugal 10 min of 5000 rpm, collects thalline.
Sample preparation and detection: take each sample thalline of 2.5g and add 100ml 20 mM Tris-HCl, pH8.0 breaks bacterium damping fluid, stir 10 ~ 20 min and make it to mix, ultrasonication is under 400W power, take 10 minutes ultrasound condition total times, open 2 seconds, close 2 seconds broken thalline.After in broken bacterium liquid, take out 20ul as whole protein sample, the broken bacterium liquid of residue is in 4 DEG C, centrifugal 10min under 12000rpm, as soluble proteins sample, carry out SDS-PAGE inspection after treatment, by bandscan software scans, from result, recombinant protein solubility expression sample improves 30% than contrast.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. one kind is effectively improved the method for recombinant protein solubility expression in intestinal bacteria, it is characterized in that: after intestinal bacteria induction, the resultant velocity reducing albumen is controlled by the supply of one or several in C, N, P nutrition source in later stage feed supplement, increase the time that albumen higher structure is folding, thus realize the object improving solubility expression of protein.
2. one kind is effectively improved the method for recombinant protein solubility expression in intestinal bacteria, it is characterized in that: there is an obvious feed supplement when intestinal bacteria high density fermentation between growing microorganism and protein induced expression and supply the rest periods, with run out of early stage supply enrich nutrient, thus be transitioned into later stage nutrition controlled abduction delivering feed supplement supply the stage.
3. effectively improve a method for recombinant protein solubility expression in intestinal bacteria, it is characterized in that: this kind of method can be expressed with other known low temperature inductions, in soluble flag's amalgamation and expression, molecular chaperones coexpression and substratum after between add some and can promote to combinationally use together with the method for albumen soluble constituents (as alcohol, sorbyl alcohol etc.) etc.
4. according to claim 1, in later stage feed supplement, C, N, P nutrition source can be synthetic medium, semisynthetic medium or complex media components, is preferably synthetic medium composition.
5., according to claim 2, the length of feed supplement supply interval, judges by saturated oxyty changing conditions and restriction nutrition source content detection mode, is preferably restriction nutrition source content detection mode.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154501A (en) * | 2015-10-26 | 2015-12-16 | 新疆大学 | Technology for fermenting halostachys caspica metallothionein and application of halostachys caspica metallothionein |
| CN105543144A (en) * | 2016-02-02 | 2016-05-04 | 武汉爱博泰克生物科技有限公司 | Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof |
| CN110819582A (en) * | 2019-11-28 | 2020-02-21 | 华派生物工程集团有限公司 | Fermentation medium and culture method for expressing foot-and-mouth disease virus-like particle antigen by using escherichia coli |
| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
-
2013
- 2013-10-29 CN CN201310517277.7A patent/CN104561200A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 姚鹏等: "低温诱导堆型艾美耳球虫3-1E基因的可溶性表达", 《北京农学院学报》 * |
| 袁辉等: "重组人酸性成纤维细胞生长因子改构体发酵的补料-分批策略研究", 《生物工程学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154501A (en) * | 2015-10-26 | 2015-12-16 | 新疆大学 | Technology for fermenting halostachys caspica metallothionein and application of halostachys caspica metallothionein |
| CN105154501B (en) * | 2015-10-26 | 2018-12-28 | 新疆大学 | A kind of caspian halostachys metallothionein zymotechnique and its application |
| CN105543144A (en) * | 2016-02-02 | 2016-05-04 | 武汉爱博泰克生物科技有限公司 | Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof |
| CN110819582A (en) * | 2019-11-28 | 2020-02-21 | 华派生物工程集团有限公司 | Fermentation medium and culture method for expressing foot-and-mouth disease virus-like particle antigen by using escherichia coli |
| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
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