CN104628832A - Method for purifying regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid - Google Patents
Method for purifying regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid Download PDFInfo
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Abstract
The invention belongs to the technical field of protein purification, and relates to the field of downstream protein purification of biological medicine and bioengineering, and discloses a method for purifying a regrouped chicken colibacillosis outer membrane protein inclusion body and carrying out renaturation by utilizing high-efficiency renaturation liquid. By adopting a three-step washing purification technological method of the inclusion body proteins, mycoproteins can be well separated from the inclusion body, the renaturation rate of the proteins can be greatly improved by virtue of a renaturation solution and a dropwise feeding one-step renaturation technological method, the renaturation process and needed equipment can be simplified, the yield rate of the target protein can be greatly increased, and the production cost of the regrouped chicken colibacillosis outer membrane protein can be reduced. The regrouped chicken colibacillosis outer membrane protein purification method is simple and easy, and the column-passing treatment is not needed.
Description
Technical field
What the present invention relates to is biological medicine and biotechnology downstream protein purification art, relate to a kind of purifying of recombination chicken escherichia coli outer membrane protein inclusion body and highly efficient renaturation liquid more specifically to the technique of its renaturation.
Background technology
Gram negative bacterium outer membrane protein plays an important role to bacterium opposing objectionable impurities or environment; it can Cell protection from or reduce the impact of multiple harmful substances; as proteolytic enzyme, microbiotic, toxin and phage etc., and the existence of maintenance gram negative bacterium under the stress conditions such as high osmotic pressure, acidity is played an important role.In addition, nonspecific ionic channel that outer membrane protein is formed absorbs nutritive substance to gram negative bacterium and excretion own metabolism refuse also plays keying action.Outer membrane protein is also referred to as porin, is the main moiety of adventitia, and the important outer membrane protein of intestinal bacteria has OmpC, OmpF and PhoE etc., and they are across lipid bilayer.In host, the kind of outer membrane protein and the change of concentration all can make the form of scavenger cell and function change; In vitro, outer membrane protein has interactional ability between complement system.It both can be used as adjuvant, can be used as again that immunostimulation is former to be applied, and thus can be applicable to the research and development of high-titer antibody and vaccine.Avian Escherichia coli bacterial outer membrane protein molecular weight is the protein of 35 ~ 45KD, has the protein general character, to thermally-stabilised, can preserve the long period for 2-8 DEG C, can preserve its activity for a long time, easily be neutralized by nuclease for 20 DEG C, can by proteolytic enzyme deactivation.Outside genetically engineered recombination chicken intestinal bacteria, membranin C is biological products that are nontoxic, harmless, noresidue, certain effect is had to suppressing viral copying and improve immunity of organisms aspect, therefore, be expected to be applied in poultry cultivation industry, to solving the subproblem existed in current China aquaculture, for China's avian health escorts, ensure for food safety provides green.
Intestinal bacteria are the expression systems being widely used for expressing foreign protein at present the most, but intestinal bacteria great expression recombinant protein usually causes its aggregate and forms albumen precipitation-inclusion body.Inclusion body protein does not have biological activity, needs complicated dissolving, renaturation, the product obtaining function that purifying comes.
Current protein renaturation has two kinds of methods usually.One is dilution method, and dilution is dissolved with the sex change liquid of albumen, and when in solution, denaturant concentration is enough low, albumen starts renaturation; But dialysis method, with means removing denaturing agents such as dialysis, ultrafiltration.Dilution method operating liquid amount is too large, and protein concn is too little simultaneously, for follow-up purifying protein is added to the difficulties, increases production cost.Dialysis method is higher to equipment requirements, adds the input of equipment, and the operating time is longer simultaneously, may cause protein precipitation.At present, the rate of recovery of the renaturation of inclusion body is probably 15-25%, and the target protein that major part is valuable is because can not renaturation and can not utilizing.The reason that renaturation yield is lower is, lacks the cofactor of some protein folding in the renaturation process of recombinant protein, or environment is uncomfortable and cannot form correct secondary key etc.
Therefore, if one can be found can to improve protein renaturation rate, and the method that effectively can reduce renaturation cost becomes a key difficult problem of gene recombinant protein drug manufacture.
Summary of the invention
In order to overcome the existing outer membrane protein inclusion body operation steps that renaturing inclusion bodies method needs are complicated aborning, cycle is long, and the shortcoming that the rate of recovery is low, provide a kind of purifying of recombination chicken escherichia coli outer membrane protein inclusion body and highly efficient renaturation liquid to the method for its renaturation.
The purifying of a kind of recombination chicken escherichia coli outer membrane protein of the present invention inclusion body and highly efficient renaturation liquid, to the method for its renaturation, comprise the following steps:
(1) extraction of inclusion body protein: 4 DEG C, 4000 ~ 6000rpm, 10 ~ 20min, collected by centrifugation thalline, PBS washs thalline, 4 DEG C, 6000 ~ 8000rpm, 10 ~ 20min, by thalline ultrasonication, power 400 watts, ultrasonic 5 seconds, interval 25 seconds, 20 ~ 35 minutes working hours, described thalline is restructuring chicken colibacillosis; Then 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation obtains avian Escherichia coli bacterial outer membrane protein inclusion body protein;
(2) purifying of inclusion body protein: be first resuspended in the first washings by mass volume ratio 1:10 ~ 1:30 by inclusion body, described washings is: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO
42mmol/L, the tritonX-100 of 0.2% ~ 1.4%, pH 8.0 ~ 8.5; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Then by mass volume ratio 1:10 ~ 1:30, inclusion body is resuspended in the second washings kind, described second washings is: 1 ~ 3mol/L urea; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Then be resuspended in the 3rd washings by mass volume ratio 1:20 ~ 1:50 by inclusion body, described 3rd washings is: 0.6 ~ 2mol/L NaCl, 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Finally by mass volume ratio 1:30 ~ 1:50, inclusion body is resuspended in 0.5 ~ 2% sodium lauryl sulphate (Sodium lauryl sulfate is called for short SLS), 4 DEG C, 8000 ~ 10000rpm, 20 ~ 30min high speed frozen centrifugation, collecting precipitation;
(3) dissolving of inclusion body protein and renaturation, be that the precipitation denaturing agent that step (2) obtains by 1:10 ~ 1:40 is resuspended by mass volume ratio, described denaturing agent is: 5 ~ 7mol/L Guanidinium hydrochloride, 1 ~ 2mmol/L EDTA, 20 ~ 50mmol/L Tris-Base, 6 ~ 10mmol/L DTT (i.e. DL-Dithiothreitol, Chinese name: dithiothreitol (DTT)), pH 8.0 ~ 8.5; Room temperature is placed to precipitation and dissolves gradually; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation, collects supernatant liquor; Under 4 DEG C of conditions, sex change liquid is dropwise added in renaturation buffer, described renaturation buffer is: 0.2 ~ 0.8mol/L arginine, 0.5 ~ 2mmol/L EDTA, 0.3 ~ 0.9mmol/L Sleep-promoting factor B, 60 ~ 100mmol/L Tris-Base, 15 ~ 30% glycerine, pH 8.0 ~ 8.5, stirs 24 ~ 48h simultaneously.
In described step (3), sex change liquid adds mode in renaturation buffer is dropping, and the volume ratio of sex change liquid and renaturation buffer is 1:20 ~ 1:50.
The present invention has following beneficial effect:
The present invention can significantly improve renaturation yield, and equipment requirements is simple.The ratio of present method appropriate change inclusion body washings, component and washing methods carry out purifying inclusion body; By renaturation agent, the ionic strength and refolding method etc. of appropriate change renaturation buffer, improve the renaturation yield of recombination chicken escherichia coli outer membrane protein inclusion body, simplify the step of renaturation, saved the time, thus greatly reduce the expense of the production of recombinant protein.To large-scale commercial production, there is directive significance.
This recombination chicken escherichia coli outer membrane protein simple purification method is easy, without the need to carrying out post process.Three step washing purification techniques of inclusion body protein of the present invention, tropina is well separated with inclusion body, pass through the processing method of renaturation solution and a drop application of sample renaturation again, the renaturation yield of protein is improved greatly, simplify renaturation technique and required equipment simultaneously, thus greatly improve the yield rate of target protein, reduce the production cost of recombination chicken escherichia coli outer membrane protein.
Embodiment
To achieve these goals, design a kind of purifying of recombination chicken escherichia coli outer membrane protein inclusion body and highly efficient renaturation liquid to the method for its renaturation, concrete processing step is as follows:
After the extraction IPTG abduction delivering of 1, inclusion body protein, collected by centrifugation thalline, by thalline ultrasonication, high speed frozen centrifugation, obtains avian Escherichia coli bacterial outer membrane protein inclusion body protein;
2, the purifying of inclusion body protein is according to certain mass volume ratio, inclusion body is resuspended in respectively washings 1, washings 2 and washings 3 and washs, eventually pass SLS sedimentation albumen, high speed frozen centrifugation, collecting precipitation;
3, the dissolving of inclusion body protein and renaturation are by certain mass volume ratio by resuspended for precipitation denaturing agent, and room temperature is placed to precipitation and dissolves gradually; Then high speed frozen centrifugation, collects supernatant liquor; Under 4 DEG C of conditions, sex change liquid is dropwise added in renaturation buffer, stirs 24 ~ 48h simultaneously.
Below in conjunction with specific embodiment, the invention will be further described, but do not limit the present invention.
Step 1:
Cultivate recombination bacillus coli, 37 DEG C, 200rpm abduction delivering recombinant protein makes it form inclusion body.The extraction of inclusion body protein, 4 DEG C, 5000rpm, 15min, collected by centrifugation thalline, PBS washs thalline, 4 DEG C, 8000rpm, 10min.Be resuspended in fragmentation in PBS damping fluid, power 400 watts, ultrasonic 5 seconds, interval 25 seconds, working hour 20 ~ 35min by ultrasonic for 10g wet thallus, then 4 DEG C, 8000rpm, 20min high speed frozen centrifugation obtains avian Escherichia coli bacterial outer membrane protein inclusion body protein;
Step 2:
The purifying of inclusion body protein, is first resuspended in the first washings (NaCl137mmol/L, KCl 2.7mmol/L, Na by mass volume ratio 1:10 by inclusion body
2hPO
410mmol/L, KH
2pO
42mmol/L, tritonX-100, the pH8.5 of 0.8%), 4 DEG C, 8000rpm, 20min high speed frozen centrifugation; Then by mass volume ratio 1:10, inclusion body is resuspended in the second washings (1.5mol/L urea), 4 DEG C, 8000rpm, 20min high speed frozen centrifugation; Then by mass volume ratio 1:20, inclusion body is resuspended in the 3rd washings (1mol/L NaCl), 4 DEG C, 8000rpm, 20min high speed frozen centrifugation, collecting precipitation; Finally by mass volume ratio 1:30, inclusion body is resuspended in 0.5%SLS, 4 DEG C, 10000rpm, 25min high speed frozen centrifugation, collecting precipitation;
Step 3:
The dissolving of inclusion body protein and renaturation are that 1:20 is by precipitation denaturing agent (6mol/L Guanidinium hydrochloride, the 2mmol/LEDTA of step 2 by mass volume ratio, 50mmol/L Tris-Base, 6mmol/L DTT, pH8.5) resuspended, room temperature is placed to precipitation and dissolves gradually; 4 DEG C, 8000rpm, 20min high speed frozen centrifugation, collects supernatant liquor; Under 4 DEG C of conditions, make sex change liquid dropwise add (0.2mol/L arginine, 0.5mmol/L EDTA, 0.3mmol/L Sleep-promoting factor B, 60mmol/L Tris-Base, 15 glycerine, pH8.5) in renaturation buffer, stir 24 ~ 48h simultaneously.Results renaturation recombination chicken escherichia coli outer membrane protein.Measure through SDS-PAGE, purity reaches more than 90%, and renaturation yield is 36%.
Claims (2)
1. the purifying of recombination chicken escherichia coli outer membrane protein inclusion body and highly efficient renaturation liquid are to a method for its renaturation, it is characterized in that comprising the following steps:
(1) extraction of inclusion body protein: 4 DEG C, 4000 ~ 6000rpm, 10 ~ 20min, collected by centrifugation thalline, PBS washs thalline, 4 DEG C, 6000 ~ 8000rpm, 10 ~ 20min, by thalline ultrasonication, power 400 watts, ultrasonic 5 seconds, interval 25 seconds, 20 ~ 35 minutes working hours, described thalline is restructuring chicken colibacillosis; Then 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation obtains avian Escherichia coli bacterial outer membrane protein inclusion body protein;
(2) purifying of inclusion body protein: be first resuspended in the first washings by mass volume ratio 1:10 ~ 1:30 by inclusion body, described washings is: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO
42mmol/L, the tritonX-100 of 0.2% ~ 1.4%, pH 8.0 ~ 8.5; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Then by mass volume ratio 1:10 ~ 1:30, inclusion body is resuspended in the second washings kind, described second washings is: 1 ~ 3mol/L urea; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Then be resuspended in the 3rd washings by mass volume ratio 1:20 ~ 1:50 by inclusion body, described 3rd washings is: 0.6 ~ 2mol/L NaCl, 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation; Finally by mass volume ratio 1:30 ~ 1:50, inclusion body is resuspended in 0.5 ~ 2% sodium lauryl sulphate, 4 DEG C, 8000 ~ 10000rpm, 20 ~ 30min high speed frozen centrifugation, collecting precipitation;
(3) dissolving of inclusion body protein and renaturation, be that the precipitation denaturing agent that step (2) obtains by 1:10 ~ 1:40 is resuspended by mass volume ratio, described denaturing agent is: 5 ~ 7mol/L Guanidinium hydrochloride, 1 ~ 2mmol/L EDTA, 20 ~ 50mmol/L Tris-Base, 6 ~ 10mmol/L dithiothreitol (DTT), pH 8.0 ~ 8.5; Room temperature is placed to precipitation and dissolves gradually; 4 DEG C, 8000 ~ 10000rpm, 10 ~ 20min high speed frozen centrifugation, collects supernatant liquor; Under 4 DEG C of conditions, sex change liquid is dropwise added in renaturation buffer, described renaturation buffer is: 0.2 ~ 0.8mol/L arginine, 0.5 ~ 2mmol/L EDTA, 0.3 ~ 0.9mmol/L Sleep-promoting factor B, 60 ~ 100mmol/L Tris-Base, 15 ~ 30% glycerine, pH 8.0 ~ 8.5, stirs 24 ~ 48h simultaneously.
2. the purifying of the recombination chicken escherichia coli outer membrane protein inclusion body according to claim l and highly efficient renaturation liquid are to the method for its renaturation, it is characterized in that, in step (3), sex change liquid adds mode in renaturation buffer is dropping, and the volume ratio of sex change liquid and renaturation buffer is 1:20 ~ 1:50.
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Cited By (3)
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| CN110272476A (en) * | 2019-07-05 | 2019-09-24 | 苏州大学 | The method of one step heating dissolution recombinant spider silk protein inclusion body |
| CN111172132A (en) * | 2020-02-20 | 2020-05-19 | 北京理工大学 | Preparation method and application of recombinant polyurethane plastic degrading enzyme |
| CN112210019A (en) * | 2020-10-15 | 2021-01-12 | 广东省科学院生物工程研究所 | Purification method of transmembrane region of membrane protein containing single transmembrane region |
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| CN102633875A (en) * | 2012-04-27 | 2012-08-15 | 天津生机集团股份有限公司 | Renaturation liquid for recombining chicken alpha interferon inclusion body as well as preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110272476A (en) * | 2019-07-05 | 2019-09-24 | 苏州大学 | The method of one step heating dissolution recombinant spider silk protein inclusion body |
| CN111172132A (en) * | 2020-02-20 | 2020-05-19 | 北京理工大学 | Preparation method and application of recombinant polyurethane plastic degrading enzyme |
| CN112210019A (en) * | 2020-10-15 | 2021-01-12 | 广东省科学院生物工程研究所 | Purification method of transmembrane region of membrane protein containing single transmembrane region |
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