CN112062820A - Renaturation and purification method of colibacillus recombinant outer membrane protein A inclusion body protein - Google Patents
Renaturation and purification method of colibacillus recombinant outer membrane protein A inclusion body protein Download PDFInfo
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Abstract
The invention relates to a renaturation purification method of escherichia coli recombinant outer membrane protein A inclusion body protein, which comprises the following steps: (1) collecting rOmpA inclusion body protein sediment of induction expression; (2) denaturation of rOmpA inclusion body protein with 8M urea solution; (3) 5-fold dilution renaturation with 100mM Tris-HCl buffer containing 15mM beta-cyclodextrin and pH8.5; (4) purification by renaturation rOmpA affinity chromatography; (5) and (3) putting the purified rOmpA into a dialysis bag, dialyzing in PBS buffer solution with pH7.6 at 4 ℃ for more than 12h, centrifuging and collecting supernatant to obtain the purified recombinant rOmpA protein. The invention renatures and purifies the expressed rOmpA inclusion body protein, and can obtain the soluble rOmpA protein with the purity of 95 percent; the renaturation method used by the invention is simple, convenient and quick, effectively solves the renaturation purification problem of the escherichia coli rOmpA inclusion body protein, and reduces the cost for large-scale production and utilization of rOmpA.
Description
Technical Field
The invention belongs to the protein engineering direction in the field of bioengineering, and relates to a renaturation purification method of escherichia coli recombinant outer membrane protein A inclusion body protein.
Background
Escherichia coli (e.coli) is a common gram-negative opportunistic pathogen that causes a variety of infections in humans and animals, and is highly infectious, causing infections that are difficult to treat. Due to the appearance of a large number of clinically resistant strains, the effect of antibiotic treatment is very little. Therefore, there is increasing interest in the study of vaccination against e.coli infections using vaccines. Previous studies have shown that escherichia coli recombinant outer membrane protein a (abbreviated as rmpa) can induce not only good immunity against e.coli infection, but also cross immunity against other bacteria of the family enterobacteriaceae, and is a vaccine candidate antigen for preventing e.coli and other related enterobacteriaceae bacterial infections. However, the expressed rOmpA mainly exists in the form of insoluble inclusion bodies, and how to renaturate and purify the insoluble inclusion body form rOmpA is a problem to be solved urgently for developing and applying the rOmpA.
The inclusion body protein in-vitro renaturation method has a plurality of methods, such as a dilution renaturation method, a dialysis renaturation method, an ultrafiltration renaturation method, a reverse micelle renaturation method developed in recent years, an on-column chromatography renaturation method, a low molecular additive assisted renaturation method, a molecular chaperone assisted renaturation method and the like, and various in-vitro renaturation methods can promote the protein renaturation to a certain degree. However, in vitro renaturation studies of E.coli rOmpA inclusion body proteins have been reported. The rOmpA inclusion body dissolving renaturation and purification method which is simple and convenient to research, high-efficiency and easy to amplify and produce is the key of further industrialized production and utilization of rOmpA.
Disclosure of Invention
The invention aims to provide a renaturation purification method of an inclusion body protein of an escherichia coli recombinant outer membrane protein A, which solves the problems that the inclusion body protein of the escherichia coli recombinant outer membrane protein A is insoluble and is not beneficial to later development and utilization.
The invention is realized by the following technical scheme: a renaturation purification method of colibacillus recombination outer membrane protein A inclusion body protein comprises the following steps:
(1) collecting the induced and expressed escherichia coli rOmpA inclusion body protein precipitate;
(2) denaturation of rOmpA inclusion body protein with 8M urea solution;
(3) 5-fold dilution renaturation with 100mM Tris-HCl buffer pH8.5 containing 15mM beta-cyclodextrin;
(4) the renatured rOmpA was purified by affinity chromatography;
(5) and (3) placing the renatured and purified rOmpA into a dialysis bag, dialyzing for more than 12h at 4 ℃ by using PBS buffer solution with pH7.6, centrifuging and collecting supernatant to obtain the purified renatured rOmpA protein.
Further, the method comprises the following specific steps:
(1) collecting the inclusion body protein precipitate: after the recombinant bacterium OmpA-pET32a/BL21 is induced to express, the bacterium is collected by centrifugation, the bacterium is washed twice by using buffer solution A, and the ratio of the wet weight of the bacterium precipitate to the buffer solution A is 1 g: 50mL, suspending by using buffer solution B, carrying out ultrasonic crushing on ice bath for 10min at the power of 150W, wherein the crushing/interval time is 5s/5s, then centrifuging for 10min at 6200g, and collecting rOmpA inclusion body protein precipitate;
(2) rioppa inclusion body proteolysis: the collected rmappa inclusion body protein pellet was washed once with buffer C and once more with buffer D, the ratio of wet weight of inclusion body protein pellet to buffer C, D was 1 g: 20mL, washing each time, slowly shaking at 2-8 ℃ for 30min, centrifuging for 10min by 6200g to retain precipitate, shaking at 4-40 ℃ by using buffer solution E, standing for more than 0.5h to dissolve rOmpA inclusion body protein precipitate, wherein the ratio of the wet weight of the inclusion body protein precipitate to the buffer solution E is 1 g: 20 mL;
(3) solubilized rmompa renaturation: buffer F was used to remove 5:1, slowly shaking and renaturing the dissolved rOmpA solution at 4 ℃ for 24 hours, centrifuging the solution for 10min at 6200g to remove precipitates, and collecting supernatant, namely the renatured rOmpA;
(4) renaturation of rOmpA affinity chromatography purification: firstly, using a buffer solution G to balance a chromatographic column, then loading a sample, washing non-specific binding protein by using a buffer solution H until no protein component is detected in an effluent liquid, and finally eluting a target protein by using a buffer solution I, wherein the eluted protein is the renaturated and purified rOmpA;
(5) diluting with buffer solution I to desired concentration, dialyzing in dialysis buffer solution J at 4 deg.C for more than 12 hr, centrifuging at 6200g for 10min, collecting supernatant, detecting protein molecular weight and purity by SDS-PAGE method, and detecting protein concentration by ultraviolet spectrophotometer.
Further, in the above-mentioned case,
and (3) buffer solution A: 50mM Tris-HCl pH8.0, 100mM NaCl;
and (3) buffer solution B: 50mM Tris-HCl pH8.0, 100mM NaCl, 1% Triton X-100;
and (3) buffer C: 50mM Tris-HCl pH8.0, 100mM NaCl, 0.5% Triton X-100, 2M urea;
and (3) buffer solution D: 100mM Tris-HCl pH8.0, 300mM NaCl, 3% Triton X-100;
and (3) buffer solution E: 8M Urea, 100mM Tris-HCl pH8.0, 100mM NaCl;
and (3) buffer solution F: 15mM beta-cyclodextrin, 100mM Tris-HCl, pH 8.5;
buffer G: 50mM Tris-HCl, 250mM NaCl, pH 8.8;
buffer solution H: 50mM Tris-HCl, 250mM NaCl, 20mM imidazole pH 8.8;
buffer I: 50mM Tris-HCl, 250mM NaCl, 350mM imidazole pH 8.8;
buffer J: PBS buffer.
Furthermore, the buffer solution J is prepared by weighing 8g of NaCl, 0.2g of KCl and Na2HPO41.44g and KH2PO40.24g, dissolved and adjusted to pH7.6 with 800mL of deionized water, then added to 1000mL of deionized water and autoclaved for use.
Adopt above-mentioned technical scheme's positive effect: the invention washes and dissolves the inclusion body protein to obtain the dissolved rOmpA inclusion body protein, then adds 100mM Tris-HCl buffer solution containing 15mM beta-cyclodextrin to dilute, and then obtains the renaturation rOmpA protein, the renaturation rate can reach 79.62%, and then the soluble rOmpA protein with the purity of 95% can be obtained by affinity chromatography purification; the renaturation method used by the invention is simple, convenient and quick, effectively solves the renaturation purification problem of the escherichia coli rOmpA inclusion body protein, and reduces the cost for large-scale production and utilization of rOmpA.
Drawings
FIG. 1 shows the result of SDS-PAGE detection of expressed rOmpA, in which M is PageRulerTMPrestained Protein Ladder; sample (A)Product 1 is a bacterial ultrasonication product before induction expression of recombinant bacteria OmpA-pET32a/BL21(DE 3); the sample 2 is a bacterial ultrasonication product after induction expression of recombinant bacteria OmpA-pET32a/BL21(DE 3); sample 3 is the centrifugation supernatant of sample 2; sample 4 is the centrifugation pellet of sample 2;
FIG. 2 shows the SDS-PAGE results of rOmpA inclusion body protein after washing, in which M is PageRulerTMPrestained Protein Ladder; sample 1 is a precipitate obtained by crushing the induced expression thallus and centrifuging the crushed thallus for 10min at 6200 g; sample 2 is the centrifugation sediment of sample 1 washed by buffer C; sample 3 is the centrifugal precipitate of sample 2 washed with buffer solution D;
FIG. 3 shows the SDS-PAGE result after rOmpA renaturation purification, in which M is PageRulerTMPrestained Protein Ladder; sample 1 is the supernatant after centrifugation of renaturated protein; samples 2, 3, 4, and 5 are centrifugation supernatants of renaturation proteins purified by affinity chromatography and dialyzed.
Detailed Description
Sources of the biological material in the present invention:
1. OmpA-pET32a/BL21 recombinant bacteria: hu R, Fan ZY, Zhang H, Tong CY, Chi JQ, Wang N, Li RT, Chen L, Ding ZF, Chen LX, Tang W, Zhou X, Pu LJ, Zhu ZB, Cui YD. outer Membrane Protein A (OmpA) transferred immunological detection against Enterobacteriaceae Infection in Mice. Israel Journal of vascular Medicine,2013,68(1), 48-55.
The following embodiments are further described, but should not be construed as limiting, the technical solutions of the present invention:
example 1
1. E.coli rOmpA inclusion body induction expression
Inoculating activated OmpA-pET32a/BL21(DE3) recombinant bacteria to LB-Amp at a ratio of 1:100+Culturing in liquid culture medium at 37 deg.C under shaking at 180rpm until bacterial liquid OD600nmAdding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM to continuously culture for 4h when the value reaches 0.6-0.8, centrifuging for 10min at 6200g to collect thalli, washing the thalli twice by using buffer solution A, wherein the ratio of wet weight of thalli precipitate to buffer solution A is 1 g: 50mL, then resuspending the cells in buffer B, sonicating in ice bathThe thallus is broken for 10min, the power is 150W, and the breaking/spacing time is 5s/5 s. Results rOmpA was correctly expressed and was present in 6200g pellets centrifuged for 10min, mainly as insoluble inclusion bodies (FIG. 1).
2. Expressed rOmpA Inclusion body protein washes
Centrifuging at 6200g centrifugal force for 10min, collecting the inclusion body precipitate, respectively washing with buffer C and buffer D once, wherein the ratio of the wet weight of the inclusion body protein precipitate to the buffer C, D is 1 g: 20mL, slowly shaking each time at 2-8 deg.C for 30min, centrifuging 6200g for 10min to obtain precipitate, washing to reduce total protein amount and reduce impurity protein ratio (FIG. 2)
3. rOmpA inclusion body dissolving, renaturation and purification
Under the condition of room temperature, shaking the buffer solution E and standing for 0.5h to dissolve rOmpA inclusion bodies, wherein the ratio of the wet weight of the inclusion body protein precipitate to the buffer solution E is 1 g: centrifuging at 6200g for 10min to collect supernatant, diluting with buffer solution F at 5:1, slowly shaking at 4 deg.C for 24 hr, centrifuging at 6200g for 10min to remove precipitate, and collecting supernatant. The absorbance of the protein dissolved in the urea solution and the absorbance of the renaturated protein are respectively measured by a Nanodrop2000/2000C ultramicro spectrophotometer, and the renaturation rate is calculated to be 79.62 +/-11.52. The OmpA protein expressed by the pET-32a vector has a 6 XHis tag, and the renaturation protein is purified by an affinity chromatography purification column. And (3) balancing the chromatographic column by using a balance buffer solution G, filtering the renaturated protein solution into the well balanced chromatographic column by using a filter head with the diameter of 0.22 mu m until the renaturated protein solution is naturally drained, repeatedly loading the percolation solution, washing the hybrid protein by using a washing buffer solution H after 2 times until no protein component is detected in the effluent liquid, eluting by using an elution buffer solution I and collecting the protein, wherein the collected protein is the renaturated and purified rOmpA protein.
4. Purified rOmpA dialysis exchange solution
The collected renaturation purified rOmpA protein is diluted to the required concentration by using a buffer solution I, placed in a dialysis zone, stirred and rotary dialyzed for more than 12h at 4 ℃ by using a buffer solution J, centrifuged for 10min at 6200g, supernatant is collected, and a sample is sampled for SDS-PAGE electrophoretic analysis, and the result shows that only one electrophoresis zone of the rOmpA protein can be obtained, and the purity can reach 95% (figure 3).
The results show that the rOmpA inclusion body protein dissolved by the renaturation 8M urea buffer solution is diluted by the renaturation buffer solution of 100mM Tris-HCl (pH 8.5) containing 15mM beta-cyclodextrin, and then the rOmpA protein is purified by an affinity chromatography column, so that the escherichia coli rOmpA inclusion body protein can be successfully renatured and purified, and the soluble rOmpA protein with the purity as high as 95 percent can be obtained.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. A renaturation purification method of colibacillus recombination outer membrane protein A inclusion body protein is characterized in that: the method comprises the following steps:
(1) collecting the induced and expressed escherichia coli rOmpA inclusion body protein precipitate;
(2) denaturation of rOmpA inclusion body protein with 8M urea solution;
(3) 5-fold dilution renaturation with 100mM Tris-HCl buffer pH8.5 containing 15mM beta-cyclodextrin;
(4) the renatured rOmpA was purified by affinity chromatography;
(5) and (3) placing the renatured and purified rOmpA into a dialysis bag, dialyzing for more than 12h at 4 ℃ by using PBS buffer solution with pH7.6, centrifuging and collecting supernatant to obtain the purified renatured rOmpA protein.
2. The renaturation and purification method of recombinant outer membrane protein A inclusion body protein of Escherichia coli according to claim 1, characterized in that: the method comprises the following specific steps:
(1) collecting the inclusion body protein precipitate: after the recombinant bacterium OmpA-pET32a/BL21 is induced to express, the bacterium is collected by centrifugation, the bacterium is washed twice by using buffer solution A, and the ratio of the wet weight of the bacterium precipitate to the buffer solution A is 1 g: 50mL, suspending by using buffer solution B, carrying out ultrasonic crushing on ice bath for 10min at the power of 150W, wherein the crushing/interval time is 5s/5s, then centrifuging for 10min at 6200g, and collecting rOmpA inclusion body protein precipitate;
(2) rioppa inclusion body proteolysis: the collected rmappa inclusion body protein pellet was washed once with buffer C and once more with buffer D, the ratio of wet weight of inclusion body protein pellet to buffer C, D was 1 g: 20mL, washing each time, slowly shaking at 2-8 ℃ for 30min, centrifuging for 10min by 6200g to retain precipitate, shaking at 4-40 ℃ by using buffer solution E, standing for more than 0.5h to dissolve rOmpA inclusion body protein precipitate, wherein the ratio of the wet weight of the inclusion body protein precipitate to the buffer solution E is 1 g: 20 mL;
(3) solubilized rmompa renaturation: buffer F was used to remove 5:1, slowly shaking and renaturing the dissolved rOmpA solution at 4 ℃ for 24 hours, centrifuging the solution for 10min at 6200g to remove precipitates, and collecting supernatant, namely the renatured rOmpA;
(4) renaturation of rOmpA affinity chromatography purification: firstly, using a buffer solution G to balance a chromatographic column, then loading a sample, washing non-specific binding protein by using a buffer solution H until no protein component is detected in an effluent liquid, and finally eluting a target protein by using a buffer solution I, wherein the eluted protein is the renaturated and purified rOmpA;
(5) diluting with buffer solution I to desired concentration, dialyzing in dialysis buffer solution J at 4 deg.C for more than 12 hr, centrifuging at 6200g for 10min, collecting supernatant, detecting protein molecular weight and purity by SDS-PAGE method, and detecting protein concentration by ultraviolet spectrophotometer.
3. The renaturation and purification method of inclusion body protein of recombinant outer membrane protein A of Escherichia coli according to claim 2, characterized in that:
and (3) buffer solution A: 50mM Tris-HCl pH8.0, 100mM NaCl;
and (3) buffer solution B: 50mM Tris-HCl pH8.0, 100mM NaCl, 1% Triton X-100;
and (3) buffer C: 50mM Tris-HCl pH8.0, 100mM NaCl, 0.5% Triton X-100, 2M urea;
and (3) buffer solution D: 100mM Tris-HCl pH8.0, 300mM NaCl, 3% Triton X-100;
and (3) buffer solution E: 8M Urea, 100mM Tris-HCl pH8.0, 100mM NaCl;
and (3) buffer solution F: 15mM beta-cyclodextrin, 100mM Tris-HCl, pH 8.5;
buffer G: 50mM Tris-HCl, 250mM NaCl, pH 8.8;
buffer solution H: 50mM Tris-HCl, 250mM NaCl, 20mM imidazole pH 8.8;
buffer I: 50mM Tris-HCl, 250mM NaCl, 350mM imidazole pH 8.8;
buffer J: PBS buffer.
4. The method for renaturation purification of inclusion body protein of E.coli rOmpA according to claim 3, wherein: the buffer solution J is prepared by weighing 8g of NaCl, 0.2g of KCl and Na2HPO41.44g and KH2PO40.24g, dissolved and adjusted to pH7.6 with 800mL of deionized water, then added to 1000mL of deionized water and autoclaved for use.
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Cited By (4)
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| CN113278048A (en) * | 2021-05-26 | 2021-08-20 | 武汉华美生物工程有限公司 | Purification method of membrane protein |
| CN114957378A (en) * | 2022-04-15 | 2022-08-30 | 深圳重链生物科技有限公司 | Renaturation method of hydrophobic inclusion body antigen protein |
| CN115785193A (en) * | 2022-11-25 | 2023-03-14 | 天津鸿宇泰生物科技有限公司 | Renaturation buffer solution and method for renaturation of inclusion body protein |
| CN116874552A (en) * | 2023-09-08 | 2023-10-13 | 成都华任康生物科技有限公司 | Purification method of target protein, kit and related application thereof |
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| CN113278048A (en) * | 2021-05-26 | 2021-08-20 | 武汉华美生物工程有限公司 | Purification method of membrane protein |
| CN113278048B (en) * | 2021-05-26 | 2023-03-10 | 武汉华美生物工程有限公司 | Purification method of membrane protein |
| CN114957378A (en) * | 2022-04-15 | 2022-08-30 | 深圳重链生物科技有限公司 | Renaturation method of hydrophobic inclusion body antigen protein |
| CN114957378B (en) * | 2022-04-15 | 2024-02-13 | 深圳重链生物科技有限公司 | Renaturation method of hydrophobic inclusion body antigen protein |
| CN115785193A (en) * | 2022-11-25 | 2023-03-14 | 天津鸿宇泰生物科技有限公司 | Renaturation buffer solution and method for renaturation of inclusion body protein |
| CN115785193B (en) * | 2022-11-25 | 2023-11-21 | 天津鸿宇泰生物科技有限公司 | Renaturation buffer solution and method for renaturation of inclusion body protein |
| CN116874552A (en) * | 2023-09-08 | 2023-10-13 | 成都华任康生物科技有限公司 | Purification method of target protein, kit and related application thereof |
| CN116874552B (en) * | 2023-09-08 | 2023-12-08 | 成都华任康生物科技有限公司 | Purification method of target protein, kit and related application thereof |
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