CN112062820B - Renaturation purification method of E.coli recombinant outer membrane protein A inclusion body protein - Google Patents
Renaturation purification method of E.coli recombinant outer membrane protein A inclusion body protein Download PDFInfo
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Abstract
The invention relates to a renaturation purification method of an escherichia coli recombinant outer membrane protein A inclusion body protein, which comprises the following steps: (1) collection of expressed rOmpA inclusion body protein precipitate induced; (2) denaturing the 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) purifying renatured rOmpA by affinity chromatography; (5) The purified rOmpA is placed in a dialysis bag, dialyzed for more than 12 hours at 4 ℃ in PBS buffer solution with pH of 7.6, and the supernatant is collected after centrifugation, thus obtaining the purified renaturated rOmpA protein. The expressed rOmpA inclusion body protein is subjected to renaturation and purification, so that the soluble rOmpA protein with the purity of up to 95% can be obtained; the renaturation method used by the invention is simple, convenient and quick, effectively solves the renaturation and purification problems of the E.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 an E.coli recombinant outer membrane protein A inclusion body protein.
Background
Escherichia coli (E.coli) is a common gram-negative pathogenic bacterium causing a variety of infections in humans and animals, and has a high infection rate, and the resulting infection is difficult to treat. The occurrence of a large number of drug-resistant strains in clinic makes the treatment of antibiotics have little effect. Thus, research into the use of vaccination to prevent E.coli infections has received increasing attention. Previous researches show that the recombinant outer membrane protein A (rOmpA for short) of the escherichia coli can induce good immunity against E.coli infection, can induce cross immunity against other bacteria of the enterobacteriaceae, and is a vaccine candidate antigen for preventing E.coli and other related enterobacteriaceae bacteria infection. However, expressed rOmpA exists mainly in insoluble inclusion body form, and how to renaturate and purify the insoluble inclusion body form rOmpA is a problem to be solved in the development and application of rOmpA.
The inclusion body protein in vitro renaturation method is a plurality of methods, such as dilution renaturation method, dialysis renaturation method, ultrafiltration renaturation method, a reverse micelle renaturation method developed in recent years, column chromatography renaturation method, low molecular additive assisted renaturation method, molecular chaperone assisted renaturation method and the like, and various in vitro renaturation methods can promote protein renaturation to a certain extent. However, there are few reports on in vitro renaturation studies of E.coli rOmpA inclusion body proteins. The rOmpA inclusion body dissolving and renaturation and purification method which is simple and convenient to study, high-efficiency and easy for large-scale production is a key of further industrialized production and utilization of rOmpA.
Disclosure of Invention
The invention aims to provide a renaturation purification method of escherichia coli recombinant outer membrane protein A inclusion body protein, which solves the problems that the existing escherichia coli recombinant outer membrane protein A inclusion body protein 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 an escherichia coli recombinant outer membrane protein a inclusion body protein, comprising the following steps:
(1) Collecting the precipitation of the expressed E.coli rOmpA inclusion body protein;
(2) Denaturing 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) Purifying the renatured rOmpA by using affinity chromatography;
(5) And (3) placing the renaturated and purified rOmpA in a dialysis bag, dialyzing for more than 12 hours at the temperature of 4 ℃ by using PBS buffer solution with the pH of 7.6, centrifuging, and collecting the supernatant to obtain the purified renaturated rOmpA protein.
Further, the method comprises the following specific steps:
(1) Collection of inclusion body protein precipitate: after recombinant OmpA-pET32a/BL21 was induced and expressed, the cells were collected by centrifugation, washed twice with buffer A, and the ratio of the wet weight of the cell pellet to that of buffer A was 1g:50mL, suspending with buffer B, ultrasonic crushing with ice bath for 10min, power of 150W, crushing/interval time of 5s/5s, centrifuging with 6200g for 10min, and collecting rOmpA inclusion body protein precipitate;
(2) rOmpA inclusion body protein lysis: the collected rOmpA inclusion body protein was precipitated, washed once with buffer C and once with buffer D, and the ratio of the wet weight of inclusion body protein precipitate to that of buffer C, D was 1g:20mL, slowly shaking for 30min at 2-8 ℃ each time, centrifuging for 10min with 6200g to obtain precipitate, shaking at 4-40 ℃ by using buffer E, standing for more than 0.5h, and dissolving rOmpA inclusion body protein precipitate, wherein the ratio of the wet weight of the inclusion body protein precipitate to the buffer E is 1g:20mL;
(3) Renaturation of solubilized rOmpA: buffer F was used at 5:1, slowly shaking and renaturating for 24 hours at 4 ℃, centrifuging for 10min at 6200g to remove sediment, and collecting supernatant, namely renaturated rOmpA;
(4) Purifying the renatured rOmpA by affinity chromatography: firstly, using a buffer solution G to balance a chromatographic column, then loading, and then using a buffer solution H to wash nonspecific binding proteins until no protein component of effluent liquid is detected, and finally using a buffer solution I to elute target proteins, wherein the eluted proteins are renaturated purified rOmpA;
(5) Diluting with buffer solution I to desired concentration, dialyzing in dialysis buffer solution J at 4deg.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, the method comprises the steps of,
buffer a:50mM Tris-HCl pH8.0, 100mM NaCl;
buffer B:50mM Tris-HCl pH8.0, 100mM NaCl,1%Triton X-100;
buffer C:50mM Tris-HCl pH8.0, 100mM NaCl,0.5%Triton X-100,2M urea;
buffer D:100mM Tris-HCl pH8.0, 300mM NaCl,3%Triton X-100;
buffer E:8M urea, 100mM Tris-HCl pH8.0, 100mM NaCl;
buffer F:15mM beta-cyclodextrin, 100mM Tris-HCl, pH 8.5;
buffer G:50mM Tris-HCl,250mM NaCl,pH8.8;
buffer H:50mM Tris-HCl,250mM NaCl,20mM imidazole pH8.8;
buffer I:50mM Tris-HCl,250mM NaCl,350mM imidazole pH8.8;
buffer J: PBS buffer.
Further, the buffer solution J is prepared by weighing 8g of NaCl, 0.2g of KCl and Na 2 HPO 4 1.44g and KH 2 PO 4 0.24g, dissolved with 800mL deionized water and adjusted to pH7.6, deionized water was added to 1000mL, and autoclaved for use.
The technical scheme has the advantages that: washing and dissolving inclusion body protein to obtain dissolved rOmpA inclusion body protein, adding 100mM Tris-HCl buffer solution containing 15mM beta-cyclodextrin for dilution to obtain renaturation rOmpA protein, wherein the renaturation rate can reach 79.62%, and purifying by affinity chromatography to obtain soluble rOmpA protein with the purity of up to 95%; the renaturation method used by the invention is simple, convenient and quick, effectively solves the renaturation and purification problems of the E.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 of expressed rOmpA, in which M is PageRuler TM Prestained Protein Ladder; sample 1 is a thallus ultrasonic disruption product before recombinant strain OmpA-pET32a/BL21 (DE 3) induced expression; sample 2 is a recombinant strain OmpA-pET32a/BL21 (DE 3) induced and expressed thallus ultrasonic disruption product; sample 3 is the centrifuge supernatant of sample 2; sample 4 is a centrifugal pellet of sample 2;
FIG. 2 shows the result of SDS-PAGE after washing of rOmpA inclusion body protein, M is PageRuler TM Prestained Protein Ladder; sample 1 is a precipitate which is centrifugated for 10min at 6200g after the induced expression thalli are crushed; sample 2 is the centrifugal precipitate of sample 1 after washing with buffer C; sample 3 is a centrifugal precipitate of sample 2 after washing with buffer solution D;
FIG. 3 shows the result of SDS-PAGE after rOmpA renaturation purification, in which M is PageRuler TM Prestained Protein Ladder; sample 1 is the supernatant after renaturation protein centrifugation; samples 2, 3, 4 and 5 are centrifugal supernatants after purifying renaturation proteins by affinity chromatography and dialyzing.
Detailed Description
The source of the biological material in the invention is as follows:
1. OmpA-pET32a/BL21 recombinant: 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) Conferred Immunoprotection against Enterobacteriaceae Infection in Mice. Israel Journal of Veterinary Medicine,2013,68 (1), 48-55.
The following examples are given to further illustrate the technical aspects of the present invention, but should not be construed as limiting the invention:
example 1
1. Coli rOmpA inclusion body induced expression
The activated OmpA-pET32a/BL21 (DE 3) recombinant bacteria were inoculated into LB-Amp at a ratio of 1:100 + Shake culturing in liquid culture medium at 37deg.C at 180rpm until the bacterial liquid OD 600nm The value reaches between 0.6 and 0.8, IPTG with the final concentration of 0.1mM is added for continuous culture for 4 hours, 6200g is centrifugated for 10min to collect thalli, the thalli is washed twice by a buffer solution A, and the proportion of the wet weight of the thalli sediment to the buffer solution A is 1g:50mL, then bufferedThe thallus is resuspended in liquid B, the thallus is crushed by ice bath ultrasonic for 10min, the power is 150W, and the crushing/interval time is 5s/5s. As a result, rOmpA was expressed correctly and was present mainly in the form of insoluble inclusion bodies in the pellet centrifuged at 6200g for 10min (FIG. 1).
2. Washing of expressed rOmpA inclusion body protein
Collecting inclusion body precipitate by centrifugation at 6200g for 10min, washing with buffer solution C and buffer solution D for one time, wherein the ratio of wet weight of inclusion body protein precipitate to buffer solution C, D is 1g:20mL, shaking slowly at 2-8deg.C for 30min each time, centrifuging at 6200g for 10min to obtain precipitate, washing to reduce total protein amount, and failing to reduce impurity protein ratio (FIG. 2)
3. rOmpA inclusion body solubilization, renaturation and purification
Under the condition of room temperature, shaking by using a buffer solution E, standing for 0.5h to dissolve rOmpA inclusion bodies, wherein the ratio of the wet weight of inclusion body protein precipitation to the buffer solution E is 1g:20mL,6200g centrifugation for 10min to collect denatured protein supernatant, buffer F was used to dilute denatured protein solution at a ratio of 5:1, slow shaking renaturation was performed at 4℃for 24h,6200g centrifugation for 10min to remove precipitate, and supernatant was collected. The absorbance of the protein dissolved in the urea solution and the absorbance of the renaturated protein are respectively measured by a Nanodrop2000/2000C ultra-micro spectrophotometer, and the renaturation rate is 79.62 +/-11.52. The pET-32a vector expresses OmpA protein with a 6 XHis tag and the renaturated protein was purified using an affinity chromatography purification column. Balancing the chromatographic column with a balancing buffer solution G, filtering renaturation protein solution with a 0.22 μm filter head until the renaturation protein solution naturally flows out of the chromatographic column, repeatedly loading the sample by a flow-through penetrating solution, washing the impurity protein with an impurity washing buffer solution H after 2 times until no protein component in the effluent is detected, eluting with an eluting buffer solution I, and collecting the protein, wherein the collected protein is renaturation purified rOmpA protein.
4. rOmpA dialysis liquid exchange after purification
The collected renatured purified rOmpA protein was diluted to a desired concentration using buffer I, placed in a dialysis zone, spin-dialyzed with buffer J at 4℃for 12 hours or more with stirring, centrifuged at 6200g for 10min, and the supernatant was collected and sampled for SDS-PAGE analysis, which showed that only one electrophoresis zone of rOmpA protein was available in 95% purity (FIG. 3).
The results show that the rOmpA inclusion body protein dissolved in the renaturation 8M urea buffer is diluted with the renaturation buffer containing 100mM Tris-HCl (pH 8.5) containing 15mM beta-cyclodextrin, and then the rOmpA protein is purified by using an affinity chromatography column, so that the rOmpA inclusion body protein of escherichia coli can be successfully renatured and purified, and the soluble rOmpA protein with the purity as high as 95% can be obtained.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. A renaturation purification method of an E.coli recombinant outer membrane protein A inclusion body protein is characterized in that: the method comprises the following steps:
(1) Collecting the precipitation of the expressed E.coli rOmpA inclusion body protein;
(2) Denaturing 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) Purifying the renatured rOmpA by using affinity chromatography;
(5) And (3) placing the renaturated and purified rOmpA in a dialysis bag, dialyzing at least 12h at 4 ℃ by using PBS buffer solution with pH of 7.6, centrifuging, and collecting the supernatant to obtain the purified renaturated rOmpA protein.
2. The method for renaturation purification of E.coli recombinant outer membrane protein A inclusion body protein according to claim 1, wherein: the method comprises the following specific steps:
(1) Collection of inclusion body protein precipitate: after recombinant OmpA-pET32a/BL21 was induced and expressed, the cells were collected by centrifugation, washed twice with buffer A, and the ratio of the wet weight of the cell pellet to that of buffer A was 1g:50mL, suspending with buffer B, ultrasonic crushing with ice bath for 10min, power 150W, crushing/interval time of 5s/5s, centrifuging with 6200g for 10min, and collecting rOmpA inclusion body protein precipitate;
(2) rOmpA inclusion body protein lysis: the collected rOmpA inclusion body protein was precipitated, washed once with buffer C and once with buffer D, and the ratio of the wet weight of inclusion body protein precipitate to that of buffer C, D was 1g:20mL, each time washed at 2-8 ℃ slowly shaking for 30min, centrifuging for 10min with 6200g to obtain precipitate, shaking at 4-40 ℃ by using buffer E, standing for more than 0.5h to dissolve rOmpA inclusion body protein precipitate, wherein the ratio of the wet weight of inclusion body protein precipitate to the buffer E is 1g:20mL;
(3) Renaturation of solubilized rOmpA: buffer F was used at 5:1, slowly shaking and renaturating for 24 hours at 4 ℃, centrifuging for 10min at 6200g to remove sediment, and collecting supernatant, namely renaturated rOmpA;
(4) Purifying the renatured rOmpA by affinity chromatography: firstly, using a buffer solution G to balance a chromatographic column, then loading, and then using a buffer solution H to wash nonspecific binding proteins until no protein component of effluent liquid is detected, and finally using a buffer solution I to elute target proteins, wherein the eluted proteins are renaturated purified rOmpA;
(5) Diluting with buffer solution I to desired concentration, dialyzing in dialysis buffer solution J at 4deg.C for 12 hr or more, centrifuging with 6200 and g for 10min, collecting supernatant, detecting protein molecular weight and purity by SDS-PAGE method, and detecting protein concentration by ultraviolet spectrophotometer;
the buffer solution A:50mM Tris-HCl pH8.0, 100mM NaCl;
buffer B:50mM Tris-HCl pH8.0, 100mM NaCl,1%Triton X-100;
buffer C:50mM Tris-HCl pH8.0, 100mM NaCl,0.5%Triton X-100,2M urea;
buffer D:100mM Tris-HCl pH8.0, 300mM NaCl,3%Triton X-100;
buffer E: 8. 8M urea, 100mM Tris-HCl pH8.0, 100mM NaCl;
buffer F:15mM beta-cyclodextrin, 100mM Tris-HCl, pH 8.5;
buffer G:50mM Tris-HCl,250mM NaCl,pH8.8;
buffer H:50mM Tris-HCl,250mM NaCl,20mM imidazole pH8.8;
buffer I:50mM Tris-HCl,250mM NaCl,350mM imidazole pH8.8;
buffer J: PBS buffer.
3. The method for renaturation purification of E.coli recombinant outer membrane protein A inclusion body protein according to claim 2, characterized in that: the buffer solution J is prepared by weighing 8g of NaCl, 0.2g of KCl and Na 2 HPO 4 1.44g and KH 2 PO 4 0.24g, using 800mL deionized water to dissolve and adjust the pH to 7.6, adding deionized water to 1000mL, and sterilizing under high pressure for later use.
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