Purification and concentration method of escherichia coli expression protein
Through centrifugal crushing, ceramic membrane filtration, elution, decolorization and reverse osmosis concentration, various problems of E. coli protein purification and concentration in the prior art are solved, and efficient and environmentally friendly protein purification and concentration are achieved, suitable for different concentrations. Escherichia coli expression protein solution.
Patent Information
- Application Number
- CN202411712564.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-21
AI Technical Summary
The existing purification and concentration methods of E. coli protein have difficulty in process amplification, difficulty in loading the chromatography column, requirements for sample loading, difficulty in determining the column efficiency, difficulty in repeated use of chromatography columns, contamination during adsorption and elution of heterogeneous proteins, etc. question.
A high concentration and high purity protein concentrate is obtained through a physical separation process using a method including centrifugal crushing, ceramic membrane filtration, elution, decolorization and reverse osmosis concentration.
It has achieved efficient and environmentally friendly protein purification and concentration, with high protein recovery rate and no less than 98.5%, and is suitable for E. coli expression protein solutions at different concentrations.
Smart Images
Figure CN119490558A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of protein separation and purification, and in particular to a method for purifying and concentrating Escherichia coli expressed protein. Background Art
[0002] There are several common methods for purifying and concentrating Escherichia coli proteins, such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc., which have the following disadvantages: (1) Difficulty in process scale-up: Molecular sieve chromatography cannot follow the principle of linear scale-up. Even if the principle of constant column bed height is followed, how to adjust the process flow rate is also a problem that needs to be faced; (2) Difficulty in filling the chromatography column; (3) There are requirements for the sample loading amount; (4) Difficulty in measuring column efficiency; (5) Difficulty in repeatedly using the chromatography column; (6) In addition to specific adsorption, some impurity proteins will still be adsorbed due to the misidentification of molecules and non-selective forces between molecules. In addition, the ligands in the elution process will inevitably fall off and enter the separation system; (7) The carrier is expensive, has low mechanical strength, and is difficult to prepare the ligand. Some ligands themselves must be separated and purified, and the coupling conditions between the ligand and the carrier are fierce.
[0003] For the purified concentrate of protein solution, the main evaluation contents include not only the process steps, but also the concentration, purity and recovery rate of the concentrate. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a method for purifying and concentrating proteins expressed in Escherichia coli. The method is simple to operate, has low production cost, and high purification and concentration efficiency. A protein concentrate with higher concentration and higher purity can be obtained. The protein recovery rate is high and the protein is not inactivated. The method has important industrial application prospects.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The present invention provides a method for purifying and concentrating an Escherichia coli expressed protein, comprising the following steps:
[0007] 1) The fermentation broth of Escherichia coli fermentation culture is centrifuged and crushed to collect the crude protein liquid;
[0008] 2) filtering the crude protein solution in step 1) through a ceramic membrane and eluting with an eluent, and collecting the eluted product;
[0009] The eluent comprises pure water and disodium hydrogen phosphate, and the conductivity of the eluent is 0.5-20ms / cm;
[0010] 3) The eluted product in step 2) is decolorized and concentrated by reverse osmosis to obtain a protein concentrate.
[0011] Preferably, the concentration of protein in the crude protein solution in step 1) does not exceed 5 g / L.
[0012] Preferably, the centrifugal crushing in step 1) is performed by centrifugation at a speed of 10000-20000 rpm for 10-30 min.
[0013] Preferably, the pore size of the ceramic membrane core in step 2) does not exceed 150 nm.
[0014] Preferably, the decolorization in step 3) is performed using a nanofiltration device.
[0015] More preferably, the molecular weight of the nanofiltration membrane core used in the nanofiltration device does not exceed 600Da.
[0016] Preferably, the decolorization in step 3) is performed in two steps.
[0017] Preferably, the operating pressure of the reverse osmosis concentration equipment used for the reverse osmosis concentration in step 3) is not less than 70 bar.
[0018] Preferably, the concentration of the protein concentrate in step 3) is not less than 20 g / L.
[0019] Beneficial effects of the present invention:
[0020] 1. The purification and concentration process of the present invention is a physical separation process, which does not add any additional chemical substances, does not introduce new pollution sources, and is highly efficient and environmentally friendly. It can also concentrate the protein solution from an initial concentration of no more than 0.6 g / L to 20 g / L or more, with a purity of no less than 98.5%.
[0021] 2. The process of the present invention is relatively simple, and the recovery rate of the protein can reach 95% or more, with high recovery efficiency and without affecting the activity of the protein.
[0022] 3. The purification and concentration method of the present invention is suitable for E. coli expression protein solutions of different concentrations, which can be as low as 0.5 g / L or as high as 4.5 g / L, and has strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The figure is a flow chart of the purification and concentration method of the present invention. DETAILED DESCRIPTION
[0024] The present invention provides a method for purifying and concentrating an Escherichia coli expressed protein, comprising the following steps:
[0025] 1) The fermentation broth of Escherichia coli fermentation culture is centrifuged and crushed to collect the crude protein liquid;
[0026] 2) filtering the crude protein solution of step 1) through a ceramic membrane and eluting with an eluent, and collecting the eluted product;
[0027] The eluent includes pure water and disodium hydrogen phosphate, and the conductivity of the eluent is 0.5-20ms / cm;
[0028] 3) The eluted product of step 2) is decolorized and concentrated by reverse osmosis to obtain a protein concentrate.
[0029] In the present invention, it is found that for the elution of Escherichia coli expressed protein, the conductivity of the eluent in the above range has a better effect on the elution of the protein. For example, the conductivity of the eluent can be any value of 0.5ms / cm, 1ms / cm, 2ms / cm, 5ms / cm, 7ms / cm, 8ms / cm, 10ms / cm, 12ms / cm, 15ms / cm, 18ms / cm, 20ms / cm, etc., without particular limitation.
[0030] In a preferred embodiment of the present invention, the protein concentration in the crude protein solution in step 1) does not exceed 5 g / L. For example, the protein concentration can be any value of 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, etc., without particular limitation.
[0031] In a preferred embodiment of the present invention, the centrifugal crushing in step 1) is performed at a speed of 10000-20000 rpm for 10-30 min. For example, the centrifugal crushing can be performed at a speed of 12000 rpm for 15 min.
[0032] In a preferred embodiment of the present invention, the pore size of the ceramic membrane core in step 2) does not exceed 150 nm. For example, the pore size can be any value of 80 nm, 100 nm, 120 nm, 150 nm, etc., without special restrictions. Ceramic membrane filtration can remove large particle impurities, suspended matter, colloids and other substances to obtain a relatively clear solution. And using an eluent can improve the recovery rate of the protein.
[0033] In a preferred embodiment of the present invention, the concentration of disodium hydrogen phosphate in step 2) is not particularly limited, and may be solid disodium hydrogen phosphate or a disodium hydrogen phosphate solution, and the concentration of the solution may be 0.0001-0.1 mol / L, for example, the concentration may be 0.0001 mol / L, 0.0005 mol / L, 0.001 mol / L, 0.005 mol / L, 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, etc. The present invention finds that the use of a low-conductivity eluent can more effectively elute the protein and improve the recovery rate of the protein.
[0034] In a preferred embodiment of the present invention, the decolorization in step 3) is performed by a nanofiltration device. The decolorization removes most of the pigments of the raw materials themselves, the pigments produced by the metabolism of the bacteria during the fermentation process, and the colored substances produced by the reaction of the fermentation liquid components. Pure water can be added for washing during the decolorization process.
[0035] In a more preferred embodiment of the present invention, the molecular weight of the nanofiltration membrane core used in the nanofiltration device does not exceed 600Da. For example, it can be any value among 400Da, 500Da, 600Da, etc., without special limitation.
[0036] In a preferred embodiment of the present invention, the decolorization in step 3) is performed in two steps. Various pigments or colored substances can be removed more completely by performing two steps of decolorization.
[0037] In a preferred embodiment of the present invention, the operating pressure of the reverse osmosis concentration equipment used for reverse osmosis RO concentration in step 3) is not less than 70 bar.
[0038] In a preferred embodiment of the present invention, the concentration of the protein concentrate in step 3) is not less than 20 g / L. In the present invention, the concentration of the obtained protein concentrate can be 20 g / L, 21 g / L, 22 g / L, 23 g / L, 25 g / L, etc.
[0039] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0040] Example 1
[0041] Combined with Figure 1 The purification process shown is to inoculate the E. coli seed liquid containing the expression gene into a sterilized fermentation tank to start the main fermentation process. During the fermentation, the pH is controlled at 7, the temperature is 15-20°C, the DO (dissolved oxygen) is monitored in real time, and the feeding, ventilation and other conditions are adjusted in time to obtain the E. coli fermentation liquid.
[0042] The above-mentioned E. coli fermentation broth was transferred to a centrifuge tube and centrifuged at 12000r / min for 15min to allow the E. coli to settle at the bottom of the centrifuge tube and remove metabolic impurities to obtain a broken E. coli solution containing expressed protein, i.e., a crude protein solution. The protein concentration in the crude protein solution was measured to be 0.5g / L.
[0043] 18L of the above protein crude liquid was loaded into a ceramic membrane raw material tank, and a ceramic membrane with a pore size of 100nm was selected as a filtration carrier. Large particle impurities, suspended matter, colloids and other substances were removed by pressure filtration with a booster pump to obtain a clarified liquid, and pure water and a solution of sodium dihydrogen phosphate were added as eluents (the conductivity of the eluent was 2ms / cm) for elution to obtain a ceramic membrane dialyzate;
[0044] The ceramic membrane dialysate is then passed through a nanofiltration membrane (molecular weight of the membrane core is 500Da) for the first decolorization treatment to remove most of the pigments in the raw materials themselves, the pigments produced by bacterial metabolism during the fermentation process, and the colored substances generated by the reaction of the fermentation liquid components. During the decolorization process, 2L of pure water is added for washing to obtain a decolorized dialysate.
[0045] The above-mentioned primary decolorized dialysate is then passed through a nanofiltration membrane (molecular weight of the membrane core is 500Da) for secondary decolorization to further remove the pigment of the raw material itself, the pigment produced by the metabolism of the bacteria during the fermentation process, and the colored substances generated by the reaction of the fermentation liquid components. During the secondary decolorization process, 2L of pure water is added for washing to obtain a secondary decolorized dialysate.
[0046] The secondary decolorized dialysate obtained above was fed into the RO system for reverse osmosis concentration at a pressure of 70 bar to finally obtain a protein concentrate.
[0047] In this example, the E. coli fermentation solution was directly obtained from the market, and the protein concentration in the crude protein solution obtained after centrifugal crushing was 0.5 g / L.
[0048] The concentrations of the various protein solutions obtained during the separation and purification process and the process yields are shown in Table 1 below.
[0049] Table 1
[0050]
[0051] Example 2
[0052] The separation and purification method of this embodiment is the same as that of embodiment 1. The E. coli fermentation solution is directly obtained from the market, and the protein concentration in the crude protein solution obtained after centrifugal crushing is 3 g / L.
[0053] The concentrations of the various protein solutions obtained during the separation and purification process and the process yields are shown in Table 2 below.
[0054] Table 2
[0055]
[0056] Example 3
[0057] The separation and purification method of this embodiment is the same as that of embodiment 1. The E. coli fermentation solution is directly obtained from the market, and the protein concentration in the crude protein solution obtained after centrifugal crushing is 4.25 g / L.
[0058] The concentrations of the various protein solutions obtained during the separation and purification process and the process yields are shown in Table 3 below.
[0059] Table 3
[0060]
[0061]
[0062] Example 4
[0063] The separation and purification method of this embodiment is the same as that of embodiment 1. The E. coli fermentation solution is directly obtained from the market, and the protein concentration in the crude protein solution obtained after centrifugal crushing is 4.25 g / L.
[0064] The concentrations of the various protein solutions obtained during the separation and purification process and the process yields are shown in Table 4 below.
[0065] Table 4
[0066]
[0067] Example 5
[0068] The difference between this embodiment and embodiment 2 is that the conductivity of the eluent is 15 ms / cm. The other steps remain unchanged. The protein concentration in the ceramic membrane dialysate was measured to be 2.55 g / L, and the protein recovery rate was 99.1%.
[0069] Comparative Example 1
[0070] The difference between this comparative example and Example 2 is that the conductivity of the eluent is 300 ms / cm. The other steps remain unchanged. The protein concentration in the ceramic membrane dialysate is measured to be 2.25 g / L, and the protein recovery rate is 87.5%.
[0071] Comparative Example 2
[0072] The difference between this comparative example and Example 1 is that the conductivity of the eluent is 500 ms / cm. The other steps remain unchanged. The protein concentration in the ceramic membrane dialysate was measured to be 0.33 g / L, and the protein recovery rate was 77.2%.
[0073] It can be concluded from the above embodiments and comparative examples that the concentration of the protein solution obtained by the present invention is greatly improved, the protein recovery rate is high, and the concentration can be increased to 4 times or more compared to the concentration of the initial protein solution. The method is relatively simple to operate, the post-processing is simple, the protein is inactive, and there will be no agglutination or denaturation, precipitation, etc. that lead to protein loss. In addition, no other chemical components will be introduced during the treatment process to affect the efficacy of use, and it has important industrial application prospects. Moreover, a low conductivity eluent can be used to obtain a higher protein recovery rate, which may be related to the fact that the low conductivity eluent can avoid protein aggregation.
[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for purifying and concentrating an Escherichia coli expressed protein, characterized in that: The following steps are involved: 1) The fermentation broth of Escherichia coli fermentation culture is centrifuged and crushed to collect the crude protein liquid; 2) filtering the crude protein solution in step 1) through a ceramic membrane and eluting with an eluent, and collecting the eluted product; The eluent comprises pure water and disodium hydrogen phosphate, and the conductivity of the eluent is 0.5-20ms / cm; 3) The eluted product in step 2) is decolorized and concentrated by reverse osmosis to obtain a protein concentrate.
2. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The protein concentration in the protein solution in step 1) does not exceed 5 g / L.
3. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The centrifugal crushing in step 1) is performed at a speed of 10000-20000 rpm for 10-30 min.
4. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The pore size of the ceramic membrane core in step 2) does not exceed 150 nm.
5. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The decolorization in step 3) is performed using a nanofiltration device.
6. The method for purifying and concentrating Escherichia coli expressed protein according to claim 5, characterized in that: The molecular weight of the nanofiltration membrane core used in the nanofiltration device does not exceed 600Da.
7. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The decolorization in step 3) is a two-step decolorization.
8. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The operating pressure of the reverse osmosis concentration equipment used for the reverse osmosis concentration in step 3) is not less than 70 bar.
9. The method for purifying and concentrating Escherichia coli expressed protein according to claim 1, characterized in that: The concentration of the protein concentrate in step 3) is not less than 20 g / L.