Purification method for removing endotoxin from prokaryotic expression recombinant protein

Abstract

The invention discloses a purification method for removing endotoxin in prokaryotic expression recombinant protein, which belongs to the technical field of bioengineering, and comprises the following steps: adding 0.1% Triton X-114 in an ultrasonication stage, carrying out ice-bath ultrasonication, after the recombinant protein is combined with a Ni-NTA affinity column, washing with 50 column volumes of a combination buffer solution containing 0.1% Triton X-114, and carrying out elution by using an elution buffer solution containing 0.1% Triton X-114 in the presence of an elution buffer solution containing 0.1% Triton X-114 in the presence of an elution buffer solution containing 0.1% Triton X-114 in the presence of an elution buffer solution containing 0.1% Triton X-114; and washing with a 4-DEG C binding buffer solution with 20 column volumes, and subsequently eluting the target protein with imidazole with different gradients. Then, regulating the pH value of the purified recombinant protein solution to 2-4 or more than 10 through 100mM hydrochloric acid or sodium hydroxide, dissociating and decomposing endotoxin, carrying out oscillation treatment at room temperature for 2-4 hours, then removing the endotoxin in the recombinant protein through ultrafiltration, retaining an endotube protein solution, and detecting the content of protein and the endotoxin in the recombinant protein; the method is easy and convenient to operate and low in cost, the endotoxin content in the recombinant protein prepared through prokaryotic expression can be reduced by 400 times on the premise that immune recognition of the recombinant protein and serum is not affected, and the phenomenon that strong cross reaction occurs between immune serum and escherichia coli due to high endotoxin residues in the recombinant protein is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a purification method for removing endotoxin from a prokaryotically expressed recombinant protein. Background Art

[0002] Escherichia coli is a typical prokaryotic expression system of Gram-negative bacteria and one of the most effective heterologous protein producers. Using this system to express proteins is the simplest, fastest and lowest cost. However, as a Gram-negative bacterium, during the preparation of recombinant proteins, Escherichia coli will release a large amount of lipopolysaccharide (LPS) in the cell membrane and cause residues in the protein solution. LPS is the main structural component on the cell membrane of Gram-negative bacteria, covering about three-quarters of the outer membrane. LPS is composed of polysaccharide O antigen (O-PS), core polysaccharide (C-OS) and lipid A. Lipid A is the smallest structural unit in LPS and the most conserved part. It is also the active center of LPS and determines the pathogenicity of bacteria. Its components are phospholipids composed of a hydrophilic skeleton composed of aminoglucosaccharides and a hydrophobic fatty acid chain. So far, the extraction structures of lipid A of various bacteria have been determined, and the main difference lies in the difference in fatty acids and phosphate groups. Lipid A, as the main concentrated part of LPS biological activity, activates complex signal pathways by interacting with receptor protein molecules of target cells, ultimately leading to a series of pathological reactions in the body. Excessive stimulation of natural immunity by lipid A can lead to organ damage, severe shock, and may even cause death in mammals and humans.

[0003] The recombinant protein expressed by E. coli is accompanied by residual endotoxin during the purification process. Since the body has an immune recognition effect on the residual LPS, it acts on mononuclear macrophages in the body and produces a variety of cytokines. The appropriate amount of cytokines can activate the immune system. Therefore, the recombinant protein expressed by E. coli contains residual endotoxin in the E. coli cell membrane. The endotoxin produces an immune response in the subsequent immunization process, and its protein serum will also have extensive cross-reactions with E. coli.

[0004] In the field of vaccine preparation, the removal of endotoxin is a necessary process, and its removal process is complete and mature. It is also clearly stipulated in the Chinese Pharmacopoeia that the endotoxin in biological products must be removed or the endotoxin content must reach the endotoxin content standard specified in the drug before it can be used. In the field of food safety testing, endotoxin has not received widespread attention, and there are few related reports. The present invention aims to reduce the impact of endotoxin on protein antigen epitopes and the occupancy of the immune system by removing endotoxin from recombinant proteins, more effectively prepare monoclonal antibodies using recombinant proteins, and promote the development of the field of food microbial immunoassay. Summary of the invention

[0005] The purpose of the present invention is to provide a purification method for removing endotoxin of prokaryotic expression recombinant protein, which is used to quickly and at low cost reduce the endotoxin content of recombinant protein. To achieve the above purpose, the present invention optimizes the fragmentation mode, the purification mode, the comparison of conventional endotoxin removal methods, the optimization of ultrafiltration method, etc. of the recombinant protein OmpA, and proposes a purification method for removing endotoxin of prokaryotic expression recombinant protein based on the detection methods of Coomassie brilliant blue quantitative protein, endotoxin content detection, Coomassie staining, silver staining, immunoblotting, and ELISA.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A purification method for removing endotoxin of prokaryotic expressed recombinant protein, characterized in that: 0.1% Triton X-114 is added in the ultrasonic crushing stage for ice bath ultrasonic crushing, after the recombinant protein is bound to a Ni-NTA affinity column, it is first washed with 50 column volumes of a binding buffer containing 0.1% Triton X-114, then washed with 20 column volumes of a 4°C binding buffer, and then the target protein is eluted with different gradients of imidazole; then, the pH of the purified recombinant protein solution is adjusted to 2-4 or >10 by 100mM hydrochloric acid or sodium hydroxide, endotoxin is dissociated and decomposed, oscillation treatment is performed at room temperature for 2-4 hours, and then the recombinant protein solution is repeatedly ultrafiltered until the pH returns to neutrality to further remove endotoxin in the recombinant protein, the endotoxin solution is retained, the endotoxin content is detected by a horseshoe crab kit, the protein content is determined by a Coomassie brilliant blue method, and the effect of the method on protein immunity is evaluated by ELISA and Western-blot.

[0008] Furthermore, the method is specifically as follows:

[0009] (1) Inoculate the seed solution of pET-32a-OmpA expression strain E. coli BL21 overnight, inoculate the seed solution into LB medium at a 1% inoculum amount, and culture at 37°C, 180 r / min shaking for 2.5 h until the cell density OD600 is 0.6-0.8, add 0.2 mM IPTG to the bacterial solution, and culture at 37°C, 180 r / min shaking for 12 h;

[0010] (2) The induced recombinant protein bacterial solution was centrifuged at 4°C, 5000r / min for 20min, the bacterial cells were resuspended in Binding Buffer and added with 0.1% Triton X-114 for ice bath ultrasonic disruption, and then centrifuged at 4°C, 12000r / min for 20min, and the supernatant was collected; the supernatant was added to the Ni column, and after the protein was bound to the column, it was first washed with 50 column volumes of binding buffer containing 0.1% Triton X-114, and then washed with 20 column volumes of 4°C binding buffer, and then the target protein was eluted with different gradients of imidazole. The eluate with a single band was collected, dialyzed and concentrated for later use;

[0011] (3) Use 100 mM HCl and 100 mM NaOH to adjust the pH of the recombinant protein to slightly acidic or alkaline, place it on a horizontal shaker at room temperature for 2-4 h for treatment, pour the treated recombinant protein solution into a 30 kDa ultrafiltration tube for ultrafiltration at 4°C, 4000 x g, and 30 min. Recover the concentrate, re-dilute it, and repeatedly ultrafilter and centrifuge it until the pH returns to neutral.

[0012] Furthermore, the molecular retention rate of the ultrafiltration tube selected in step (3) should be greater than the molecular size of the endotoxin monomer and controlled to be less than half the size of the target protein.

[0013] The following beneficial effects can be obtained through the above technical solution:

[0014] Compared with conventional endotoxin removal methods, this technical solution has great advantages: compared with single methods such as affinity medium adsorption method and chemical method, this solution has better endotoxin removal effect on recombinant protein, consumables are simple and easy to obtain, the method is convenient and effective, and there is no significant adverse effect on the immune effect of recombinant protein; compared with affinity medium adsorption method or chromatography method, this solution is significantly less time-consuming and inexpensive. Under the premise of not affecting the immune recognition of recombinant protein and serum, the endotoxin content in recombinant protein prepared by prokaryotic expression can be reduced by 400 times, and the phenomenon of strong cross-reaction between immune serum and Escherichia coli caused by high residual endotoxin in recombinant protein can be reduced.

[0015] This method can remove endotoxins through simple operations, reduce the occupancy of endotoxins on the immune system of immunized mice, reduce immunotoxicity, reduce the impact of endotoxins on the exposure of recombinant protein surface antigen epitopes, improve immune specificity, and help find antigens with better immune recognition and higher specificity. It lays the foundation for the further development of immunoassays in the field of food safety testing and has good research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Examination of recombinant protein eluates prepared by different fragmentation and purification methods

[0017] Figure 2 Effects of different disruption and purification methods on endotoxin content of recombinant protein (Limulus amebocyte lysate method)

[0018] Figure 3 Recombinant proteins prepared by different disruption and purification methods were stained, silver stained, and immunoblotted

[0019] Figure 4 Effects of different disruption and purification methods on the titer of recombinant proteins

[0020] Figure 5 Effects of different endotoxin removal methods on endotoxin content and protein retention rate of recombinant proteins

[0021] Figure 6 Recombinant proteins prepared by different endotoxin removal methods were stained, silver stained, and immunoblotted

[0022] Figure 7 Effects of different endotoxin removal methods on the titer of recombinant proteins

[0023] Figure 8 Effect of optimizing ultrafiltration method on endotoxin content and protein retention rate of recombinant protein (Limulus amebocyte lysate method)

[0024] Fig. 9 Optimization of ultrafiltration method for immunoblotting of recombinant proteins

[0025] Fig.10 Effect of optimizing ultrafiltration method on the titer of recombinant protein

[0026] Fig.11 Effect of pH treatment time on endotoxin removal

[0027] Fig.12 Comparison of the effects of optimized ultrafiltration and conventional ultrafiltration

[0028] Fig.13 Verification of the effect of composite method on the removal of endotoxin from recombinant protein

[0029] Fig.14 Recombinant proteins prepared by composite method are stained, silver stained, and immunoblotted DETAILED DESCRIPTION

[0030] The following is combined with Figure 1-14 The present invention is further described as follows:

[0031] A purification method for removing endotoxin from recombinant protein, the specific technical scheme is as follows:

[0032] 1. Recombinant protein induction

[0033] The pET-32a-OmpA expression strain E. coli BL21 was inoculated with seed liquid and cultured overnight. The seed liquid was inoculated into LB medium according to 1% inoculum, and cultured in a shaking incubator at 37°C and 180 r / min for 2.5 h to make the cell density OD600 be 0.6-0.8. 0.2 mM IPTG was added to the bacterial liquid, and cultured in a shaking incubator at 37°C and 180 r / min for 12 h.

[0034] 2. Recombinant protein was broken and purified using Triton X-114

[0035] (1) The overnight bacterial culture was centrifuged at 4°C, 5000 r / min for 20 min. The cells were resuspended in PBS and a protease inhibitor was added. The cells were ultrasonically disrupted in an ice bath without or with 0.1% Triton X-114. After disruption, the cells were centrifuged at 4°C, 12000 r / min for 20 min and the supernatant was collected.

[0036] (2) The supernatant was added to a Ni column and purified using two methods: ① directly eluting the target protein with different gradients of imidazole; ② first washing with 50 column volumes of binding buffer containing 0.1% Triton X-114, then washing with 20 column volumes of 4°C binding buffer, and subsequently eluting the target protein with different gradients of imidazole.

[0037] (3) After collecting the elution solution, SDS-PAGE gel electrophoresis analysis is performed; according to the gel image results, after collecting the recombinant protein with a single band, sufficient elution solution is added for dialysis and concentration to complete the preparation of the recombinant protein.

[0038] like Figure 1 As shown in the figure, in the ultrasonic fragmentation step, compared with the common fragmentation method, the method of adding 0.1% Triton X-114 and then fragmenting does not affect the binding of the target protein to the Ni column, and can normally elute a pure and single target band; in the purification step, compared with the common purification method, the washing step in the purification method using Triton X-114 washing does not elute the target protein, has no effect on the subsequent imidazole elution step, and can still prepare a pure and single target band. Therefore, the fragmentation method of adding 0.1% Triton X-114 and the purification method using Triton X-114 washing can be used for the preparation of recombinant proteins.

[0039] like Figure 2As shown, the endotoxin content of the original protein was 411956EU / mL, and the endotoxin content of the protein prepared by the optimized crushing method was reduced to 351422EU / mL, and the endotoxin removal rate was 14.69%; the endotoxin content of the protein prepared by the optimized purification method was reduced to 5940EU / mL, and the endotoxin removal rate was 98.56%; the endotoxin content of the protein prepared by the combined use of the optimized crushing method and the optimized purification method was reduced to 5715EU / mL, and the endotoxin removal rate was 98.61%. The above data show that the optimized crushing method has a certain degree of promoting effect on the removal of endotoxins from recombinant proteins, and the optimized purification method has a significant effect on the removal of endotoxins from recombinant proteins. Therefore, considering the composite scheme is conducive to the preparation of recombinant proteins with lower endotoxin content.

[0040] like Figure 3-4 As shown in the test staining diagram, each method can effectively prepare a single-band target protein; the silver staining results are consistent with the endotoxin content results. After the optimized purification method removes a large amount of endotoxin, the lower lipopolysaccharide band disappears, while the recombinant protein prepared by the optimized fragmentation method has a large amount of endotoxin remaining, and the endotoxin band can still appear; the immunoblotting results are consistent with the above results. The target bands of the recombinant proteins prepared by each method are clearly imprinted, and the lower endotoxin bands of the groups with good endotoxin removal effects are significantly lighter; in the ELISA results, the titer of the group with effective endotoxin removal is reduced from the original 243K to 81K. In summary, it is determined that endotoxin has an impact on immune recognition, leading to the occurrence of nonspecific reactions and cross-reactions. Removing endotoxins can significantly reduce the adverse effects of endotoxins on immune recognition.

[0041] 3. Comparison of conventional endotoxin removal methods

[0042] (1) In this experiment, the molecular weight of the recombinant protein OmpA is about 49 kDa, and the molecular weight of endotoxin is 10-15 kDa, so a pyrogen-free 30 kDa ultrafiltration tube was selected. Add 2 mL of protein sample, centrifuge at 4°C, 4000 x g, 30 min, and collect the concentrate in the ultrafiltration tube. Add pyrogen-free water to the original volume of the solution, and repeat the above operation 3 times.

[0043] (2) Affinity media adsorption method: Mix the Endotoxin Removal Beads thoroughly, use a non-pyrogenic pipette to draw 2 mL of slurry and add it to the chromatography column. Open the lower outlet to remove the protective solution. Wash with 3 mL of regeneration solution (equilibrium solution containing 1% Triton X-114), control the flow rate at 0.25 mL / min, or less than 10 drops per minute, and control the temperature at 2-8°C. Repeat at least twice to ensure that there is no endotoxin in the column.

[0044] Use 3 mL of equilibration solution (20 mM phosphate, 0.15 M NaCL, pH 7.4) to equilibrate the inner wall of the column tube and the filler, drain it, with a flow rate of about 0.5 mL / min, and control the temperature at 2-8°C. Repeat at least twice.

[0045] Add the sample to the balanced Endotoxin Removal Beads, adjust the flow rate to 0.25mL / min, or less than 10 drops per minute, and start collecting the effluent when about 1mL of the effluent flows out. After it dries up, add 1mL of the equilibration solution and continue collecting. Detect the endotoxin content in the sample and the sample recovery rate.

[0046] (3) Extraction method: prepare n-butanol-butyl acetate (9:1, V / V), mix with the protein solution in a volume ratio of 1:5, stir for 15 min, let stand for 30 min, discard the upper organic phase, use a pipette to draw the lower aqueous phase and place it in a new centrifuge tube, repeat the operation three times to obtain the lower aqueous phase.

[0047] (4) Compare the three methods for the removal of endotoxin from recombinant proteins, determine the protein content, and evaluate the effects of the methods on immune recognition.

[0048] like Figure 5 The results showed that the endotoxin content of the recombinant protein treated by ordinary crushing and ordinary purification methods was 779051EU / mL, and the endotoxin content of the protein treated by ultrafiltration was reduced to 14569EU / mL, with an endotoxin removal rate of 98.13% and a protein retention rate of 83.3%; the endotoxin level of the protein treated by affinity medium adsorption was reduced to 12542EU / mL, with an endotoxin removal rate of 98.39% and a protein retention rate of 91%; the endotoxin level of the protein treated by extraction was reduced to 16360EU / mL, with an endotoxin removal rate of 97.9% and a protein retention rate of 75.6%. All three methods can effectively reduce the endotoxin level of the recombinant protein and retain a large amount of protein.

[0049] like Figure 6-7 The results showed that the protein bands obtained by the three methods were of consistent thickness and the protein retention rate was high. In the silver staining image, the treated recombinant proteins had no lower endotoxin bands, which was consistent with the phenomenon after endotoxin removal. The immunoblotting showed that the proteins obtained by the three methods could still be identified with serum, while the lower endotoxin reaction bands disappeared. In the ELISA results, the titer of the group that effectively removed endotoxin was reduced from 243K to 81K.

[0050] 4. Ultrafiltration Optimization

[0051] (1) Use 100 mM HCl and NaOH to adjust the pH of the recombinant protein OmpA solution to 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. Place on a shaker at room temperature for 2 h.

[0052] (2) Considering that the size of the recombinant protein OmpA is 49 kDa, a 30 kDa ultrafiltration tube was used to ultrafilter the treated recombinant protein solution at 4°C, 4000 x g, and 30 min. The concentrate was recovered, re-diluted, and the operation was repeated until the pH returned to neutral.

[0053] (3) The pH of the recombinant protein OmpA solution was adjusted to 2 using 100 mM HCl and NaOH, and the solution was placed on a shaker at room temperature for 0.25, 0.5, 0.75, 1.2, 4, 6, and 8 h to verify the effect of treatment time on endotoxin removal.

[0054] (4) The upper phase and lower phase prepared by the conventional ultrafiltration method were compared and silver staining was performed to verify the actual removal effect of the optimized ultrafiltration method.

[0055] like Figure 8-12 It shows that in the method where the recombinant protein is first subjected to pH adjustment and then ultrafiltration, the endotoxin content of the protein obtained is further reduced when the pH is adjusted in the range of 2-4 or >10. Compared with the protein obtained by the ordinary ultrafiltration method, the endotoxin content is reduced by 90%, and the protein retention rate is maintained at 70%-80% during the pH adjustment process, which is generally stable. Immunoblotting shows that the immune recognition of the prepared protein is not affected by the pH factor adjustment, the target band is recognized normally, the lower endotoxin band disappears, and the titer determined by ELISA is 81K, which has not decreased further compared with the ordinary ultrafiltration method. The treatment time after pH adjustment was optimized. The silver staining results showed that the lower endotoxin band became lighter when the pH was adjusted to 2 for 15 minutes. When the treatment time reached more than 2 hours, the lower endotoxin band became significantly lighter. Therefore, it is believed that the removal effect is better when treated at room temperature for 2-4 hours. Compared with the original recombinant protein OmpA, the ordinary ultrafiltration method can remove free or loosely bound endotoxins, so the upper and lower phases obtained by ultrafiltration contain a large amount of endotoxins. In the optimized ultrafiltration method, adjusting the pH can decompose the endotoxin monomer molecules and dissociate them into smaller molecules, which is beneficial to the separation of protein and endotoxin in the next step of ultrafiltration. The decomposed endotoxins do not show any bands in silver staining and immunoblotting.

[0056] 5. Evaluation of the effect of composite method in removing endotoxin

[0057] (1) The induced bacterial solution was centrifuged, the supernatant was discarded, the precipitate was resuspended in Binding Buffer, 0.1% TritonX-114 was added, and a suitable ultrasonic probe was selected to ultrasonically disrupt the solution at 300W for 3s on and 3s off for 1h. The disrupted solution was centrifuged and the supernatant was taken. The supernatant was passed through the column, and after the recombinant protein was bound to the column, it was washed with 50 column volumes of binding buffer containing 0.1% Triton X-114, and then washed with 20 column volumes of 4°C binding buffer. Subsequently, the target protein was eluted with different gradients of imidazole. After running on the gel, the eluate containing only the single target protein was collected, dialyzed and concentrated for later use.

[0058] (2) Use 100mM HCl and 100mM NaOH to adjust the pH of the recombinant protein to 2-4 or >10, and place it on a horizontal shaker at room temperature for 2-4 hours for treatment. The treated recombinant protein solution is placed in a 30kDa ultrafiltration tube for ultrafiltration at 4°C, 4000xg, 30min, and the concentrated solution is recovered. After re-diluting, the ultrafiltration and centrifugation are repeated until the pH returns to neutral and the protein is recovered.

[0059] like Figure 13-14 It shows that the endotoxin content of the original protein is 411956EU / mL; the endotoxin content of the protein prepared only by the optimized crushing method and the optimized purification method is reduced to 5715EU / mL, and the endotoxin removal rate is 98.61%; the original sample is only adjusted to pH 2, shaken at room temperature for 2h and then repeatedly ultrafiltered, and the endotoxin content of the prepared protein is reduced to 4537EU / mL, and the endotoxin removal rate is 98.9%; the endotoxin content of the prepared protein treated by the composite method is reduced to 1120EU / mL, and the endotoxin removal rate is 99.73%. The protein bands in the test staining are single, and only the untreated recombinant protein original sample can appear endotoxin bands in the silver staining. The immunoblot also shows that a large number of endotoxin reaction bands appear in the untreated protein sample, while the endotoxin bands in the lower layer of the treated protein sample are all lightened and disappeared. The above results show that the method established by the present invention can quickly remove the endotoxin content of the recombinant protein at low cost and convenience, and does not affect the immune recognition of the protein.

[0060] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A purification method for removing endotoxin from prokaryotically expressed recombinant proteins, characterized in that: During the ultrasonic stage, 0.1% Triton X-114 was added for ice-bath ultrasonic disruption. After the recombinant protein bound to the Ni-NTA affinity column, it was first washed with 50 column volumes of binding buffer containing 0.1% Triton X-114, and then washed with 20 column volumes of 4°C binding buffer. The target protein was subsequently eluted with different gradients of imidazole. Subsequently, the pH of the purified recombinant protein solution was adjusted to 2-4 or >10 with 100 mM hydrochloric acid or sodium hydroxide to dissociate and decompose endotoxins. The solution was shaken at room temperature for 2-4 hours, and then the recombinant protein solution was repeatedly ultrafiltered until the pH returned to neutral to further remove endotoxins from the recombinant protein. The endotoxin solution was retained, and the endotoxin content was detected by a horseshoe crab kit, the protein content was determined by the Coomassie Brilliant Blue method, and the effect of the method on protein immunity was evaluated by ELISA and Western-blot.

2. The method for purifying a recombinant protein to remove endotoxin according to claim 1, characterized in that: The method is specifically as follows: (1) Take pET-32a- OmpA Expression strain E. coli BL21 was inoculated with seed solution and cultured overnight. The seed solution was inoculated into LB medium at 1% inoculum and cultured at 37°C, 180 r / min on a shaker for 2.5 h until the cell density OD600 was 0.6-0.

8. 0.2 mM IPTG was added to the bacterial solution and cultured at 37°C, 180 r / min on a shaker for 12 h. (2) The induced recombinant protein bacterial solution was centrifuged at 4°C, 5000 r / min for 20 min, the bacteria were resuspended in Binding Buffer and added with 0.1% Triton X-114 for ice-bath ultrasonic disruption, and then centrifuged at 4°C, 12000 r / min for 20 min, and the supernatant was collected; the supernatant was added to the Ni column, and after the protein was bound to the column, it was first washed with 50 column volumes of binding buffer containing 0.1% Triton X-114, and then washed with 20 column volumes of 4°C binding buffer, and then the target protein was eluted with different gradients of imidazole, and the eluate with a single band was collected, dialyzed and concentrated for later use; (3) Use 100 mM HCl and 100 mM NaOH to adjust the pH of the recombinant protein to slightly acidic or alkaline, and place it on a horizontal shaker at room temperature for 2-4 h for treatment. Pour the treated recombinant protein solution into a 30 kDa ultrafiltration tube for ultrafiltration at 4°C, 4000 x g, and 30 min. Recover the concentrate, re-dilute it, and repeatedly ultrafilter and centrifuge it until the pH returns to neutral.

3. The method for purifying a recombinant protein to remove endotoxin according to claim 2, characterized in that: The molecular retention rate of the ultrafiltration tube selected in step (3) should be greater than the molecular size of the endotoxin monomer and controlled to be less than half the size of the target protein.

4. The method for purifying a recombinant protein to remove endotoxin according to claim 2, characterized in that: In step (3), 100 mM HCl and NaOH were used to adjust the pH of the recombinant protein OmpA solution to 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively, and the solution was placed on a shaker at room temperature for 2 h. The size of the recombinant protein OmpA is 49 kDa, and a 30 kDa ultrafiltration tube was used to ultrafilter the treated recombinant protein solution at 4°C, 4000 xg, and 30 min. The concentrated solution was recovered, and the operation was repeated after redilution until the pH returned to neutral. The pH of the recombinant protein OmpA solution was adjusted to 2 using 100 mM HCl and NaOH, and the solution was placed on a shaker at room temperature for 0.25, 0.5, 0.75, 1.2, 4, 6, and 8 h to verify the effect of the treatment time on the removal of endotoxins. The upper phase and lower phase prepared by the conventional ultrafiltration method were compared and silver stained to verify the actual removal effect of the optimized ultrafiltration method.

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