Escherichia coli protein purification method
Through gene optimization, low-temperature induction and multi-step purification process, the problems of inclusion body formation, protein degradation and low purity when expressing proteins in E. coli are solved, and the purity, activity and recovery rate of the protein are significantly improved.
Patent Information
- Application Number
- CN202411818526.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art has problems with inclusion body formation, protein degradation, low expression amount and low purity when expressing proteins in E. coli, resulting in complex purification process and affecting protein quality.
Cell cracking through gene optimization, low temperature induction, ultrasonic crushing method, or high -pressure average method, use high concentration urea or sink salt to dissolve the packaging body, and restore the protein structure through dialysis and gradient elution method. Layers, ion exchange layers, and gel filter layers are added, and antioxidants and auxiliary factor are added to maintain protein stability.
It significantly improves the purity, activity and recovery rate of target protein, reducing the risk of protein loss and degradation, and improving the final quality of purification efficiency and protein.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of bioengineering, and in particular to a method for purifying Escherichia coli protein. Background Art
[0002] With the rapid development of genetic engineering technology, recombinant proteins are increasingly used in drug development, vaccine production, enzyme catalysis, etc. Among them, Escherichia coli has become the most commonly used recombinant protein expression system due to its fast growth rate, simple culture conditions and low cost.
[0003] However, although the E. coli system has been widely used, the following problems often occur during protein expression, which complicates the purification process and affects the quality of the protein.
[0004] 1. Inclusion body formation:
[0005] Target proteins often accumulate in the form of inclusion bodies in E. coli. Inclusion bodies are non-functional protein complexes that aggregate inside cells and are difficult to extract directly from cells and difficult to dissolve. Traditional methods usually rely on high concentrations of denaturants (such as urea, guanidine salts, etc.) for resolubilization, but in this process the protein easily loses its native conformation and biological activity. In order to refold the protein, extremely complex refolding steps are often required, and the success rate is low.
[0006] 2. Protein degradation:
[0007] During protein expression and purification, endogenous proteases in cells will degrade the target protein, reducing the final protein yield and quality. In order to avoid protein degradation, protease inhibitors are usually added to the lysis buffer, but this also increases the complexity of the purification process.
[0008] 3. Low expression and low purity:
[0009] For some difficult-to-express proteins, common purification methods (such as affinity chromatography and ion exchange chromatography) are difficult to achieve the desired purity and often require multiple rounds of purification and time consumption. Although traditional affinity chromatography methods can initially remove impurities, they often cannot effectively remove low molecular weight contaminants or other non-specific binders, resulting in low purity of the target protein.
[0010] In summary, the deficiencies in the prior art are:
[0011] For inclusion body proteins, the folding efficiency is low after melting, and the protein is easily inactivated;
[0012] For low-expression proteins, the expression level is low and they are easily contaminated during purification;
[0013] Traditional single chromatography methods cannot effectively remove nonspecific binders, resulting in low purification efficiency;
[0014] Therefore, a new solution to the above problems needs to be proposed. Summary of the invention
[0015] The purpose of the present invention is to provide a method for purifying Escherichia coli protein, which significantly improves the purity, activity and recovery rate of the target protein by optimizing expression conditions, protein solubilization, purification process and adding auxiliary factors, so as to solve the technical problems raised in the background technology.
[0016] To achieve the above object, the present invention provides the following technical solution: a method for purifying Escherichia coli protein, comprising at least the following steps:
[0017] Gene optimization: codon optimization and gene sequence adjustment of target protein genes to improve the expression and solubility of target proteins;
[0018] Expression condition regulation: Under low temperature conditions of 16℃-20℃, use 0.1mM-0.5mM IPTG to induce the expression of the target protein, and add low concentrations of metal ions or coenzymes to the culture medium to enhance the natural folding of the protein;
[0019] Cell lysis: Use ultrasonic disruption or high-pressure homogenization to lyse cells, and add 1mM PMSF and other protease inhibitors to the lysate to prevent degradation of the target protein;
[0020] Inclusion body dissolution and folding: The inclusion body protein is dissolved by high concentration of urea or guanidine salt, and the concentration of urea or guanidine salt is gradually reduced by dialysis to restore the protein to its native conformation. Folding auxiliary factors are added during the dialysis process to promote correct folding;
[0021] Multi-step purification: Multiple purification steps using affinity chromatography, ion exchange chromatography and gel filtration chromatography are used to purify the target protein.
[0022] Furthermore, the affinity chromatography uses a Ni-NTA affinity chromatography column and a gradient elution method to improve the purity of the target protein.
[0023] Furthermore, the ion exchange chromatography uses a DEAE-Sepharose or CM-Sepharose column, and the pH value and salt concentration gradient of the elution buffer are optimized.
[0024] Furthermore, the gel filtration chromatography uses size exclusion chromatography (SEC) to further remove polymers and low molecular weight impurities in the target protein to ensure that the protein is a monomer.
[0025] Furthermore, antioxidants (such as DTT, β-mercaptoethanol) are added to prevent protein oxidative degradation, and appropriate cofactors or metal ions (such as Zn 2+ Mg 2+ etc.) to maintain the stability and activity of the target protein.
[0026] Furthermore, the recombinant protein is an enzyme protein, an antibody protein, a vaccine antigen or a structural protein.
[0027] Furthermore, the protein folding auxiliary factors in the step include heavy metal ions, coenzymes or low-concentration reducing agents.
[0028] Furthermore, the multi-step purification includes ion exchange chromatography and gel filtration chromatography after affinity chromatography to improve the purity and recovery rate of the target protein.
[0029] Furthermore, the purification method also includes using freeze-drying technology or low-temperature storage (-80°C) to preserve the protein for a long time, and adding a protective agent such as glycerol to prevent protein denaturation during freezing and thawing.
[0030] Furthermore, the method is applicable to the expression and purification of different types of recombinant proteins from E. coli, including but not limited to enzymes, antibodies, vaccine antigens and structural proteins.
[0031] Compared with the prior art, the present invention has the following beneficial effects:
[0032] 1. Optimization of cell expression system: Through gene optimization and induction condition regulation, the solubility of target protein is significantly improved and the formation of inclusion bodies is reduced. By introducing auxiliary folding factors or coenzymes, the natural folding and stability of target protein are enhanced.
[0033] 2. Innovation in inclusion body treatment: A "staged dissolution" strategy is adopted for inclusion body proteins. First, the inclusion bodies are dissolved with high concentrations of urea or guanidine salts, and then the protein structure is gradually restored through dialysis and gradient elution methods, and correct folding is promoted through dynamic folding auxiliary factors.
[0034] 3. Multi-step purification process: A multiple purification combination of affinity chromatography, ion exchange chromatography and gel filtration chromatography is used to optimize the buffer, elution conditions and gradient of different chromatography steps to improve the purity and recovery rate of the target protein.
[0035] 4. Protein stability control: Various methods are used to ensure that the protein remains stable during the purification process, including the use of antioxidants, metal ions and cofactors to reduce the risk of protein degradation. DETAILED DESCRIPTION
[0036] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0037] Embodiment 1:
[0038] Expression and purification of recombinant antibody Fab fragments
[0039] Target protein: recombinant human antibody Fab fragment.
[0040] Expression system:
[0041] Use pET-28a(+) vector to insert the target antibody gene (with His tag) and transform BL21(DE3) Escherichia coli strain.
[0042] Expression conditions:
[0043] The low temperature induction method was used to induce expression with 0.2 mM IPTG at 16°C for 18 hours. Luria-Bertani (LB) medium was used with a culture volume of 1 L, and induction was performed when the bacterial concentration reached OD600=0.6.
[0044] Cell lysis and inclusion body solubilization:
[0045] The cells were lysed by ultrasonication, and 1 mM PMSF (protease inhibitor) was added to the lysate for protection. After lysis, the cell debris was removed by centrifugation and the inclusion bodies were collected. 8 M urea was used to dissolve the inclusion bodies, which were dissolved slowly at 30 °C. Afterwards, the urea concentration was gradually reduced by dialysis, and 0.1 mM Zn was added. 2+ To promote antibody folding.
[0046] Purification process:
[0047] Affinity chromatography:
[0048] A Ni-NTA affinity column was used for preliminary purification. The elution buffer used was 50 mM NaH2PO4, 300 mM NaCl, 200 mM Mimidazole, pH 8.0. After elution, low molecular weight impurities were further removed by dialysis.
[0049] Ion exchange chromatography:
[0050] A secondary purification was performed using DEAE-Sepharose and eluted with a gradient salt solution (0-1 M NaCl) to remove impurities.
[0051] Gel filtration chromatography:
[0052] The protein was further purified using a Superdex200 gel filtration column to ensure that it was in a monomeric state.
[0053] Quality Control:
[0054] Purity was analyzed by SDS-PAGE and Western Blot to confirm the correctness of the target antibody Fab fragment. Antigen binding test was performed for activity.
[0055] Example 2: Expression and purification of recombinant enzyme
[0056] Target protein: Recombinant Escherichia coli β-glucosidase.
[0057] Expression system:
[0058] The β-glucosidase gene (with a His tag) was inserted into the pET-32a vector and transformed into the BL21 (DE3) Escherichia coli strain.
[0059] Expression conditions:
[0060] The expression was induced with 0.5 mM IPTG at 37°C for 4 hours. LB medium was used with a culture volume of 500 mL. Induction was performed when the bacterial concentration reached OD600 = 0.6.
[0061] Cell lysis and inclusion body solubilization:
[0062] The cells were lysed by high pressure homogenization, and 1 mM PMSF and 1 μg / mL DNase were added to the lysate to reduce the contamination of the cell lysate. After lysis, the inclusion bodies were collected by centrifugation and dissolved with 6 M urea.
[0063] Purification process:
[0064] Affinity chromatography:
[0065] A Ni-NTA affinity column was used for preliminary purification, and the eluent was 50 mM NaH2PO4, 300 mM NaCl, 250 mM Mimidazole, pH 7.4. The eluate was collected and dialyzed to remove impurities.
[0066] Ion exchange chromatography:
[0067] Further purification was performed using CM-Sepharose, and the elution buffer was a 0-1 M NaCl gradient salt solution, pH 7.0.
[0068] Gel filtration chromatography:
[0069] The protein was further separated using a Superdex75 column.
[0070] Quality Control:
[0071] Purity and enzyme activity were confirmed by SDS-PAGE and activity assay (by hydrolysis reaction of substrate pNP-β-D-glucopyranoside).
[0072] Example 3: Expression and purification of recombinant lysozyme
[0073] Target protein: Recombinant human lysozyme (Lysozyme).
[0074] Expression system:
[0075] The human lysozyme gene (with GST tag) was inserted into the pGEX-6P-1 vector and transformed into the BL21(DE3) E. coli strain.
[0076] Expression conditions:
[0077] The expression was induced with 0.3 mM IPTG at 18°C for 16 hours. The culture medium was 2xYT, the culture volume was 500 mL, and the induction was performed when the bacterial concentration reached OD600 = 0.7.
[0078] Cell lysis and inclusion body solubilization:
[0079] The cells were lysed by ultrasonication, and 1 mM PMSF was added to the lysate for protease inhibition. The inclusion bodies were collected by centrifugation, dissolved with 6 M urea, and dialysis was used to remove urea, and coenzymes and reducing agents were added to help the protein recover activity.
[0080] Purification process:
[0081] GST affinity chromatography:
[0082] A Glutathione Sepharose 4B affinity chromatography column was used for preliminary purification, and PBS buffer was used for washing and elution, and the eluate contained 10 mM GSH.
[0083] GST excision:
[0084] The eluted GST-lysozyme fusion protein was added to the GST digestion solution to remove the GST tag, and the uncut part was collected for purification.
[0085] Ion exchange chromatography:
[0086] Further purification was performed using DEAE-Sepharose with a 0-1 M NaCl gradient elution.
[0087] Quality Control:
[0088] Purity analysis was performed using SDS-PAGE, and Western Blot confirmed that the molecular weight of the protein was consistent with the expected value. The activity of the protein was confirmed by enzyme activity assay (cleavage experiment using Murat's liquid substrate).
[0089] The three embodiments are compared with the prior art, see Table 1:
[0090] Table 1
[0091]
[0092]
[0093] It can be seen that the advantages compared with the existing technology are:
[0094] Optimized expression conditions:
[0095] Low temperature induction (such as 16°C, 18°C) can effectively reduce protein misfolding and inclusion body formation, improve protein solubility and activity. Compared with conventional high temperature induction methods, this low temperature induction can improve protein quality and yield.
[0096] Improved inclusion body solubilization method:
[0097] High concentration urea was used to dissolve the inclusion bodies, combined with Zn 2+ Assisted folding can significantly improve the correct folding and activity recovery of antibody Fab fragments. Compared with simple urea dissolution, the addition of cofactors can enhance protein activity.
[0098] Multi-step purification process:
[0099] A variety of purification methods were used, including affinity chromatography, ion exchange chromatography and gel filtration chromatography. Through the multi-step purification process, the purity and activity of the protein were significantly improved, avoiding the impurities and low purity problems that may occur in the existing technology, and ensuring the final quality of the protein.
[0100] Comprehensive quality control:
[0101] The correctness and activity of the protein are ensured by SDS-PAGE, Western Blot and specific activity assays (such as antigen binding experiments and enzyme activity assays). The technical solution is more accurate in activity verification and can effectively verify the functionality of the target protein.
[0102] Refinement of cell lysis and protein protection:
[0103] The high-pressure homogenization method (in Example 2) was combined with the ultrasonic disruption method (in Examples 1 and 3), and protease inhibitors and DNA enzymes were added to significantly reduce the contamination of the cleavage product and maintain the stability of the protein.
[0104] Summarize:
[0105] The above examples demonstrate the expression and purification methods of different types of recombinant proteins (antibody Fab fragments, enzymes, lysozymes). By optimizing the expression system, cell lysis, inclusion body treatment, purification process and protein stability control, the high purity, high activity and stability of the protein are ensured, proving the technical advantages of the present invention. By optimizing the expression system, inclusion body dissolution and multi-step purification, the purity, activity and recovery rate of the target protein are significantly improved, and the problems of protein inactivation and degradation in traditional methods are avoided.
[0106] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
Claims
1. A method for purifying Escherichia coli protein, characterized in that: At least the following steps are included: Gene optimization: codon optimization and gene sequence adjustment of target protein genes to improve the expression and solubility of target proteins; Expression condition regulation: Under low temperature conditions of 16℃-20℃, use 0.1mM-0.5mM IPTG to induce the expression of the target protein, and add low concentrations of metal ions or coenzymes to the culture medium to enhance the natural folding of the protein; Cell lysis: Use ultrasonic disruption or high-pressure homogenization to lyse cells, and add 1mM PMSF and other protease inhibitors to the lysate to prevent degradation of the target protein; Inclusion body dissolution and folding: The inclusion body protein is dissolved by high concentration of urea or guanidine salt, and the concentration of urea or guanidine salt is gradually reduced by dialysis to restore the protein to its native conformation. Folding auxiliary factors are added during the dialysis process to promote correct folding; Multi-step purification: Multiple purification steps using affinity chromatography, ion exchange chromatography and gel filtration chromatography are used to purify the target protein.
2. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The affinity chromatography adopts a Ni-NTA affinity chromatography column and uses a gradient elution method to improve the purity of the target protein.
3. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The ion exchange chromatography uses a DEAE-Sepharose or CM-Sepharose column, and the pH value and salt concentration gradient of the elution buffer are optimized.
4. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The gel filtration chromatography uses size exclusion chromatography to further remove polymers and low molecular weight impurities in the target protein to ensure that the protein is a monomer.
5. The method for purifying Escherichia coli protein according to claim 1, characterized in that: During the purification process, antioxidants are added to prevent protein oxidative degradation, and appropriate cofactors or metal ions are added to maintain the stability and activity of the target protein.
6. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The recombinant protein is an enzyme protein, an antibody protein, a vaccine antigen or a structural protein.
7. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The protein folding auxiliary factors in the step include heavy metal ions, coenzymes or low concentration reducing agents.
8. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The multi-step purification includes ion exchange chromatography and gel filtration chromatography after affinity chromatography to improve the purity and recovery rate of the target protein.
9. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The purification method also includes using freeze-drying technology or low-temperature storage (-80°C) to preserve the protein for a long time, and adding a protective agent such as glycerol to prevent protein denaturation during freezing and thawing.
10. The method for purifying Escherichia coli protein according to claim 1, characterized in that: The method is suitable for expressing and purifying different types of recombinant proteins from E. coli, including but not limited to enzymes, antibodies, vaccine antigens and structural proteins.