Method for extracting and purifying CRM197 protein

Abstract

The invention provides a method for extracting and purifying CRM197 protein, and belongs to the technical field of vaccine preparation. Specifically, CRM197 protein is expressed in escherichia coli cytoplasm by adopting a method without adding signal peptide and fusion tag, thalli are treated by an osmotic pressure method (repeated freezing and thawing + hypertonic solution + hypotonic solution) so as to extract soluble expression CRM197 protein in cytoplasm, and then two-step column chromatography (anion column chromatography + hydrophobic column chromatography) is carried out to obtain the CRM197 protein in the cytoplasm. The target protein with relatively high purity is obtained. The CRM197 protein extraction and purification method provided by the invention is simple, efficient and easy to operate, does not need the assistance of complex equipment, and has a cost advantage.

Description

Technical Field

[0001] The invention provides a method for extracting and purifying CRM197 protein, belonging to the technical field of vaccine preparation. Background Art

[0002] CRM197, a non-toxic mutant of diphtheria toxin (glycine 52 mutated to glutamic acid), retains full immunogenicity and is non-toxic. It has become a core carrier protein for polysaccharide conjugate vaccines and is widely used in vaccines against pneumococci and meningococci. Corynebacterium diphtheriae is the natural host for CRM197 production, but production efficiency is low, costs are high, and biosafety requirements are high. Consequently, attempts have been made to heterologously express CRM197 in a variety of other organisms, such as Bacillus subtilis, Pichia pastoris, Pseudomonas fluorescens, and Escherichia coli. Escherichia coli is a preferred host for heterologous expression of CRM197 due to its clear genetic background, high expression yield, low production costs, and high safety.

[0003] The CRM197 protein often appears as inclusion bodies when expressed in E. coli, which is not conducive to subsequent protein purification and large-scale industrial production. Therefore, people often use various means to promote the expression of CRM197 in E. coli in a soluble form. One commonly used method is to add a signal peptide to the N-terminus of the original amino acid sequence of CRM197. The signal peptide guides the CRM197 protein to the cell periplasm with a better oxidative environment for expression, promotes the correct folding of disulfide bonds, and reduces the formation of inclusion bodies. However, the periplasmic volume only accounts for 15% of the cell, which limits the yield. In addition, this technology also has the risk of incomplete signal peptide cleavage, resulting in uneven target protein structure. In addition, adding a fusion tag is also an important means to promote protein soluble expression, but there is also the risk of incomplete tag cleavage and increased impurities.

[0004] Therefore, expressing CRM197 in the cytoplasm of E. coli without adding additional amino acid sequences is very attractive. However, this method still needs to solve the problems of inclusion body expression and the difficulty of purifying CRM197 protein in the later stage. Due to the presence of disulfide bonds and strong hydrophobicity, natural CRM197 protein may exist in various forms such as monomers, polymers and inclusion bodies when expressed in the cytoplasm. Polymers and inclusion bodies will interfere with the purification of monomeric CRM197 protein, causing great difficulties in protein purification. Most existing technologies use a three-step column purification method (patent number CN106350527B) and composite fillers (Yulia Alexandrovna Khodak et al., BioTech (Basel). 2023 Jan 11;12(1):9. doi: 10.3390 / biotech12010009) to purify CRM197 protein. These technologies generally have problems such as complicated purification steps, high costs, and low purity of the target protein after purification. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide a method suitable for extracting and purifying CRM197 protein soluble expressed in the cytoplasm of engineered bacteria: the present invention adopts a method without signal peptide and fusion tag addition to express CRM197 protein in the cytoplasm of engineered bacteria, and repeatedly freeze-thaws and treats the bacteria with osmotic pressure to extract the CRM197 protein in the cytoplasm into the supernatant, and then purifies the target protein through two-step column chromatography using an anion chromatography column (NanoGel-50Q) and a hydrophobic chromatography column (Cytiva Butyl Sepharose 4 FF), thereby obtaining a monomeric CRM197 protein with higher purity.

[0006] Specifically, the present invention provides a method for extracting and purifying CRM197 protein soluble in the cytoplasm of an engineered bacterium, characterized in that the CRM197 protein is extracted by an osmotic pressure method and purified by a two-step column chromatography method, wherein:

[0007] (1) Extraction of CRM197 protein by osmotic pressure method:

[0008] S1. After the culture, the cells were collected by centrifugation and repeatedly frozen and thawed between -80°C and room temperature.

[0009] S2. Resuspend the cells in hypertonic solution at a ratio of 20-40 mL / g wet bacteria. Incubate in an ice-water bath for 10-30 min. Centrifuge the cells at 8000-12000 rpm for 10-30 min. Collect the pellet and the supernatant to obtain Solution I.

[0010] S3. Resuspend the bacterial pellet obtained in the previous step in hypotonic solution at a ratio of 20-40 mL / g wet bacteria. Incubate in an ice-water bath for 10-30 min. Centrifuge the cells at 8000-12000 rpm for 10-30 min. Collect the supernatant as Solution II.

[0011] S4. Mixing solution I and solution II to obtain a CRM197 extract;

[0012] Wherein, the hypertonic solution is: 30-100mM Tris, 5-10mM EDTA, 20% sucrose, pH=8.0;

[0013] Wherein, the hypotonic solution is ultrapure water;

[0014] (2) Purification of CRM197 protein by two-step column chromatography:

[0015] Including anion column chromatography and hydrophobic column chromatography:

[0016] A. Anion column chromatography:

[0017] A1) An anion chromatography column was equilibrated with ion chromatography buffer A (20 mM Tris–HCl, pH 8.0).

[0018] A2) Loading the CRM197 extract onto the equilibrated chromatography column;

[0019] A3) performing gradient elution using an eluent consisting of ion chromatography buffer A and ion chromatography buffer B, wherein ion chromatography buffer B is 20 mM Tris–HCl + 0.5 M NaCl, pH 8.0; wherein the eluent comprises the following two gradients: 10% B and 30% B, where B represents ion chromatography buffer B and the percentage is by volume, and the remainder of the eluent is ion chromatography buffer A; collecting the 30% B elution product for hydrophobic column chromatography;

[0020] B. Hydrophobic column chromatography:

[0021] B1) Equilibrate the hydrophobic chromatography column with hydrophobic chromatography buffer A (20 mM Tris–HCl + 2 M NaCl, pH 8.0).

[0022] B2) For the 30%B elution product from the anion column chromatography, first adjust its conductivity with sodium chloride to be consistent with the conductivity of the hydrophobic chromatography buffer A, and then load it onto the equilibrated hydrophobic chromatography column;

[0023] B3) Gradient elution was performed using an eluent consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B, wherein hydrophobic chromatography buffer B was 20 mM Tris–HCl, pH 8.0; wherein the eluent contained the following two gradients: 50% B and 90% B, where B represents hydrophobic chromatography buffer B and the percentage is by volume, and the remainder of the eluent is hydrophobic chromatography buffer A; the 90% B elution product was collected to obtain the purified CRM197 protein product.

[0024] Preferably, the hypertonic solution is: 50 mM Tris, 10 mM EDTA, 20% sucrose, pH=8.0.

[0025] Preferably, in step S2 and / or S3, the ice-water bath and centrifugation parameters are as follows: after incubating in the ice-water bath for 20 minutes, the bacteria are centrifuged at 12000 rpm for 10 minutes.

[0026] Preferably, in anion column chromatography, the eluent gradient is: 10% B, 20% B, 30% B, 100% B.

[0027] Preferably, in the hydrophobic column chromatography, the eluent gradient is: 40% B, 50% B, 90% B, 100% B.

[0028] Preferably, the engineered bacteria are Escherichia coli, including but not limited to BL21-DE3, or BL21 (DE3), which is an engineered strain of Escherichia coli for recombinant protein expression, wherein DE3 represents the DE3 fragment of λ phage integrated into the Escherichia coli genome, which carries the T7 RNA polymerase gene and is regulated by the lacUV5 promoter.

[0029] Preferably, the amino acid sequence of the CRM197 protein is shown in SEQ ID NO: 1.

[0030] Preferably, the number of repeated freeze-thaw cycles is 1 to 5 times, more preferably 3 times.

[0031] Preferably, the anion chromatography column is a NanoGel 50Q chromatography column.

[0032] Preferably, the hydrophobic chromatography column is a Cytiva Butyl Sepharose 4 FF chromatography column.

[0033] Beneficial effects of the present invention:

[0034] (1) The present invention uses repeated freeze-thaw cycles and osmotic pressure to extract CRM197 protein, and then purifies it through two-step column chromatography to obtain a highly pure target protein. Through repeated research, the present invention has developed parameters suitable for extracting and purifying CRM197 protein using osmotic pressure plus two-step column chromatography, which increases the purity of the target protein from 62.79% to 93.01%. Compared with other existing technologies, the purification method of the present invention is simple, efficient, easy to operate, does not require complex equipment, and has certain cost advantages.

[0035] (2) The present invention is suitable for extracting and purifying CRM197 protein that is soluble and expressed in the cytoplasm. The amino acid sequence of the CRM197 protein does not contain any additional amino acid sequences such as signal peptides, thereby avoiding the problems of incomplete signal peptide cleavage and heterogeneous target protein structure from the source. In addition, the present invention abandons the process of expressing the CRM197 protein in the periplasm, thereby avoiding the problem of limited target protein production due to the periplasmic volume accounting for only 15% of the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : SDS-PAGE pattern of the sample extracted by high pressure homogenization method in Example 2;

[0037] Figure 2 : SDS-PAGE pattern of the anion column chromatography purified sample in Example 2;

[0038] Figure 3 : SDS-PAGE pattern of the sample purified by two-step column chromatography in Example 2;

[0039] Figure 4 : SEC-HPLC profile of the sample purified by two-step column chromatography in Example 2;

[0040] Figure 5 : SDS-PAGE pattern of the sample extracted by osmotic pressure method in Example 3;

[0041] Figure 6 : SDS-PAGE pattern of the sample purified by anion column chromatography in Example 3;

[0042] Figure 7 : SDS-PAGE pattern of the sample purified by two-step column chromatography in Example 3;

[0043] Figure 8 : SEC-HPLC profile of the two-step column chromatography purified sample in Example 3. DETAILED DESCRIPTION

[0044] In order to more accurately understand and master the object, technical scheme and superiority of the present application, the following will combine specific embodiments to make a detailed description of the present application. Please note that the embodiments set forth herein only represent some application examples of the present application, and do not encompass all embodiments.

[0045] Example 1: Expression of CRM197 Protein

[0046] Target protein sequence: the amino acid sequence of the CRM197 protein used in this example is shown as SEQ ID NO: 1, which is characterized in that it does not contain any natural or heterologous signal peptide sequence, nor other additional amino acid sequence, only 1 methionine is added at the N terminus of the CRM197 protein sequence as a start codon for protein expression. The target protein sequence shown as SEQ ID NO: 1 is codon-optimized (different nucleic acid sequences encoding the target protein are obtained and compared for their expression, and a nucleic acid sequence with better expression is screened out), and the optimized nucleic acid sequence is used for whole gene synthesis.

[0047] SEQ ID NO: 1: MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0048] Expression vector construction: The optimized, synthesized CRM197 gene was inserted into the pET30a(+) plasmid via NdeI and HindIII. The constructed vector was transformed into DH5α competent cells, and positive clones were selected using antibiotics. Positive clones were cultured in large numbers at 37°C and 220 rpm, and the plasmid was extracted. The extracted plasmid was sequenced to verify sequence accuracy and named pFd001.

[0049] Target protein expression: Transform the plasmid pFd001 containing the CRM197 gene into competent E. coli BL21(DE3) cells. Select positive clones and culture in LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) supplemented with 50 μg / mL kanamycin. Cultivate at 37°C and 220 rpm until the OD reaches approximately 0.6. Induce protein expression by adding 0.5 mM IPTG. Lower the temperature to 16°C and continue culturing at 220 rpm for 16-20 hours.

[0050] Example 2: Extraction and purification of CRM197 protein (high-pressure homogenization + two-step column chromatography)

[0051] 1. Extraction of CRM197 protein by high-pressure homogenization:

[0052] After expressing the CRM197 protein according to Example 1, the CRM197 protein was extracted using a high-pressure homogenization method:

[0053] (1) Centrifuge (12000 rpm, 4°C, 10 min) to collect the cells;

[0054] (2) Resuspend the cells in 20 mM Tris–HCl buffer (pH 8.0) and then perform high-pressure homogenization (1200 bar, 3 cycles);

[0055] (3) Centrifuge (12000 rpm, 4°C, 10 min) and collect the homogenized supernatant (bacterial supernatant) and precipitate separately.

[0056] Detection and analysis: After completing the above operations, the solubility of CRM197 protein expression was analyzed by SDS-PAGE, and the feasibility of high-pressure homogenization method for extracting the target protein was evaluated. Figure 1 As shown, low-temperature induction at 16°C using LB liquid culture medium can cause most of the CRM197 protein to be expressed in the homogenized supernatant, forming soluble expression. This result also shows that the soluble CRM197 protein expressed in the cytoplasm can be extracted by breaking up the bacteria through high-pressure homogenization.

[0057] 2. Two-step column chromatography purification of CRM197 protein:

[0058] The homogeneous supernatant (bacterial supernatant) obtained by high-pressure homogenization extraction was purified by two-step column chromatography (anion column chromatography + hydrophobic column chromatography):

[0059] A. Anion column chromatography:

[0060] A1) An anion chromatography column (NanoGel, 50Q) was equilibrated with ion chromatography buffer A (20 mM Tris–HCl, pH 8.0);

[0061] A2) Loading the lysed supernatant onto the equilibrated chromatography column;

[0062] A3) Gradient elution was performed using an eluent consisting of ion chromatography buffer A and ion chromatography buffer B (20 mM Tris–HCl + 0.5 M NaCl, pH 8.0). Based on previous experimental data, the target protein was eluted at a conductivity of 10-20 ms / cm. Therefore, an elution gradient was set according to this conductivity. Specifically, the gradient of ion chromatography buffer B in the eluent was set to 10% B, 20% B, 30% B, 40% B, 50% B, and 100% B (where B represents ion chromatography buffer B and the percentage represents the volume fraction of ion chromatography buffer B. The remainder of the eluent was ion chromatography buffer A. Therefore, corresponding to ion chromatography buffer B, the gradient of ion chromatography buffer A was: 90% A, 80% A, 70% A, 60% A, 50% A, and 0% A. Since the sum of the volume fractions of different buffers in the eluent should be 100%, which is well known in the art, the gradient of ion chromatography buffer A was set to 10% B, 20% B, 30% B, 40% B, 50% B, and 100% B. The gradient of A can be omitted; in the gradient elution described in this application, the next gradient can be entered after the baseline of the system's ultraviolet absorbance value and the conductivity measurement value are leveled, the same below).

[0063] Detection and analysis: For the elution sample of anion column chromatography, the gradient of target protein was analyzed by SDS-PAGE. Figure 2As shown: i) After the CRM197 supernatant was purified by anion column chromatography (50Q column), the target protein was distributed in the flow-through (Flow through-1, Flow through-2), eluent (10%B-1~50%B), regeneration solution (100%B), cleaning solution (1MNaCl) and CIP (Clean In Place, i.e., online cleaning, in which 0.5M NaOH was used to clean the chromatography column). Analysis showed that the reason may be the presence of misfolded or multimeric CRM197 molecules in the supernatant. The various forms of CRM197 molecules have different charge properties, so the protein can be distributed in different components; ii) A horizontal comparison of the SDS-PAGE profiles of the elution products corresponding to the six gradient elution solutions revealed that the 30%B elution product corresponded to the highest target protein content, so the 30%B elution product was selected for the next hydrophobic column chromatography.

[0064] B. Hydrophobic column chromatography:

[0065] B1) Equilibrate a hydrophobic chromatography column (Cytiva, Butyl Sepharose 4 FF) with hydrophobic chromatography buffer A (20 mM Tris–HCl + 2 M NaCl, pH 8.0);

[0066] B2) Adjusting the NaCl concentration in the 30% B elution product to approximately 2 M NaCl using 4 M NaCl so that the sample to be tested has substantially the same conductivity as the hydrophobic chromatography buffer A, and then loading the sample onto the equilibrated hydrophobic chromatography column;

[0067] B3) Gradient elution was performed using an eluent consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B (20 mM Tris–HCl, pH 8.0). Based on previous exploratory experimental data, the target protein eluted at a conductivity of less than 100 mS / cm. Therefore, the elution gradient was set around this conductivity. Specifically, the gradient of hydrophobic chromatography buffer B in the eluent was set to 50% B, 60% B, 65% B, 75% B, 80% B, and 100% B (correspondingly, the gradient of hydrophobic chromatography buffer A was 50% A, 40% A, 35% A, 25% A, 20% A, and 0% A, where the percentages are by volume).

[0068] Detection and analysis: For the two-step column chromatography elution samples, the target protein gradient was analyzed by SDS-PAGE and the purity was tested by SEC-HPLC. The results are as follows: i) Figure 3As shown in Figure 2, the target protein was eluted from the hydrophobic chromatography column under 50% B conditions (Note: 50% B-1 and 50% B-2 were both eluted under 50% hydrophobic chromatography Buffer B conditions, but two different protein peaks appeared during collection. Therefore, two samples were collected and labeled 50% B-1 and 50% B-2, respectively. The other labels remain the same). ii) The 50% B-2 eluate sample (50% B-2 elution product) was subjected to SEC-HPLC analysis, and the results are shown in Figure 2. Figure 4 As shown, the purity of the target protein in this sample was 62.79% (due to the low purity, the yield was not further calculated).

[0069] Example 3: Extraction and purification of CRM197 protein (osmotic pressure method + two-step column chromatography)

[0070] Because the purity of the CRM197 protein obtained by direct bacterial disruption using high-pressure homogenization followed by two-step column chromatography is relatively low, to further enhance the purification effect, the present invention uses osmotic pressure to replace high-pressure homogenization to extract the target protein from the E. coli cytoplasm before the two-step column chromatography, thereby improving protein purification efficiency. Furthermore, because different protein extraction methods lead to significant differences in target protein content and impurity levels, based on preliminary exploratory experimental data, the elution gradients of anionic column chromatography and hydrophobic column chromatography were partially adjusted in this example.

[0071] 1. Extraction of CRM197 protein by osmotic pressure method:

[0072] After expressing the CRM197 protein according to Example 1, the CRM197 protein was extracted by osmotic pressure method in this example:

[0073] After the culture, the cells were collected by centrifugation and repeatedly frozen and thawed between -80°C and room temperature (0, 1, 3, and 5 times) to improve cell membrane permeability.

[0074] S2. Resuspend the cells in a hypertonic solution (50 mM Tris, 10 mM EDTA, 20% sucrose, pH 8.0) at a ratio of 20 mL / g wet bacteria. Incubate in an ice-water bath for 20 min. Centrifuge the cells at 12,000 rpm for 10 min. Collect the pellet and the supernatant to obtain Solution I.

[0075] S3. Resuspend the bacterial pellet obtained in the previous step in a hypotonic solution (ultrapure water) at a ratio of 20 mL / g wet bacteria. Incubate in an ice-water bath for 20 min. Centrifuge the cells at 12,000 rpm for 10 min and collect the supernatant as Solution II.

[0076] S4. Mix solution I and solution II to obtain a CRM197 extract.

[0077] Detection and analysis: The CRM197 extract obtained by the osmotic pressure method was analyzed by SDS-PAGE for the extraction effect of the target protein, and the CRM197 protein bands were quantitatively analyzed using ImageJ software (to avoid interference from CRM197 multimers, only non-reduced protein samples were quantitatively analyzed). The results are as follows Figure 5 As shown in Table 1, during osmotic extraction, i) if the cells were not frozen and thawed, the extraction efficiency was low, with both Solution I and Solution II containing less CRM197 protein. ii) Freezing and thawing the cells increased cell membrane permeability, significantly improving the efficiency of osmotic extraction of the target protein. Freezing and thawing once, three times, and five times significantly increased the amount of CRM197 protein extracted compared to no freezing and thawing. The best extraction efficiency was achieved with three freeze-thaw cycles, with the CRM197 extract containing the highest peak area of ​​the target protein.

[0078] Table 1: ImageJ analysis of CRM197 protein amount extracted by osmotic pressure method

[0079]

[0080] Note: The data in the table are the peak areas of the non-reduced CRM197 protein bands obtained by ImageJ analysis; the peak area of ​​the CRM197 extract is the sum of the peak areas corresponding to solution I and solution II.

[0081] 2. Two-step column chromatography purification of CRM197 protein:

[0082] The CRM197 extract that had been frozen and thawed three times was used as the loading sample and subjected to two-step column chromatography (anion column chromatography + hydrophobic column chromatography). The specific operation is as follows:

[0083] A. Anion column chromatography:

[0084] A1) An anion chromatography column (NanoGel, 50Q) was equilibrated with ion chromatography buffer A (20 mM Tris–HCl, pH 8.0);

[0085] A2) Loading the CRM197 extract onto the equilibrated chromatography column;

[0086] A3) Perform gradient elution using an eluent consisting of ion chromatography buffer A and ion chromatography buffer B (20 mM Tris–HCl + 0.5 M NaCl, pH 8.0): the gradient of ion chromatography buffer B is set as 10% B, 20% B, 30% B, and 100% B (correspondingly, the gradient of ion chromatography buffer A is 90% A, 80% A, 70% A, and 0% A, the percentages being by volume). Collect the 30% B elution product for the next hydrophobic column chromatography step.

[0087] Detection and analysis: For the sample after anion column chromatography, the elution gradient of the target protein is analyzed by SDS-PAGE. The SDS-PAGE detection results are as follows: Figure 6 As shown: i) The target protein was primarily present in the eluents (10% B, 20% B, and 30% B), while no CRM197 protein was found in the flow-through or regeneration buffer (100% B). This demonstrates that osmotic pressure analysis can potentially separate misfolded or multimeric CRM197 molecules from monomeric CRM197 molecules in the cytoplasm. ii) Comparison of the SDS-PAGE profiles of the elution products corresponding to the four gradient elutions revealed that the 30% B elution product contained the highest target protein content, so the 30% B elution product was selected for the next step of hydrophobic column chromatography.

[0088] B. Hydrophobic column chromatography:

[0089] B1) Equilibrate a hydrophobic chromatography column (Cytiva, Butyl Sepharose 4 FF) with hydrophobic chromatography buffer A (20 mM Tris–HCl + 2 M NaCl, pH 8.0);

[0090] B2) adjusting the NaCl concentration in the 30% B elution product to approximately 2 M NaCl using 4 M NaCl so that the sample to be tested has substantially the same conductivity as that of the hydrophobic chromatography buffer A, and then loading the sample onto the equilibrated hydrophobic chromatography column;

[0091] B3) Perform gradient elution using an eluent consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B (20 mM Tris–HCl, pH 8.0): the hydrophobic chromatography buffer B gradient is set to 40% B, 50% B, 90% B, and 100% B (correspondingly, the hydrophobic chromatography buffer A gradient is set to 60% A, 50% A, 10% A, and 0% A, where the percentages are by volume).

[0092] Detection and analysis: For the samples after two-step column chromatography, the elution gradient of the target protein was analyzed by SDS-PAGE, and the purity of the eluted samples from the two-step column chromatography was detected by SEC-HPLC. Figure 7 As shown: The target protein is mostly present in the 90% B elution gradient. The 90% B eluate sample (90% B elution product) was tested by SEC-HPLC, and the results are as follows Figure 8As shown, the target protein in this sample had a purity of 93.01%. The protein concentration was determined by the Bradford assay, and the final yield was 0.8 mg / g (0.8 mg of target protein per gram of wet bacteria). This demonstrates that the optimized osmotic pressure method plus two-step column chromatography protocol can significantly improve the extraction and purification of the CRM197 protein.

[0093] It should be noted that, based on the above experimental results, in actual application of the present invention: A) anion column chromatography can be set with only two gradients of 10% B and 30% B, wherein 10% B is used for washing impurities (washing away impurity proteins) and 30% B is used for eluting the target protein, without setting other gradients (for example, 20% B, 100% B, wherein 100% B is only used to verify whether the target protein is completely eluted and is not a subsequent elution gradient); B) hydrophobic column chromatography can be set with only two gradients of 50% B and 90% B, wherein 50% B is used for washing impurities and 90% B is used for eluting the target protein, without setting other elution gradients (for example, 40% B, 100% B).

[0094] The above embodiments are several specific examples, which are used to explain the present invention in detail so that those skilled in the art can understand the solutions. The specific implementation methods and the protection scope of this application are not limited thereto.

Claims

1. A method for extracting and purifying soluble CRM197 protein expressed in the cytoplasm of engineered bacteria, characterized in that: The CRM197 protein was extracted by osmotic pressure method and purified by two-step column chromatography, wherein: (1) Extraction of CRM197 protein by osmotic pressure method: S1. After the culture, the cells were collected by centrifugation and repeatedly frozen and thawed between -80°C and room temperature. S2. Resuspend the cells in hypertonic solution at a ratio of 20-40 mL / g wet bacteria. Incubate in an ice-water bath for 10-30 min. Centrifuge the cells at 8000-12000 rpm for 10-30 min. Collect the pellet and the supernatant to obtain Solution I. S3. Resuspend the bacterial pellet obtained in the previous step in hypotonic solution at a ratio of 20-40 mL / g wet bacteria. Incubate in an ice-water bath for 10-30 min. Centrifuge the cells at 8000-12000 rpm for 10-30 min. Collect the supernatant as Solution II. S4. Mixing solution I and solution II to obtain a CRM197 extract; Wherein, the hypertonic solution is: 30-100mM Tris, 5-10mM EDTA, 20% sucrose, pH=8.0; Wherein, the hypotonic solution is ultrapure water; (2) Purification of CRM197 protein by two-step column chromatography: Including anion column chromatography and hydrophobic column chromatography: A. Anion Column Chromatography: A1) balancing an anion chromatography column with ion chromatography buffer A, wherein ion chromatography buffer A is: 20 mM Tris-HCl, pH 8.0; A2) Loading the CRM197 extract onto the equilibrated chromatography column; A3) performing gradient elution using an eluent consisting of ion chromatography buffer A and ion chromatography buffer B, wherein ion chromatography buffer B is 20 mM Tris–HCl + 0.5 M NaCl, pH 8.0; wherein the eluent comprises the following two gradients: 10% B and 30% B, where B represents ion chromatography buffer B and the percentage is by volume, and the remainder of the eluent is ion chromatography buffer A; collecting the 30% B elution product for hydrophobic column chromatography; B. Hydrophobic column chromatography: B1) equilibrate the hydrophobic chromatography column with hydrophobic chromatography buffer A, wherein the hydrophobic chromatography buffer A is 20 mM Tris–HCl + 2 M NaCl, pH 8.0; B2) For the 30%B elution product from the anion column chromatography, first adjust its conductivity with sodium chloride to be consistent with the conductivity of the hydrophobic chromatography buffer A, and then load it onto the equilibrated hydrophobic chromatography column; B3) Gradient elution was performed using an eluent consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B, wherein hydrophobic chromatography buffer B was 20 mM Tris–HCl, pH 8.0; wherein the eluent contained the following two gradients: 50% B and 90% B, where B represents hydrophobic chromatography buffer B and the percentages are by volume, and the remainder of the eluent was hydrophobic chromatography buffer A; the 90% B elution product was collected to obtain the purified CRM197 protein product.

2. The method according to claim 1, characterized in that In step S2 and / or step S3, the ice-water bath and centrifugation parameters are as follows: after incubation in the ice-water bath for 20 minutes, the bacteria are centrifuged at 12000 rpm for 10 minutes.

3. The method according to claim 1, wherein The hypertonic solution comprises: 50 mM Tris, 10 mM EDTA, 20% sucrose, pH=8.

0.

4. The method according to claim 1, wherein In anion column chromatography, the eluent gradient was: 10% B, 20% B, 30% B, 100% B.

5. The method according to claim 1, wherein In hydrophobic column chromatography, the eluent gradient is: 40% B, 50% B, 90% B, 100% B.

6. The method according to claim 1, wherein The engineered bacteria is Escherichia coli.

7. The method according to claim 1, wherein The amino acid sequence of the CRM197 protein is shown in SEQ ID NO:

1.

8. The method according to claim 1, wherein The number of repeated freeze-thaw cycles is 1 to 5 times.

9. The method according to claim 8, characterized in that The number of repeated freeze-thaw cycles was 3 times.

10. The method according to claim 1, wherein The anion chromatography column is a NanoGel 50Q chromatography column.

11. The method according to claim 1, wherein The hydrophobic chromatography column is a Cytiva Butyl Sepharose 4 FF chromatography column.

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