A method for extracting and purifying CRM197 protein

Abstract

The application provides a method for extracting and purifying CRM197 protein, and belongs to the technical field of vaccine preparation. Specifically, the CRM197 protein is expressed in the cytoplasm of Escherichia coli by a method without signal peptide and fusion tag addition, and the bacterial cells are treated by an osmotic pressure method (repeated freeze-thawing + high-osmotic solution + low-osmotic solution) to extract the CRM197 protein expressed in the cytoplasm, and then two-step column chromatography (anion column chromatography + hydrophobic column chromatography) is performed to obtain the target protein with high purity. The method for extracting and purifying the CRM197 protein is simple, efficient, easy to operate, does not need complex equipment assistance, and has a cost advantage.

Description

Technical Field

[0001] This invention provides a method for extracting and purifying CRM197 protein, belonging to the field of vaccine preparation technology. Background Technology

[0002] CRM197 protein, a non-toxic mutant of diphtheria toxin (glycine at position 52 is mutated to glutamic acid), has become a core carrier protein in polysaccharide conjugate vaccines due to its intact immunogenicity and non-toxicity, and is widely used in vaccines against pneumococcus and meningococcus. Corynebacterium diphtheriae is the natural host for CRM197 protein production, but its production efficiency is low, production costs are high, and biosafety requirements are stringent. Therefore, researchers have explored heterologous expression of CRM197 protein in numerous other organisms, such as Bacillus subtilis, Pichia pastoris, Pseudomonas fluorescens, and Escherichia coli. Among these, Escherichia coli is the preferred host for heterologous expression of CRM197 protein due to its clear genetic background, high expression yield, low production cost, and relatively high safety.

[0003] CRM197 protein expression in *E. coli* often occurs as inclusion bodies, which is detrimental to subsequent protein purification and large-scale industrial production. Therefore, various methods are employed to promote the expression of CRM197 in a soluble form in *E. coli*. One common method is to add a signal peptide to the N-terminus of the original amino acid sequence of CRM197. This signal peptide guides the CRM197 protein to the periplasm, which has a better oxidative environment, promoting proper disulfide bond folding and reducing inclusion body formation. However, the periplasmic volume only accounts for 15% of the cell volume, limiting yield, and this technique also carries the risk of incomplete signal peptide cleavage, leading to heterogeneous target protein structure. Adding a fusion tag is another important method to promote soluble protein expression, but it also carries the risk of incomplete tag cleavage and increased impurities.

[0004] Therefore, expressing CRM197 in the cytoplasm of *E. coli* without adding extra amino acid sequences is highly attractive. However, this method still needs to address issues such as inclusion body expression and the difficulty in purifying the CRM197 protein later. Due to its disulfide bonds and strong hydrophobicity, the natural CRM197 protein may exist in various forms during cytoplasmic expression, including monomers, polymers, and inclusion bodies. Polymers and inclusion bodies interfere with the purification of monomeric CRM197 protein, causing significant difficulties in protein purification. Most existing technologies employ a three-step column purification method (patent number CN106350527B) and composite packing material (Yulia Alexandrovna Khodaket al., BioTech (Basel. 2023 Jan 11;12(1):9. doi: 10.3390 / biotech12010009) for CRM197 protein purification. These technologies generally suffer from cumbersome purification steps, high costs, and low purity of the target protein after purification. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a method suitable for extracting and purifying soluble CRM197 protein expressed in the cytoplasm of engineered bacteria. The present invention employs a method without signal peptide or fusion tag addition to express CRM197 protein in the cytoplasm of engineered bacteria. The bacterial cells are subjected to repeated freeze-thaw cycles and osmotic pressure treatment to extract CRM197 protein from the cytoplasm into the supernatant. The target protein is then purified by a two-step column chromatography process using an anion exchange chromatography column (NanoGel-50Q) and a hydrophobic chromatography column (Cytiva Butyl Sepharose 4 FF), thereby obtaining a high-purity monomeric CRM197 protein.

[0006] Specifically, this invention provides a method for extracting and purifying soluble CRM197 protein expressed in the cytoplasm of engineered bacteria, characterized by: extracting CRM197 protein using osmotic pressure and purifying CRM197 protein using a two-step column chromatography method, wherein:

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

[0008] S1. After the culture is completed, the bacterial cells are collected by centrifugation, and the bacterial cells are repeatedly frozen and thawed between -80℃ and room temperature;

[0009] S2. Resuspend the bacterial cells in a hypertonic solution at a ratio of 20-40 mL / g wet bacteria, incubate in an ice-water bath for 10-30 min, and then centrifuge the bacterial cells at 8000-12000 for 10-30 min. Collect the bacterial cell precipitate on one hand and the supernatant on the other hand to obtain solution I.

[0010] S3. Resuspend the bacterial precipitate obtained in the previous step in a hypotonic solution at a ratio of 20-40 mL / g wet bacteria, incubate in an ice-water bath for 10-30 min, centrifuge the bacterial cells at 8000-12000 for 10-30 min, and collect the supernatant as solution II.

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

[0012] The hypertonic solution is composed of 30-100 mM Tris, 5-10 mM EDTA, 20% sucrose, and pH=8.0.

[0013] The hypotonic solution is ultrapure water;

[0014] (2) Two-step column chromatography method for purifying CRM197 protein:

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

[0016] A. Anion chromatography:

[0017] A1) The anion exchange chromatography column was equilibrated using ion exchange buffer A, which was 20 mM Tris–HCl at pH 8.0.

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

[0019] A3) Gradient elution was performed using an ion chromatography buffer consisting of ion chromatography buffer A and ion chromatography buffer B. Ion chromatography buffer B was 20 mM Tris–HCl + 0.5 M NaCl, pH 8.0. The elution buffer contained two gradients: 10% B and 30% B, where B represents ion chromatography buffer B, and the percentage is a volume ratio. The remaining amount in the elution buffer was ion chromatography buffer A. The 30% B elution product was collected for hydrophobic column chromatography.

[0020] B. Hydrophobic column chromatography:

[0021] B1) Equilibrate the hydrophobic chromatography column using hydrophobic chromatography buffer A, which is 20 mM Tris–HCl + 2 M NaCl, pH 8.0;

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

[0023] B3) Gradient elution was performed using an elution buffer consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B, which was 20 mM Tris–HCl at pH 8.0. The elution buffer contained two gradients: 50% B and 90% B, where B represents hydrophobic chromatography buffer B, and the percentage is a volume ratio. The remaining amount in the elution buffer was hydrophobic chromatography buffer A. The 90% B elution product was collected to obtain the purified CRM197 protein.

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

[0025] Preferably, in steps S2 and / or S3, the ice-water bath and centrifugation parameters are as follows: after incubating in an ice-water bath for 20 minutes, the bacterial cells are centrifuged at 12,000 rpm for 10 minutes.

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

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

[0028] Preferably, the engineered bacteria is Escherichia coli, including but not limited to BL21-DE3, or BL21(DE3), which is an engineered Escherichia coli strain used for recombinant protein expression. DE3 indicates that the DE3 fragment of λ phage is integrated into the Escherichia coli genome. This fragment 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 freeze-thaw cycles is 1 to 5 times, and more preferably 3 times.

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

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

[0033] The beneficial effects of this invention are:

[0034] (1) This invention uses repeated freeze-thaw cycles of bacterial cells and osmotic pressure to extract CRM197 protein, which is then purified by two-step column chromatography to obtain a high-purity target protein. Through repeated research, this invention has developed parameter conditions suitable for the extraction and purification of CRM197 protein using osmotic pressure + two-step column chromatography, thereby increasing the purity of the target protein from 62.79% to 93.01%. Compared with other existing technologies, the purification method of this invention is simple, efficient, easy to operate, requires no complex equipment, and has certain cost advantages.

[0035] (2) This invention is applicable to the extraction and purification of soluble CRM197 protein expressed in the cytoplasm. The amino acid sequence of the CRM197 protein does not have additional amino acid sequences such as signal peptides added, thus avoiding the problems of incomplete cleavage of signal peptides and non-uniform structure of target protein from the source. Furthermore, this invention abandons the process route of expressing CRM197 protein in the periplasm, thereby avoiding the problem of limited target protein yield due to the periplasm volume accounting for only 15% of the cell. Attached Figure Description

[0036] Figure 1 SDS-PAGE spectra of samples extracted by high-pressure homogenization in Example 2;

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

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

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

[0040] Figure 5 SDS-PAGE spectra of samples extracted by osmotic pressure method in Example 3;

[0041] Figure 6 SDS-PAGE spectra of the anion exchange column chromatography purified sample in Example 3;

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

[0043] Figure 8 SEC-HPLC chromatogram of the sample purified by two-step column chromatography in Example 3. Detailed Implementation

[0044] To more accurately understand and grasp the objectives, technical solutions, and advantages of this invention, the invention will be described in detail below with reference to specific embodiments. Please note that the embodiments described herein represent only some application examples of this invention 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 embodiment is shown in SEQ ID NO: 1. Its characteristic is that it does not contain any natural or heterologous signal peptide sequences, nor any other additional amino acid sequences. Protein expression is achieved by adding one methionine residue as a start codon to the N-terminus of the CRM197 protein sequence. The target protein sequence shown in SEQ ID NO: 1 was codon-optimized (different nucleic acid sequences encoding the target protein were obtained and their expression was compared, and the nucleic acid sequence with better expression levels was selected). The entire genome was synthesized based on the optimized nucleic acid sequence.

[0047] SEQ ID NO: 1: MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGA SRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTV TGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNT VEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0048] Expression vector construction: The optimized and synthesized CRM197 gene was inserted into the pET30a(+) plasmid using NdeI and HindIII. The constructed vector was transformed into DH5α competent cells, and positive clones were screened using antibiotics. Positive clones were cultured in large quantities at 37℃ and 220 rpm, and plasmids were extracted. After the extracted plasmid was sequenced and verified to be correct, it was named pFd001.

[0049] Target protein expression: The plasmid pFd001, correctly ligated with the CRM197 gene, was transformed into E. coli competent cells BL21(DE3). Positive clones were picked and cultured in LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing 50 μg / mL kanamycin at 37°C and 220 rpm until the OD reached approximately 0.6. 0.5 mM IPTG was then added to induce protein expression. The temperature was lowered to 16°C, and the cells were cultured at 220 rpm for another 16-20 h.

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

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

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

[0053] (1) Collect bacterial cells by centrifugation (12000 rpm, 4℃, 10 min);

[0054] (2) The cells were resuspended in 20 mM Tris–HCl buffer (pH 8.0) and then homogenized by high pressure (1200 bar, 3 cycles).

[0055] (3) Centrifuge (12000 rpm, 4℃, 10 min) and collect the homogenized supernatant (bacterial rupture supernatant) and precipitate respectively.

[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 for extracting the target protein was evaluated. Figure 1 As shown, low-temperature induction at 16°C using LB liquid medium can induce the expression of most CRM197 protein in the homogenized supernatant, forming soluble expression. This result also demonstrates that soluble CRM197 protein can be extracted from the cytoplasm by high-pressure homogenization.

[0057] II. Two-step column chromatography method for purifying CRM197 protein:

[0058] The homogenized supernatant (bacterial lysis supernatant) obtained by high-pressure homogenization was purified using a two-step column chromatography method (anion exchange column chromatography + hydrophobic column chromatography):

[0059] A. Anion chromatography:

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

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

[0062] A3) Gradient elution was performed using an elution buffer 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 eluted at a conductivity of 10–20 ms / cm. Therefore, the elution gradient was set according to this conductivity. Specifically, the gradient of ion chromatography buffer B in the elution buffer was set as 10%B, 20%B, 30%B, 40%B, 50%B, 100%B (where B represents ion chromatography buffer B, and the percentage is the volume fraction of ion chromatography buffer B; the remaining amount in the elution buffer is ion chromatography buffer A, so the gradient of ion chromatography buffer A corresponding to ion chromatography buffer B is: 90%A, 80%A, 70%A, 60%A, 50%A, 0%A; since it is well known in the art that the sum of the volume fractions of different buffers in the elution buffer should be 100%, the ion chromatography buffer... The gradient of A can be omitted; in the gradient elution described in this application, once the baselines of the system's UV absorbance and conductivity measurements are leveled out, the next gradient can be started (the same applies below).

[0063] Detection and Analysis: For samples eluted by anion exchange column chromatography, the gradient of the target protein was analyzed by SDS-PAGE. Results are as follows: Figure 2As shown: i) After purification by anion exchange column chromatography (50Q column) of the CRM197 lysis supernatant, the target protein can be distributed in flow-through buffer (Flow-through-1, Flow-through-2), elution buffer (10%B-1~50%B), regeneration buffer (100%B), washing buffer (1M NaCl), and CIP (Clean In Place, i.e., online cleaning, in which 0.5M NaOH is used to clean the chromatography column). The reason for this may be that there are misfolded or multimeric CRM197 molecules in the supernatant. The different forms of CRM197 molecules have different charges, so the protein can be distributed in different components; ii) A horizontal comparison of the SDS-PAGE spectra of the elution products corresponding to the six gradient elution buffers revealed that the target protein content was highest in the 30%B elution product. Therefore, the 30%B elution product was selected for the next step of hydrophobic column chromatography.

[0064] B. Hydrophobic column chromatography:

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

[0066] B2) The NaCl concentration in the 30% B elution product was adjusted to about 2M NaCl using 4M sodium chloride so that the sample to be tested and the hydrophobic chromatography buffer A have basically the same conductivity. Then it was loaded onto the equilibrated hydrophobic chromatography column.

[0067] B3) Gradient elution was performed using an elution buffer 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 according to this conductivity. Specifically, the gradient of hydrophobic chromatography buffer B in the elution buffer 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, and these percentages are volume ratios).

[0068] Detection and Analysis: For samples eluted by two-step column chromatography, the gradient of the target protein was analyzed by SDS-PAGE, and purity was determined by SEC-HPLC. The results are as follows: i) ... Figure 3As shown, 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, so two sample tubes were collected and labeled as 50% B-1 and 50% B-2 respectively, with the remaining labels being the same). ii) The 50% B-2 eluent sample (50% B-2 elution product) was analyzed by SEC-HPLC, and the results are shown below. Figure 4 As shown, the purity of the target protein in this sample is 62.79% (the yield was not calculated further due to the low purity).

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

[0070] Because the purity of CRM197 protein obtained after direct lysis using high-pressure homogenization followed by two-step column chromatography is low, this invention employs osmotic pressure extraction instead of high-pressure homogenization to extract the target protein from the cytoplasm of *E. coli* before the two-step column chromatography to improve protein purification. Furthermore, since different protein extraction methods result in significant differences in target protein content and impurity levels, the elution gradients for anion exchange chromatography and hydrophobic column chromatography were partially adjusted in this embodiment based on preliminary exploratory experimental data.

[0071] I. Osmotic extraction of CRM197 protein:

[0072] After expressing CRM197 protein in Example 1, CRM197 protein was extracted in this example using osmotic pressure extraction:

[0073] S1. After the culture is completed, the bacterial cells are collected by centrifugation. The bacterial cells are repeatedly frozen and thawed between -80℃ and room temperature (0 times, 1 time, 3 times, 5 times) to improve cell membrane permeability.

[0074] S2. Resuspend the bacterial cells in a hypertonic solution (50mM Tris, 10mM EDTA, 20% sucrose, pH=8.0) at a ratio of 20mL / g wet bacteria. After incubating in an ice-water bath for 20min, centrifuge the bacterial cells at 12000rpm for 10min. Collect the bacterial precipitate and the supernatant to obtain solution I.

[0075] S3. Resuspend the bacterial precipitate 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, then centrifuge the bacterial cells at 12000 rpm for 10 min and collect the supernatant as solution II.

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

[0077] Detection and Analysis: For the CRM197 extract obtained by osmotic pressure method, the extraction efficiency of the target protein was analyzed by SDS-PAGE, and the CRM197 protein bands were quantitatively analyzed using ImageJ software (to avoid interference from CRM197 polymers, only non-reducing protein samples were quantitatively analyzed). Results are as follows: Figure 5 As shown in Table 1: During osmotic extraction, i) if the bacterial cells were not freeze-thawed, the extraction efficiency was low, and both solutions I and II contained relatively little CRM197 protein; ii) freeze-thawing of the bacterial cells increased cell membrane permeability, significantly improving the osmotic extraction efficiency of the target protein. Freeze-thaw cycles of 1, 3, and 5 times significantly increased the extraction yield of CRM197 protein compared to no freeze-thaw. The best extraction effect was observed with 3 freeze-thaw cycles, resulting in the highest peak area of ​​the target protein band in the CRM197 extract.

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

[0079]

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

[0081] II. Two-step column chromatography method for purifying CRM197 protein:

[0082] The CRM197 extract, which had undergone three freeze-thaw cycles, was used as the sample for two-step column chromatography (anion exchange chromatography + hydrophobic column chromatography). The specific procedures are as follows:

[0083] A. Anion chromatography:

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

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

[0086] A3) Gradient elution was performed using an ion chromatography buffer 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 was set to 10% B, 20% B, 30% B, and 100% B (correspondingly, the gradient of ion chromatography buffer A was 90% A, 80% A, 70% A, and 0% A, and these percentages are volume ratios), and 30% of the eluted product was collected for the next hydrophobic column chromatography step.

[0087] Detection and Analysis: For samples after anion exchange column chromatography, the elution gradient of the target protein was analyzed by SDS-PAGE. The SDS-PAGE results are as follows: Figure 6 As shown: i) The target protein was mainly present in the elution buffers (10%B, 20%B, 30%B), while CRM197 protein was not observed in the flow-through and regeneration buffers (100%B), proving that osmotic pressure chromatography may be able to separate misfolded or multimeric CRM197 molecules from monomeric CRM197 molecules in the cytoplasm. ii) A horizontal comparison of the SDS-PAGE spectra of the elution products corresponding to the four gradient elution buffers revealed that the 30%B elution product had the highest content of the target protein. Therefore, the 30%B elution product was selected for the next step of hydrophobic column chromatography.

[0088] B. Hydrophobic column chromatography:

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

[0090] B2) The NaCl concentration in the 30% B elution product was adjusted to about 2M NaCl using 4M sodium chloride so that the sample to be tested and the hydrophobic chromatography buffer A have basically the same conductivity. Then it was loaded onto the equilibrated hydrophobic chromatography column.

[0091] 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): the hydrophobic chromatography buffer B gradient was set to 40%B, 50%B, 90%B, 100%B (correspondingly, the hydrophobic chromatography buffer A gradient was 60%A, 50%A, 10%A, 0%A, the percentages being volume ratios).

[0092] 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 was determined by SEC-HPLC. The SDS-PAGE results are shown below. Figure 7 As shown: the target protein is mostly present in the 90% B elution gradient. The 90% B elution buffer sample (90% B elution product) was analyzed by SEC-HPLC, and the results are as follows. Figure 8As shown, the target protein purity in this sample reached 93.01%, and the protein concentration was determined using the Bradford method, with a final yield of 0.8 mg / g (0.8 mg of target protein can be obtained from 1 gram of wet bacteria). This demonstrates that the optimized osmotic pressure method combined with two-step column chromatography significantly improves the extraction and purification efficiency of CRM197 protein.

[0093] It should be noted that, based on the above experimental results, in practical applications of this invention: A) Anion exchange column chromatography can use only two gradients, 10% B and 30% B, where 10% B is used for washing away impurities (removing contaminating proteins) and 30% B is used for eluting the target protein, without needing to set other gradients (e.g., 20% B, 100% B, where 100% B is only used to verify whether the target protein has been completely eluted and is not a usable elution gradient for subsequent applications); B) Hydrophobic column chromatography can use only two gradients, 50% B and 90% B, where 50% B is used for washing away impurities and 90% B is used for eluting the target protein, without needing to set other elution gradients (e.g., 40% B, 100% B).

[0094] The above embodiments are several specific examples used to illustrate the present invention in detail so that those skilled in the art can understand the solution. 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 *Escherichia coli*, characterized in that, The amino acid sequence of the CRM197 protein is shown in SEQ ID NO:

1. The CRM197 protein was extracted using osmotic pressure and purified using a two-step column chromatography method, wherein: (1) Extraction of CRM197 protein by osmotic pressure method: S1. After the culture is completed, the bacterial cells are collected by centrifugation, and the bacterial cells are repeatedly frozen and thawed between -80℃ and room temperature, and the number of times the freeze-thaw is repeated is 3; S2. Resuspend the bacterial cells in a hypertonic solution at a ratio of 20-40 mL / g wet bacteria, incubate in an ice-water bath for 10-30 min, and then centrifuge the bacterial cells at 8000-12000 rpm for 10-30 min. Collect the bacterial cell precipitate and the supernatant to obtain solution I. S3. Resuspend the bacterial precipitate obtained in the previous step in a hypotonic solution at a ratio of 20-40 mL / g wet bacteria, incubate in an ice-water bath for 10-30 min, centrifuge the bacterial cells at 8000-12000 for 10-30 min, and collect the supernatant as solution II. S4. Mix solution I and solution II to obtain CRM197 extract; The hypertonic solution is composed of 30-100 mM Tris, 5-10 mM EDTA, 20% sucrose, and pH=8.

0. The hypotonic solution is ultrapure water; (2) Two-step column chromatography method for purifying CRM197 protein: Including anion exchange chromatography and hydrophobic column chromatography: A. Anion chromatography: A1) The anion exchange chromatography column was equilibrated using ion exchange buffer A, wherein the anion exchange chromatography column was a NanoGel 50Q column, and the ion exchange buffer A was 20 mM Tris–HCl, pH 8.0; A2) Load the CRM197 extract onto the equilibrated chromatography column; A3) Gradient elution was performed using an ion chromatography buffer consisting of ion chromatography buffer A and ion chromatography buffer B. Ion chromatography buffer B was 20 mM Tris–HCl + 0.5 M NaCl, pH 8.

0. The elution buffer contained two gradients: 10% B and 30% B, where B represents ion chromatography buffer B, and the percentage is a volume ratio. The remaining amount in the elution buffer was ion chromatography buffer A. The 30% B elution product was collected for hydrophobic column chromatography. B. Hydrophobic column chromatography: B1) Equilibrate the hydrophobic chromatography column using hydrophobic chromatography buffer A, wherein the hydrophobic chromatography column is a Cytiva ButylSepharose 4 FF chromatography column, and the hydrophobic chromatography buffer A is 20 mM Tris–HCl + 2 M NaCl, pH 8.0; B2) For the elution product of 30%B in anion exchange column chromatography, first use sodium chloride to adjust its conductivity to be consistent with that of hydrophobic chromatography buffer A, and then load it onto the equilibrated hydrophobic chromatography column. B3) Gradient elution was performed using an elution buffer consisting of hydrophobic chromatography buffer A and hydrophobic chromatography buffer B, which was 20 mM Tris–HCl at pH 8.

0. The elution buffer contained two gradients: 50%B and 90%B, where 50%B represents hydrophobic chromatography buffer B and the percentage is a volume ratio. The remaining amount in the elution buffer was hydrophobic chromatography buffer A. The 90%B elution product was collected to obtain the purified CRM197 protein.

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 incubating in an ice-water bath for 20 min, the bacterial cells are centrifuged at 12000 rpm for 10 min.

3. The method according to claim 1, characterized in that, The hypertonic solution consisted of 50 mM Tris, 10 mM EDTA, 20% sucrose, and pH 8.

0.

4. The method according to claim 1, characterized in that, In anion exchange column chromatography, the eluent gradient is: 10%B, 20%B, 30%B, 100%B.

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

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