A method for renaturation of CRM197 inclusion body protein

Abstract

The invention discloses a denaturation method for CRM197 inclusion body protein, and relates to the technical field of bioengineering. The invention comprises the following steps: S1, dissolving the CRM197 inclusion body protein with a mixed denaturing solution of an alkali solution and a denaturant, and then centrifuging to obtain a supernatant; S2, performing a primary chromatography elution on the supernatant to obtain an eluted protein; S3, performing a secondary chromatography elution on the eluted protein after ultrafiltration to obtain a pure CRM197 protein. The invention solves the technical problem that the currently used urea denaturant has a high concentration, which leads to the generation of impurity precipitation, resulting in low purity, low yield and long time consumption of the obtained CRM197 protein.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a denaturation method of CRM197 inclusion body protein. Background Art

[0002] CRM197 ( Cross reacting material , diphtheria toxin mutant) is a mutant of diphtheria toxin that has lost its toxicity. It is a mutant in which a base G in the nucleotide sequence of wild-type DT is mutated to A, resulting in the 52nd amino acid Gly changing to Glu. Structurally, CRM197 has a complete DT functional structure. However, experiments have shown that CRM197 cannot produce toxic effects on cells.

[0003] CRM197 has no enzyme activity and toxicity, but has the immunogenicity of DT. Therefore, CRM197 is used as a protein carrier to couple with polysaccharides to prepare polysaccharide conjugate vaccines, and corresponding products have been launched on the market.

[0004] At present, CRM197 is generally produced by secretion of lysogenized Corynebacterium diphtheriae, which contains a tox mutant gene encoding DT through infection with beta phage. The advantage of this system is that the target protein is expressed in a secreted form outside the cell, and the low content of impurities is conducive to purification production. The disadvantage is that the fermentation conditions of Corynebacterium diphtheriae are harsh, the culture medium components are complex, and the price is expensive.

[0005] The E. coli expression system has the advantages of short production cycle, low cost, easy introduction of exogenous DNA, simple expression of foreign protein, and rapid production of large amounts of target protein. It is the preferred system for expressing recombinant proteins. However, the CRM197 protein currently expressed by this system exists in the form of inclusion bodies and needs to go through the stages of denaturation and renaturation.

[0006] Extreme pH values, high temperatures, detergents, high concentrations of inorganic salts or organic solvents can all be used to dissolve inclusion bodies. Currently, most people use 6.0 mol / L guanidine hydrochloride or 8.0 mol / L urea in combination with a reducing agent to dissolve inclusion bodies. However, urea dissolves inclusion bodies slowly and weakly. When the action time is long or the temperature is high, it will cleave to form cyanate, which will cause free aminoformylation of the polypeptide chain to covalently modify the amino group of the recombinant protein. Guanidine hydrochloride denaturation has the disadvantages of high cost, easy precipitation under acidic conditions, and removal after renaturation may cause a large amount of protein precipitation and interfere with protein ion exchange chromatography. Summary of the invention

[0007] In order to overcome the defects of the prior art, the present invention provides a denaturation method for CRM197 inclusion body protein, which can solve the technical problems that the currently used urea denaturant has a high concentration and thus leads to the generation of impurity precipitation, resulting in low purity, low yield and long time consumption of the obtained CRM197 protein.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] The invention provides a denaturation method for CRM197 inclusion body protein, comprising the following steps: S1, dissolving the CRM197 inclusion body protein with a mixed denaturing solution of an alkali solution and a denaturant, and then centrifuging to obtain a supernatant; S2, performing a primary chromatography elution on the supernatant to obtain an eluted protein; S3, performing a secondary chromatography elution on the eluted protein after ultrafiltration to obtain a pure CRM197 protein; in S1, the alkali solution is a sodium hydroxide solution, the denaturant is urea, in the mixed denaturing solution, the concentration of the sodium hydroxide solution is 8-12 mM, the concentration of the urea is 1.8-2.2 M, the pH of the mixed denaturing solution is 11.5-12.5, and the volume-to-mass ratio of the mixed denaturing solution to the CRM197 inclusion body protein is 19-21 mL / g.

[0010] The principle of the present invention is as follows: the present invention uses a combination of sodium hydroxide and urea for alkaline denaturation, which not only reduces the amount of urea used, but also makes the alkaline dissolution of inclusion bodies faster, thereby further improving the denaturation rate of the overall protein.

[0011] In one embodiment, the CRM197 inclusion body protein is derived from Escherichia coli.

[0012] In one embodiment, in S2, the chromatography column used for the initial chromatography elution is a Q anion exchange chromatography column.

[0013] In one embodiment, in S2, the primary chromatography elution includes gradient buffer elution and salt elution, the liquid A used for the gradient buffer elution is a mixture of 8~12 mM hydroxide solution and 1.8~2.2 M urea, the liquid B is a mixture of 4.5~5.5 wt% glycerol, 48~52 mM Tris-HCl and 0.08~0.12 mol NaCl, and the elution gradient of the gradient buffer elution is 100%A~100%B, 1.9~2.1 hours; the eluent used for the salt elution is a mixture of 18~22 mMTris-HCl and 495~505 mM NaCl; the pH of the A liquid is 11.5~12.5, and the pH of the B liquid and the eluent of the salt elution are 7.5~8.5, respectively.

[0014] In one embodiment, in S3, the ultrafiltration comprises the following steps: using a 30KD membrane package to replace the eluted protein with an equal volume of buffer 5 times, the replacement buffer is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, and the pH of the replacement buffer is 7.5-8.5.

[0015] In one embodiment, in S3, the chromatography column used for the secondary chromatography elution is a DEAE anion exchange chromatography column.

[0016] In one embodiment, in S3, the secondary chromatography elution includes salt elution, the solution A used for the salt elution is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, the solution B used for the salt elution is a mixture of 18-22 mM Tris-HCl and 490-510 mM NaCl, the elution gradient of the salt elution is 100% A-100% B, 0.9-1.1 hours; the pH of the solution A and the solution B used for the salt elution are respectively 7.5-8.5.

[0017] In one embodiment, the secondary chromatography elution and before the salt elution also include a pretreatment, and the pretreatment includes the following steps: equilibrating the chromatography column with an equilibrating buffer, the equilibrating buffer is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, and the pH of the equilibrating buffer is 7.5-8.5.

[0018] In one embodiment, in S1, the centrifugal force of the centrifugation is 10 4 g, and the centrifugation temperature was 4°C.

[0019] In one embodiment, in S1, the dissolution time is more than 2 h.

[0020] The main beneficial effects of the present invention are:

[0021] Reduce the use of denaturants, and the alkaline denaturation speed is faster. The protein renaturation is carried out by Q chromatography, and the renaturation efficiency is higher than the conventional dilution method. The correctness of the structure after renaturation is high, and it has the advantages of low material and equipment investment and short process operation time.

[0022] The present invention will be described in more detail below based on embodiments and with reference to the accompanying drawings, wherein:

[0023] Figure 1 This is an SDS-Page image of the CRM197 inclusion body protein after Q purification in the present invention, wherein 1: denatured supernatant (load solution, after ethanol precipitation), 2: flow-through, 3: purified sample;

[0024] Figure 2 This is an SDS-Page diagram of the CRM197 inclusion body protein purified by DEAE in the present invention, wherein 1: DEAE loading solution, 2: DEAE flow-through, 3: DEAE collection solution;

[0025] Figure 3 This is a graph showing the detection results of SEC-HPLC of the CRM197 purified protein in the present invention;

[0026] Figure 4 This is a WB image obtained by Western blotting detection of the recombinantly expressed CRM197 purified protein in the present invention. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0028] The present invention will be further described below in conjunction with the accompanying drawings.

[0029] Embodiment 1:

[0030] (1) Collect E. coli cells by centrifugation at 10,000 g and 4°C. Take 10 g of cells, resuspend them in 20 mM Tris-HCl (pH=8.0), and then homogenize and break the cells. Collect the precipitate by centrifugation at 10,000 g and 4°C. Wash the inclusion bodies twice with 20 mM Tris-HCl (pH=8.0) containing 1.5 M urea, collect the precipitate, and obtain relatively pure inclusion bodies.

[0031] (2) Dissolve the inclusion bodies in 10 mM NaOH + 2 M urea, pH 12 at a ratio of inclusion body weight to denaturing solution volume (W (g): V (mL)) = 1:20. Dissolve the inclusion bodies at room temperature for 3 hours, then centrifuge at 10,000 g, 4°C to collect the supernatant.

[0032] (3) Q chromatography refolding (Q impres), use the equilibration solution 10 mM sodium hydroxide + 3 M guanidine hydrochloride + 2 M sodium chloride pH 12 to equilibrate the chromatography column until the baseline is stable, then load 10 CV of the dissolved supernatant, and then rinse with a buffer with gradually decreasing denaturant concentration. The specific composition of the buffer rinse with gradually decreasing denaturant concentration is: A: 10 mM sodium hydroxide + 2 M urea, pH 12, B: 50 mM Tris-HCl, 5 wt% glycerol, 0.1 mol NaCl, pH = 8.0, 100% A → 100% B linear gradient, rinse for 2 hours, then use 20 mM Tris-HCl, 0.5 M NaCl, pH = 8.0 to elute, collect the elution peak, and the elution results are shown in Figure 1 The results are shown.

[0033] (4) Ultrafiltration: The collected eluate was replaced five times with an equal volume of buffer using a 30 KD membrane package. The replacement buffer was 20 mM Tris-HCl, 50 mM NaCl, pH = 8.0.

[0034] (5) DEAE chromatography (Capto DEAE): Use equilibration buffer 20 mM Tris-HCl + 50 mM NaCl (pH = 8.0) to equilibrate the Capto DEAE (H = 10 cm, CV = 20 ml) chromatography column, and load the ultrafiltered protein sample onto the column. Then use A: 20 mM Tris-HCl, 50 mM NaCl, pH = 8.0, B: 20 ​​mM Tris-HCl, 500 mM NaCl, pH = 8.0, 100% A → 100% B linear gradient, rinse for 1 hour, collect the eluted protein peak, and the elution results are shown in the figure. Figure 2 The results are shown in Figure 2. Quantification was performed by the extinction coefficient method, and a total of 40 mg of pure protein was harvested.

[0035] Embodiment 2:

[0036] (1) Collect E. coli cells by centrifugation at 10,000 g and 4°C. Take 10 g of cells, resuspend them in 20 mM Tris-HCl (pH=8.0), and then homogenize and break the cells. Collect the precipitate by centrifugation at 10,000 g and 4°C. Wash the inclusion bodies twice with 20 mM Tris-HCl (pH=8.0) containing 1.5 M urea, collect the precipitate, and obtain relatively pure inclusion bodies.

[0037] (2) Dissolve the inclusion bodies using 8 mM NaOH + 1.8 M urea, pH 11.5 at a ratio of inclusion body weight to denaturing solution volume (W (g): V (mL)) = 1:19. Dissolve the inclusion bodies at room temperature for 4 hours, then centrifuge at 10,000 g, 4°C to collect the supernatant.

[0038] (3) Q chromatography refolding (Q impres), use the equilibration solution 8 mM sodium hydroxide + 3 M guanidine hydrochloride + 2 M sodium chloride pH 12 to equilibrate the chromatography column until the baseline is stable, then load 10 CV of the dissolved supernatant, and then wash with a buffer with gradually decreasing denaturant concentration. The specific composition of the buffer wash with gradually decreasing denaturant concentration is: A: 8 mM sodium hydroxide + 1.8 M urea, pH 11.5, B: 48 mM Tris-HCl, 4.5 wt% glycerol, 0.08 mol NaCl, pH = 8.0, 100% A → 100% B linear gradient, wash for 1.9 hours, then use 18 mM Tris-HCl, 0.4 M NaCl, pH = 7.5 to elute, and collect the elution peak.

[0039] (4) Ultrafiltration: The collected eluate was replaced five times with an equal volume of buffer using a 30 KD membrane package. The replacement buffer was 18 mM Tris-HCl, 48 mM NaCl, pH = 7.5.

[0040] (5) DEAE chromatography (Capto DEAE): The Capto DEAE (H=10 cm, CV=20 ml) chromatography column was equilibrated with equilibration buffer 18 mM Tris-HCl + 48 mM NaCl (pH=7.5), and the ultrafiltered protein sample was loaded onto the column. Then, a linear gradient of 100%A→100%B was used, and the column was washed for 1 hour. The eluted protein peak was collected and quantified by the extinction coefficient method. A total of 38 mg of pure protein was harvested.

[0041] Example 3

[0042] (1) Collect E. coli cells by centrifugation at 10,000 g and 4°C. Take 10 g of cells, resuspend them in 20 mM Tris-HCl (pH=8.0), and then homogenize and break the cells. Collect the precipitate by centrifugation at 10,000 g and 4°C. Wash the inclusion bodies twice with 20 mM Tris-HCl (pH=8.0) containing 1.5 M urea, collect the precipitate, and obtain relatively pure inclusion bodies.

[0043] (2) Dissolve the inclusion bodies using 12 mM NaOH + 2.2 M urea, pH 12.5 at a ratio of inclusion body weight to denaturing solution volume (W (g): V (mL)) = 1:21. Dissolve the inclusion bodies at room temperature for 4 hours, then centrifuge at 10,000 g, 4°C to collect the supernatant.

[0044] (3) Q chromatography refolding (Q impres), use the equilibration solution 12 mM sodium hydroxide + 3 M guanidine hydrochloride + 2 M sodium chloride pH 12 to equilibrate the chromatography column until the baseline is stable, then load 10 CV of the dissolved supernatant, and then wash with a buffer with a gradually decreasing denaturant concentration. The specific composition of the buffer wash with a gradually decreasing denaturant concentration is: A: 12 mM sodium hydroxide + 2.2 M urea, pH 12.5, B: 52 mM Tris-HCl, 5.5 wt% glycerol, 0.12 mol NaCl, pH = 8.5, 100% A → 100% B linear gradient, wash for 2.1 hours, then use 22 mM Tris-HCl, 0.6 M NaCl, pH = 8.5 to elute, and collect the elution peak.

[0045] (4) Ultrafiltration: The collected eluate was replaced five times with an equal volume of buffer using a 30 KD membrane package. The replacement buffer was 22 mM Tris-HCl, 52 mM NaCl, pH = 8.5.

[0046] (5) DEAE chromatography (Capto DEAE): The Capto DEAE (H=10 cm, CV=20 ml) chromatography column was equilibrated with equilibration buffer 22 mM Tris-HCl + 52 mM NaCl (pH=8.5), and the ultrafiltered protein sample was loaded onto the column. Then, a linear gradient of 100%A→100%B was used, and the column was washed for 1.1 hours. The eluted protein peak was collected and quantified by the extinction coefficient method. A total of 39 mg of pure protein was obtained.

[0047] Comparative Example 1

[0048] (1) Collect the cells by centrifugation at 10,000 g and 4°C. Take 10 g of E. coli cells, resuspend them in 20 mM Tris-HCl (pH=8.0), and then homogenize and break the cells. Collect the precipitate by centrifugation at 10,000 g and 4°C. Wash the inclusion bodies twice with 20 mM Tris-HCl (pH=8.0) containing 1.5 M urea, collect the precipitate, and obtain relatively pure inclusion bodies.

[0049] (2) Dissolve the inclusion bodies in 100 mM Tris + 6 M guanidine hydrochloride, pH 8.0 at a ratio of inclusion body weight to denaturing solution volume (W:V) of 1:20 for more than 5 hours at room temperature. Centrifuge at 10,000 g at 4°C to collect the supernatant.

[0050] (3) Dilution and renaturation: ① Use the extinction coefficient method to quantify the protein in the centrifugal supernatant, and then use denatured protein diluent to dilute the protein concentration to 1 mg / mL; ② When the temperature of the renaturation solution (20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5wt% glycerol pH 8.0) drops to 6°C, start renaturation: slowly add the two barrels of diluted protein solution to the constantly stirred renaturation solution, and control the total renaturation time to about 30 h.

[0051] (4) Ultrafiltration: The renatured protein solution was ultrafiltered five times with an equal volume of replacement buffer using a 30 KD membrane package. The replacement buffer was 20 mM Tris-HCl, 50 mM NaCl, pH = 8.0.

[0052] (5) Anion exchange chromatography (QHP): The QHP (H=10 cm, CV=20 ml) chromatography column was equilibrated with equilibration buffer 20 mM Tris-HCl + 50 mM NaCl (pH=8.0), and the ultrafiltered protein sample was loaded onto the column. Then, a linear gradient of 100%A→100%B was used, and the column was washed for 1 hour, and the eluted protein peak was collected. The protein was quantified by the extinction coefficient method, and a total of 32 mg of protein was harvested.

[0053] Comparative Example 2

[0054] When dissolving the inclusion bodies, 8.0 mol / L urea solution, pH 8.0, was used to dissolve the inclusion bodies, and the remaining steps were the same as those in Comparative Example 1.

[0055] Test Example 1

[0056] The purified protein obtained in Example 1 was subjected to SEC-HPLC, and the steps were as follows:

[0057] 1. Experimental Preparation

[0058] Instruments and Materials

[0059] HPLC system: includes pump, injector, column oven, detector (ultraviolet detector UV) and data acquisition system.

[0060] SEC chromatography column: agarose gel (Sepharose).

[0061] Mobile phase: Tris-HCl buffer, pH 8.0.

[0062] Sample: purified protein obtained in Example 1.

[0063] 2. Column Preparation

[0064] Choice of elution buffer

[0065] Buffer: 20 mM Tris-HCl, pH 7.5.

[0066] Ionic strength: 0.15 M NaCl.

[0067] Filtration: Filter the mobile phase through a 0.22 μm filter to remove particulate matter.

[0068] Column equilibration

[0069] Equilibration: Connect the column to the HPLC system and flush the column with mobile phase at 0.5 mL / min until the baseline is stable.

[0070] 3. Sample Preparation

[0071] Filtration or centrifugation

[0072] Filtration: Filter samples through a 0.22 μm filter to remove particulate matter.

[0073] Centrifuge: 10,000 rpm, 10 min, collect the supernatant.

[0074] Concentration determination

[0075] Protein concentration: Use Bradford method, BCA method or UV absorption method to determine the protein concentration of the sample to ensure that the injection concentration is appropriate.

[0076] 4. Sampling and separation

[0077] Injection

[0078] Injection volume: 10 μL.

[0079] Injection: Inject the sample into the injector and start the HPLC system for separation.

[0080] Separation conditions

[0081] Flow rate: 0.5 mL / min.

[0082] Column temperature: room temperature.

[0083] Detector: Use UV detector (280 nm wavelength) to monitor protein elution.

[0084] The results are as follows Figure 3 As shown, according to Figure 3 The results showed that the peak area reached a maximum of 98.70% at 14.6 minutes, which means that the sample purity was 98.70%.

[0085] Test Example 2

[0086] SDS-PAGE electrophoresis

[0087] Preparation of gel

[0088] Gel preparation: Prepare separation gel (12%) and concentrated gel (4%). Gel filling: Pour the separation gel into the gel plate, insert the comb, and wait for the separation gel to solidify before filling the concentrated gel. Sample loading and electrophoresis

[0089] Sample treatment: Mix the purified protein prepared in Example 1 with the loading buffer (DTT) and heat at 95°C for 5 minutes to denature the protein. Loading: Load the treated purified protein and pre-stained protein marker into the loading wells. Electrophoresis: Connect the power supply, set the voltage to 120 V, and run the electrophoresis until the bromophenol blue reaches the bottom of the gel. 3. Transfer to membrane

[0090] Prepare the transfer apparatus

[0091] Select membrane: NC membrane. Assemble the transfer cassette: Place the sponge pad, filter paper, gel, membrane, filter paper and sponge pad in order, making sure there are no bubbles between the layers.

[0092] Transfer: Connect the power supply, set the voltage (100 V), and transfer time to 1 hour. Blocking and antibody incubation

[0093] Closed

[0094] Blocking: Place the transferred membrane in blocking solution (5% skim milk powder) and block for 1 hour on a shaker at room temperature.

[0095] Primary antibody incubation: Incubate the blocked membrane with diluted primary antibody (1:1000) overnight at 4°C. Washing: Wash the membrane 3 times with TBST for 5 minutes each. Secondary antibody incubation: Incubate the membrane with diluted secondary antibody (1:5000) at room temperature for 1 hour. Washing: Wash the membrane 3 times with TBST for 5 minutes each. 5. Color development and imaging

[0096] Color

[0097] Color development: Place the membrane in ECL luminescent solution and incubate for 1-2 minutes. Imaging: Use a chemiluminescent imager for imaging. The results are as follows Figure 4 As shown, according to Figure 4 The results showed that the molecular weight of the purified protein prepared in Example 1 was about 70 kDa, which was consistent with that of the purified protein of CRM197.

[0098] Test Example 3

[0099] The denaturation time, renaturation time, yield and purity used in the renaturation process of Examples 1-3 of the present invention and Comparative Examples 1-2 were recorded, and the results are shown in Table 1.

[0100] Table 1 Comparison of various results

[0101]

[0102] As shown in the results in Table 1, the purified protein obtained by the denaturation and renaturation method provided by the present invention not only has obvious advantages in yield and purity, but also greatly shortens the time used for denaturation and renaturation.

[0103] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "bottom", "top", "front", "back", "inside", "outside", "left", "right", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0104] Although the present invention is described herein with reference to specific embodiments, it should be understood that these embodiments are merely examples of the principles and applications of the present invention. It should therefore be understood that many modifications may be made to the exemplary embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims. It should be understood that the various dependent claims and features described herein may be combined in a manner different from that described in the original claims. It should also be understood that features described in conjunction with individual embodiments may be used in other described embodiments.

Claims

1. A method for renaturation of CRM197 inclusion body protein, characterized in that: The steps include: S1. Dissolve the CRM197 inclusion body protein with a mixture of alkali solution and denaturant, centrifuge, and collect the supernatant; S2, performing primary chromatography elution on the supernatant to obtain eluted protein; S3, ultrafiltration of the eluted protein followed by secondary chromatography elution to obtain pure CRM197 protein; In S1, the alkali solution is a sodium hydroxide solution, the denaturant is urea, in the mixed denaturing solution, the concentration of the sodium hydroxide solution is 8-12 mM, the concentration of the urea is 1.8-2.2 M, the pH of the mixed denaturing solution is 11.5-12.5, and the volume-mass ratio of the mixed denaturing solution to the CRM197 inclusion body protein is 19-21 mL / g; The CRM197 inclusion body protein is derived from Escherichia coli; In S2, the chromatography column used for the initial chromatography elution is a Q anion exchange chromatography column.

2. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In S2, the primary chromatography elution includes gradient buffer elution and salt elution, the liquid A used for the gradient buffer elution is a mixture of 8-12 mM sodium hydroxide solution and 1.8-2.2 M urea, the liquid B is a mixture of 4.5-5.5 wt% glycerol, 48-52 mM Tris-HCl and 0.08-0.12 mol NaCl, and the elution gradient of the gradient buffer elution is 100% A-100% B, 1.9-2.1 hours; The eluent used for the salt elution is a mixture of 18-22 mM Tris-HCl and 495-505 mM NaCl; the pH of the A solution is 11.5-12.5, and the pH of the B solution and the eluent for the salt elution are both 7.5-8.

5.

3. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In S3, the ultrafiltration comprises the following steps: using a 30 KD membrane package to replace the eluted protein with an equal volume of buffer 5 times, the replacement buffer is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, and the pH of the replacement buffer is 7.5-8.

5.

4. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In S3, the chromatography column used for the secondary chromatography elution is a DEAE anion exchange chromatography column.

5. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In S3, the secondary chromatography elution includes salt elution, the solution A used for the salt elution is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, the solution B used for the salt elution is a mixture of 18-22 mM Tris-HCl and 490-510 mM NaCl, the elution gradient of the salt elution is 100% A-100% B, 0.9-1.1 hours; the pH of the solution A and the solution B used for the salt elution are 7.5-8.5, respectively.

6. The method for renaturation of CRM197 inclusion body protein according to claim 5, characterized in that: In the secondary chromatography elution, a pretreatment is also included before the salt elution, and the pretreatment includes the following steps: balancing the chromatography column with an equilibrium buffer, the equilibrium buffer is a mixture of 18-22 mM Tris-HCl and 48-52 mM NaCl, and the pH of the equilibrium buffer is 7.5-8.

5.

7. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In S1, the centrifugal force of the centrifuge is 10 4 g, and the centrifugation temperature was 4°C.

8. The method for renaturation of CRM197 inclusion body protein according to claim 1, characterized in that: In the S1, the dissolution time is more than 2 h.

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