Method for purifying human C-reactive protein
The purification method combining strong and weak anion columns, along with optimized pH and conductivity conditions, simplifies the preparation process of C-reactive protein, solving the problems of low purity and yield in existing technologies, and achieving the preparation of high-purity and high-yield C-reactive protein.
Patent Information
- Application Number
- CN202511122351.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-12-19
AI Technical Summary
In existing technologies, the preparation methods for C-reactive protein are cumbersome, result in increased impurities, low purity and yield, high production costs, and limited sources of human blood and pleural effusion, making stable production difficult.
The sample was diluted using a combination of strong and weak anion exchange columns. The strong anion exchange column was used for coarse purification, and the diluted weak anion exchange column was used for fine purification. This method optimized pH and conductivity conditions, simplified the process, and improved purity and yield.
This achieved a C-reactive protein purity of over 98%, simplified the production process, reduced production costs, and increased yield.
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Figure CN121159664A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for purifying human C-reactive protein. Background Technology
[0002] C-reactive protein (CRP) is an acute-phase reactant protein, named for its initial discovery of binding to the C-polysaccharide of pneumococcus. CRP is an acute-phase reactant produced and released by the liver during acute infection and inflammation, and can serve as a marker of acute inflammation.
[0003] In 1930, Tillett and Fransic of the Avery Laboratory at the Rockefeller Institute in the United States discovered that the serum of patients with acute infections could precipitate C-polysaccharide on the cell wall of Streptococcus pneumoniae. In 1941, Avery et al. confirmed that the protein reacting with C-polysaccharide was a protein that appears during acute infection, and this protein was named C-reactive protein (CRP). CRP was the earliest known acute-phase protein. In 1950, CRP was purified and crystallized. Using electron microscopy and X-ray diffraction techniques to explore the structure of the CRP protein, it was discovered that CRP is a cyclic pentamer protein.
[0004] The C-reactive protein (CRP) gene is located on chromosome 1 q23 and is highly conserved in sequence. It belongs to the penetrin family and has a relative molecular mass of 115-140 kDa. It consists of five identical monomers, each spherical (composed of 206 amino acids), which form a symmetrical pentagonal ring structure (a very rare conformation in proteins) through non-covalent bonding. CRP has a sedimentation coefficient of 6.5-7.5, an isoelectric point of 5.5, is poorly soluble in water, crystals in rhomboid form, and can be precipitated by ammonium sulfate. It is heat-sensitive, being destroyed at 60°C for 30 minutes.
[0005] Under normal conditions, CRP molecules exist in the form of pentamers. They can also decompose into monomers in acidic or alkaline environments, thereby triggering certain immune responses. However, since CRP monomers are present in cell membranes rather than in serum, they are difficult to detect.
[0006] The CRP sequence is highly conserved, and the amino acid sequences of CRPs from other animals show a striking similarity to those of humans. Therefore, CRP is considered one of the most conserved proteins and is of great importance to the survival of species.
[0007] CRP possesses diverse biological functions, participating in various physiological and pathophysiological processes. CRP exhibits a high affinity for phosphatidylcholine residues and can bind to a variety of intrinsic ligands (such as plasma cell lipoproteins, cell membranes of damaged cells, small ribosome protein particles, and opsoninocytes) or exogenous ligands (such as polysaccharides, phospholipids, and components of microorganisms such as bacteria, fungi, and parasites). After binding to these ligands, CRP is recognized by CIq and can activate the classical complement activation pathway.
[0008] In recent years, it has also been found that CRP plays a role in platelet-mediated cell-viruses. On the one hand, it may activate platelets through oxidized CRP. On the other hand, CRP exerts its regulatory role in the inflammatory response by binding to platelet activating factor (PAF). PAF is known to stimulate platelet aggregation and release arachidonic acid from the platelet membrane, and inhibit PAF-induced neutrophil binding.
[0009] CRP is synthesized under the stimulation of cytokines such as IL-6, IL-2, and TNF during inflammation, infection, and tissue damage. The liver is its main site of synthesis, and small amounts are also produced locally in other tissues, such as nerve cells, monocytes, lymphocytes, and atherosclerotic plaques. CRP has a stable half-life in the blood, approximately 19 hours, and its concentration mainly depends on the amount produced by the liver. CRP cannot cross the placenta and is widely distributed in the body; it can be detected not only in blood but also in pleural effusion, ascites, pericardial fluid, and synovial fluid.
[0010] The concentration of CRP in normal human serum is very low (0.07–8.0 mg / L, with a significantly positively skewed distribution in healthy individuals). After infection, CRP concentration rises significantly, starting to increase after 6–8 hours and peaking at 24–48 hours, reaching 1000–2000 times the normal value. The increase is directly proportional to the severity of the infection. After inflammation heals, the concentration rapidly decreases, returning to normal levels within a week. CRP does not increase during viral infections, and its changes are unaffected by individual patient differences, physical condition, or medications used in treatment.
[0011] Natural sources of CRP are limited, therefore CRP is mainly prepared through the expression of exogenous recombinant proteins. Existing CRP preparation methods mostly employ two approaches. One is to fuse CRP with an affinity-purifiable tag protein to form a recombinant protein. After cell expression, the fusion protein is purified, and then the target protein and tag protein are separated by enzymatic digestion, followed by further capture and purification of the target protein. However, this method involves many steps, is cumbersome, and introduces enzymes, leading to increased impurities. The second approach involves purifying CRP from human blood or pleural effusion. However, human blood and pleural effusion have complex compositions, making it difficult to obtain high-purity CRP. Furthermore, the sources of human blood and pleural effusion are limited, hindering stable production. Summary of the Invention
[0012] The purpose of this invention is to provide a method for purifying human C-reactive protein, which reduces the burden of host protein and host DNA residues and two-step purification, while improving the yield and purity of the target protein, simplifying the production process of C-reactive protein, increasing the yield of C-reactive protein and reducing production costs.
[0013] This invention provides the following technical solution:
[0014] A method for purifying human C-reactive protein is as follows:
[0015] S1. Obtain a sample containing C-reactive protein: CHO cell fermentation broth or supernatant obtained by centrifuging CHO cell fermentation broth;
[0016] S2. Dilute the sample: Dilute the sample with a diluent of 20-100 mM Tris, 0-50 mM NaCl, and pH 8.0 to reduce the sample conductivity to 0-4.5 ms / cm and balance the sample pH to 7.8-8.2 to obtain the protein sample.
[0017] S3, Crude Purification: Protein samples obtained through a strong anion exchange column are crudely purified to obtain crude purified samples;
[0018] S4. Dilute the crude pure sample: Dilute the crude pure sample with a diluent of 20-100 mM Tris, 0-50 mM NaCl, 1-5 mM EDTA, and pH 8.0 to reduce the conductivity of the crude pure sample to 0-4.5 mS / cm and balance the pH of the crude pure sample to 7.8-8.2.
[0019] S5. Purification: The target product can be obtained by purifying the diluted crude sample using a weak anion exchange column.
[0020] Preferably, the method for obtaining samples containing C-reactive protein is as follows:
[0021] M1. Construction of CHO cells expressing human C-reactive protein: The nucleotide sequence encoding human C-reactive protein was cloned into plasmid pcDNA3.4; the recombinant plasmid pcDNA3.4-CRP was stably transfected into CHO cells;
[0022] M2, expressing human C-reactive protein: CHO in M1 was cultured at 37℃, 5% CO2, and 110 rpm with shaking. Glucose was added starting from the third day of culture, and then added every other day until the final concentration reached 6 g / L. Feeding was added starting from the sixth day of culture, and then added every two days, with each feeding volume being 5% of the total fermentation volume. Samples were collected after 2 to 4 feedings to obtain the fermentation broth.
[0023] M3. Centrifugation: Centrifuge the fermentation broth at 10000xg for 10min, filter and collect the supernatant.
[0024] Preferably, the CHO cells expressing human C-reactive protein include a foreign plasmid comprising a nucleotide sequence encoding an amino acid sequence of human C-reactive protein.
[0025] The preferred crude purity from the strong anion column is as follows:
[0026] The purification linear flow rate is 120–200 cm / h;
[0027] The purified equilibration solution was a solution of 20 mM Tris, 2 mM CaCl2, and pH 8.0.
[0028] After equilibrating 10 column volumes, the protein sample was loaded and then washed with a washing buffer of 20 mM Tris, 2 mM CaCl2, 120 mM NaCl, pH 8.0 for 10 column volumes.
[0029] The crude protein was obtained by elution with 20 mM Tris, 2 mM CaCl2, 250 mM NaCl, and pH 8.0.
[0030] Preferably, the purification chromatography packing material for the strong anion exchange column is POROS XQ, GE-QHP, GE-QFF, 50Q-HP, or HC60-Q.
[0031] Preferably, the purification process using a weak anion exchange column is as follows: the linear flow rate for purification is 120-200 cm / h.
[0032] The purified equilibration buffer was a solution of 20 mM Tris, 2 mM EDTA, and pH 8.0, and equilibration was carried out for 10 column volumes.
[0033] Then, the crude pure sample diluted in S4 was loaded. After loading, the sample was washed for 10 column volumes with a washing buffer of 20 mM Tris, 200 mM NaCl, 2 mM EDTA, and pH 8.0.
[0034] The protein was eluted with 20 mM Tris, 500 mM NaCl, 2 mM EDTA at pH 8.0 to obtain the purified target protein.
[0035] Preferably, the purification chromatography packing material for the weak anion exchange column is DEAE FF or DEAE HP.
[0036] The beneficial effects of this invention are:
[0037] 1. This application further improves the yield of the target protein and increases the purity of the target protein to over 98%, thereby improving both the yield and purity of the target protein and obtaining a C-reactive protein with high purity and good activity.
[0038] 2. By optimizing column loading conditions such as pH and conductivity, this application can improve the purity of the target protein to over 98% with only two steps of ion chromatography, simplifying the process and improving the purity of the target protein. Furthermore, diluted protein can be directly loaded onto the column without changing the medium, reducing the number of chromatography steps in existing methods, eliminating the operation of changing the medium during purification, and further improving the yield and production.
[0039] 3. Each liter of fermentation broth in this application can produce 600 mg of human C-reactive protein; using it to prepare C-reactive protein simplifies the production process of human C-reactive protein, improves the purity and yield of human C-reactive protein, and greatly reduces the purification cost of human C-reactive protein by using antibody-antigen affinity chromatography or phosphocholine affinity chromatography. Attached Figure Description
[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:
[0041] Figure 1 This is an SDS-PAGE image of human C-reactive protein;
[0042] Figure 2 The results of SEC-HPLC detection of purified human C-reactive protein. Detailed Implementation
[0043] The present application relates to the construction of CHO cells expressing human C-reactive protein, which includes a foreign plasmid comprising a nucleotide sequence encoding an amino acid sequence of human C-reactive protein.
[0044] The amino acid sequence of human C-reactive protein is as follows:
[0045] MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHI CTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP(SEQ ID NO.1).
[0046] The nucleotides encoding the amino acid sequence of human C-reactive protein, synthesized by Genscript Biotech Inc., are as follows:
[0047] (SEQ ID NO.2).
[0048] Example 1
[0049] Protein sample acquisition: Construction of CHO cells expressing human C-reactive protein: The nucleotide sequence encoding human C-reactive protein was cloned into plasmid pcDNA3.4; the recombinant plasmid pcDNA3.4-CRP was stably transfected into CHO cells; Expression of human C-reactive protein: CHO cells from M1 were cultured at 37°C, 5% CO2, and with shaking at 110 rpm. Glucose was added starting from the third day of culture, and then every other day thereafter, until the final concentration reached 6 g / L. The feeding method was as follows: CHO Feed06Supplement: Starting from day 6 of culture, feed was added every two days thereafter, with each addition being 5% of the total fermentation volume, to provide the nutrients required for cell growth and expression of human C-reactive protein. When cell viability dropped below 85%, the fermentation broth was centrifuged (10000×g, 10 min), diluted with diluent (20 mM Tris), resulting in a conductivity of 0.1 mS / cm and a pH of 7.8. The diluted solution was then centrifuged (10000×g, 10 min) and filtered (using a 0.45 μm filter membrane) to obtain the protein sample.
[0050] Purification is as follows:
[0051] Crude purification using a strong anion exchange column: The purification chromatography packing material was GE-QHP, the purification linear flow rate was 150 cm / h, and the purification equilibration buffer was 20 mM Tris, 2 mM CaCl2, pH 8.0. After equilibration for 10 column volumes, the protein sample was loaded, centrifuged, and filtered to obtain 70 ml of protein sample per ml of packing material. The washing buffer was 20 mM Tris, 2 mM CaCl2, 120 mM NaCl, pH 8.0, and the sample was washed for 10 column volumes. Elution was then performed with a solution of 20 mM Tris, 2 mM CaCl2, 250 mM NaCl, pH 8.0 to obtain approximately 18 mg of crude purified target protein.
[0052] Dilute the crude pure sample: Dilute the obtained crude pure target protein with dilution buffer (20 mM Tris, 1 mM EDTA, pH 8.0). After dilution, the conductivity is 0.1 ms / cm and the pH is 7.8. Centrifuge (10000×g, 10 min) and filter (0.45 μm filter membrane).
[0053] Weak anion exchange column purification: DEAE HP was used as the purification chromatography packing material, the purification linear flow rate was 150 cm / h, and the purification equilibration buffer was 20 mM Tris, 2 mM EDTA, pH 8.0. After equilibration for 10 column volumes, the protein sample (approximately 20 ml of fermentation broth) was loaded onto the buffer. The sample was washed with 20 mM Tris, 2 mM EDTA, 200 mM NaCl, pH 8.0 for 10 column volumes. The protein sample was then eluted with 20 mM Tris, 2 mM EDTA, 500 mM NaCl, pH 8.0, yielding approximately 10.8 mg of purified target protein.
[0054] The purity of the target protein was determined by SDS-PAGE and SEC-HPLC. The SDS-PAGE result showed a single band with no obvious impurities, and the band position was approximately 23-25 kDa. The SEC-HPLC showed two peaks with retention times of 7.629 and 8.962, respectively, and the main peak area percentage was 98.565%.
[0055] Example 2:
[0056] Protein sample acquisition: Construction of CHO cells expressing human C-reactive protein: The nucleotide sequence encoding human C-reactive protein was cloned into plasmid pcDNA3.4; the recombinant plasmid pcDNA3.4-CRP was stably transfected into CHO cells; Expression of human C-reactive protein: CHO cells from M1 were cultured at 37°C with shaking in 5% CO2. Glucose was added starting from the third day of culture, and then every other day thereafter, until the final concentration reached 6 g / L. The feeding method was as follows: CHO Feed06Supplement: Starting from day 6 of culture, feed was added every two days thereafter, with each addition being 5% of the total fermentation volume, to provide the nutrients required for cell growth and expression of human C-reactive protein. When cell viability dropped below 85%, the fermentation broth was centrifuged (10000×g, 10 min), diluted with diluent (20 mM Tris, pH 8.0), resulting in a conductivity of 4.25 ms / cm and a pH of 7.9. The diluted solution was then centrifuged (10000×g, 10 min) and filtered (using a 0.45 μm filter membrane) to obtain the protein sample.
[0057] Purification is as follows:
[0058] Crude purification using a strong anion exchange column: The purification chromatography packing material was POROS XQ, the purification linear flow rate was 120 cm / h, and the purification equilibration buffer was 20 mM Tris, pH 8.0. After equilibration for 10 column volumes, the protein sample was loaded, centrifuged, and filtered to obtain 70 ml of protein sample per ml of packing material. The washing buffer was 20 mM Tris, 120 mM NaCl, pH 8.0, for 10 column volumes. The protein sample was then eluted with 20 mM Tris, 250 mM NaCl, pH 8.0, yielding approximately 20 mg of crude purified target protein.
[0059] Dilute the crude pure sample: Dilute the obtained crude pure target protein with diluent (20 mM Tris, 2 mM EDTA, pH 8.0). After dilution, the conductivity is 3.36 ms / cm and the pH is 7.9. Centrifuge (10000×g, 10 min) and filter (0.45 μm filter membrane).
[0060] Weak anion exchange column purification: DEAE HP was used as the purification chromatography packing material, the purification linear flow rate was 120 cm / h, and the purification equilibration buffer was 20 mM Tris, 2 mM EDTA, pH 8.0. After equilibration for 10 column volumes, the protein sample (approximately 20 ml of fermentation broth) was loaded onto the buffer. The sample was washed with 20 mM Tris, 2 mM EDTA, 200 mM NaCl, pH 8.0 for 10 column volumes. The protein sample was then eluted with 20 mM Tris, 2 mM EDTA, 500 mM NaCl, pH 8.0, yielding approximately 12 mg of purified target protein.
[0061] The purity of the purified target protein was determined by SDS-PAGE and SEC-HPLC, and the results are as follows: Figure 1 and Figure 2 As shown, the purity of human C-reactive protein is 98.15%, which is greater than 98%.
[0062] Example 3:
[0063] Protein sample acquisition: Construction of CHO cells expressing human C-reactive protein: The nucleotide sequence encoding human C-reactive protein was cloned into plasmid pcDNA3.4; the recombinant plasmid pcDNA3.4-CRP was stably transfected into CHO cells; Expression of human C-reactive protein: CHO cells from M1 were cultured at 37°C with shaking in 5% CO2. Glucose was added starting from the third day of culture, and then every other day thereafter, until the final concentration reached 6 g / L. The feeding method was as follows: CHO Feed06 Supplement: Starting from day 6 of culture, feed was added every two days thereafter, with each addition being 5% of the total fermentation volume to provide the nutrients required for cell growth and expression of human C-reactive protein. When cell viability dropped below 85%, the fermentation broth was centrifuged (10000×g, 10 min), diluted with diluent (100 mM Tris, 50 mM NaCl), resulting in a conductivity of 4.5 mS / cm and a pH of 8.2. The diluted solution was then centrifuged (10000×g, 10 min) and filtered (using a 0.45 μm filter membrane) to obtain the protein sample.
[0064] Purification is as follows:
[0065] Crude purification using a strong anion exchange column: The purification chromatography packing material was HC60-Q, the linear flow rate was 200 cm / h, and the equilibration buffer was 20 mM Tris, 2 mM CaCl2, pH 8.0. After equilibration for 10 column volumes, the protein sample was loaded, centrifuged, and filtered to obtain 70 ml of protein sample per ml of packing material. The washing buffer consisted of 20 mM Tris, 2 mM CaCl2, 120 mM NaCl, pH 8.0, for 10 column volumes. Elution was then performed with a solution of 20 mM Tris, 2 mM CaCl2, 250 mM NaCl, pH 8.0, yielding approximately 14 mg of crude purified target protein.
[0066] Dilute the crude pure sample: Dilute the obtained crude pure target protein with diluent (100 mM Tris, 50 mM NaCl, 5 mM EDTA, pH 8.0). After dilution, the conductivity is 4.5 mS / cm and the pH is 8.2. Centrifuge (10000×g, 10 min) and filter (0.45 μm filter membrane).
[0067] Weak anion exchange column purification: DEAE HP was used as the purification chromatography packing material, the purification linear flow rate was 200 cm / h, and the purification equilibration buffer was 20 mM Tris, 2 mM EDTA, pH 8.0. After equilibration for 10 column volumes, the protein sample (approximately 20 ml of fermentation broth) was loaded. The sample was washed with 20 mM Tris, 2 mM EDTA, 200 mM NaCl, pH 8.0 for 10 column volumes, and then eluted with 20 mM Tris, 2 mM EDTA, 500 mM NaCl, pH 8.0, yielding approximately 8.5 mg of purified target protein.
[0068] The purity of the target protein was determined by SDS-PAGE and SEC-HPLC. SDS-PAGE showed a single band with no obvious impurities, located approximately at 23-25 kDa. SEC-HPLC showed two peaks with retention times of 7.6232 and 8.965 kDa, respectively, and the main peak area was 98.781%.
[0069] Example 4:
[0070] This example demonstrates the effect of different strong anion exchange chromatography packing materials on the purification of human C-reactive protein. Example 2 maintains the same conditions except for the use of different strong anion exchange chromatography packing materials: POROS XQ, GE-QHP, GE-QFF, 50Q-HP, or HC60-Q. 20 ml of fermentation broth was loaded onto 1 ml of each of the five strong anion exchange chromatography packing materials, and eluted under the same conditions to obtain crude purified protein. The protein content is shown in Table 2. POROS XQ was significantly superior to the other packing materials. The results are shown in the following table:
[0071] Packing name POROSXQ GE-QHP GE-Q FF 50Q-HP HC60-Q Crude protein yield / mg 20 18 12 17 14
[0072] This application addresses the problems of low yield, high production cost, and low production volume in existing methods for preparing C-reactive protein (CRP) via affinity chromatography. The proposed purification method involves obtaining the CRP fermentation broth, followed by dilution, strong anion exchange purification, and then further dilution and weak anion exchange purification to obtain the target protein (CRP). In this method, the initial strong anion exchange column purification significantly improves the purity and concentration of the target protein, greatly reducing host protein and host DNA residues and reducing the burden of the second purification step. After dilution, only a single weak anion exchange column purification step is needed to increase the purity of the target protein to over 98%, simultaneously improving the yield and purity of the target protein. This simplifies the CRP production process and increases the CRP yield. Using this purification method, 600 mg of CRP can be purified from 1 L of fermentation broth, and the chromatography medium can be reused multiple times, significantly reducing production costs.
[0073] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for purifying human C-reactive protein, as follows: S1. Obtain a sample containing C-reactive protein: CHO cell fermentation broth or supernatant obtained by centrifuging CHO cell fermentation broth; S2. Dilute the sample: Dilute the sample with a diluent of 20-100 mM Tris, 0-50 mM NaCl, and pH 8.0 to reduce the sample conductivity to 0-4.5 ms / cm and balance the sample pH to 7.8-8.2 to obtain the protein sample. S3, Crude Purification: Protein samples obtained through a strong anion exchange column are crudely purified to obtain crude purified samples; S4. Dilute the crude pure sample: Dilute the crude pure sample with a diluent of 20-100 mM Tris, 0-50 mM NaCl, 1-5 mM EDTA, and pH 8.0 to reduce the conductivity of the crude pure sample to 0-4.5 mS / cm and balance the pH of the crude pure sample to 7.8-8.
2. S5. Purification: The target product can be obtained by purifying the diluted crude sample using a weak anion exchange column.
2. The method for purifying human C-reactive protein according to claim 1, characterized in that: The method for obtaining samples containing C-reactive protein is as follows: M1. Construction of CHO cells expressing human C-reactive protein: The nucleotide sequence encoding human C-reactive protein was cloned into plasmid pcDNA3.4; the recombinant plasmid pcDNA3.4-CRP was stably transfected into CHO cells; M2, expressing human C-reactive protein: CHO in M1 was cultured at 37℃, 5% CO2, and 110 rpm with shaking. Glucose was added starting from the third day of culture, and then added every other day until the final concentration reached 6 g / L. Feed was added starting from the sixth day of culture, and then added every two days, with each feed volume being 5% of the total fermentation volume. When the cell viability dropped below 85%, the fermentation broth was collected. M3. Centrifugation: Centrifuge the fermentation broth at 10000xg for 10min, filter and collect the supernatant.
3. The method for purifying human C-reactive protein according to claim 2, characterized in that: CHO cells expressing human C-reactive protein include a foreign plasmid containing a nucleotide sequence encoding the amino acid sequence of human C-reactive protein.
4. The method for purifying human C-reactive protein according to claim 1, specifically the crude purification using a strong anion exchange column, is as follows: The purification linear flow rate is 120–200 cm / h; The purified equilibration solution was a solution of 20 mM Tris, 2 mM CaCl2, and pH 8.
0. After equilibrating 10 column volumes, the protein sample was loaded and then washed with a washing buffer of 20 mM Tris, 2 mM CaCl2, 120 mM NaCl, pH 8.0 for 10 column volumes. The crude protein was obtained by elution with 20 mM Tris, 2 mM CaCl2, 250 mM NaCl, and pH 8.
0.
5. The method for purifying human C-reactive protein according to claim 4, characterized in that: The purification chromatography packing material for strong anion exchange columns is POROS XQ, GE-QHP, GE-QFF, 50Q-HP, or HC60-Q.
6. The method for purifying human C-reactive protein according to claim 1, characterized in that: The specific purification process for the weak anion exchange column is as follows: the linear flow rate for purification is 120-200 cm / h. The purified equilibration buffer was a solution of 20 mM Tris, 2 mM EDTA, and pH 8.0, and equilibration was carried out for 10 column volumes. Then, the diluted crude pure sample in S4 was loaded. After loading, the sample was washed for 10 column volumes with a washing buffer of 20 mM Tris, 200 mM NaCl, 2 mM EDTA, and pH 8.
0. The protein was eluted with 20 mM Tris, 500 mM NaCl, 2 mM EDTA at pH 8.0 to obtain the purified target protein.
7. The method for purifying human C-reactive protein according to claim 6, characterized in that: The purification chromatography packing material for the weak anion exchange column is DEAE FF or DEAE HP.
8. A method for purifying human C-reactive protein according to any one of claims 1-7, characterized in that: It is used in the preparation of C-reactive protein.