A method for purifying heparin synthetase PmHS2
By employing a two-step purification process and ion exchange chromatography with a specific buffer solution, the problems of the complexity and high cost of purifying heparin synthase PmHS have been solved, enabling the industrial production of high-purity and high-yield heparin synthase PmHS.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MINGCHENG HUIZHONG (JIANGSU) PHARM RES CO LTD
- Filing Date
- 2024-12-31
- Publication Date
- 2026-06-30
AI Technical Summary
Existing purification methods for heparin synthase PmHS are complex to operate, have low purity, long purification cycles, and high costs, making it difficult to meet the needs of industrial production.
A two-step purification process, including ultrafiltration and ion exchange chromatography, is employed. Specific buffer solutions A, B, and C are used for purification. Nickel and cation exchange columns are combined, and pH and salt concentration are optimized to improve purity and yield.
It achieves the purification of high-purity (≥95%) heparin synthase, simplifies the operation process, reduces costs, and is suitable for industrial-scale production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme separation and purification technology, specifically relating to a purification method for heparin synthase PmHS2. Background Technology
[0002] Heparin is a naturally occurring, highly sulfated polysaccharide, primarily composed of alternating glucuronic acid and iduronic acid, belonging to the category of complex carbohydrate polymers. It is mainly found in animal tissues such as the liver, lungs, and blood vessel walls, and possesses potent anticoagulant activity, making it one of the most widely used anticoagulant drugs in clinical practice. Heparin synthase (PmHS) is an enzyme with specialized catalytic functions, catalyzing the elongation and sulfation of the heparin polysaccharide chain during specific biosynthetic processes, thus participating in the biosynthesis of heparin molecules. PmHS enzymes were initially isolated from Pasteurella multocida, hence their name. PmHS plays a crucial role in heparin synthesis. It not only catalyzes the elongation of the heparin polysaccharide chain but also highly sulfates heparin by introducing sulfate groups. These sulfate groups significantly enhance the anticoagulant activity of heparin, making it a highly effective anticoagulant drug. With the continuous deepening of research on heparin synthase (PmHS), its application prospects in medicine, biotechnology, and other fields are becoming increasingly broad. On the one hand, PmHS enzymes can serve as key enzymes in the heparin synthesis pathway, enabling the construction of efficient and controllable heparin production systems. On the other hand, by modifying and optimizing PmHS enzymes through genetic engineering and other means, novel anticoagulant drugs with higher anticoagulant activity and lower side effects can be developed. Furthermore, research on PmHS enzymes contributes to a deeper understanding of the interaction mechanisms between heparin and other biomolecules, providing a practical basis and experimental foundation for the clinical application of heparin-based drugs.
[0003] With the development of biotechnology, due to the low expression level of natural heparin synthase PmHS and its certain cytotoxicity, the key to restricting the large-scale production of heparin synthase PmHS has gradually shifted from the early problem of the production capacity of crude heparin synthase PmHS fermentation liquid to how to efficiently purify heparin synthase PmHS.
[0004] Currently, three types of heparinase have been isolated and purified from Flavobacterium heparinum: heparinase I, heparinase II (PmHS2), and heparinase III. These three enzymes have different enzymatic properties. The current purification methods for PmHS2 include the dissolution-precipitation method and the dialysis method. Although these methods are simple to operate, they have drawbacks such as solvent residue, product loss, low purity, and long purification cycles, which prevent them from being well applied to mass production.
[0005] In summary, it is of great importance to research and develop a simple, low-cost, and high-recovery purification method for heparin synthase PmHS and apply it to the industrial separation and purification of heparin synthase PmHS. This is a key technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention utilizes optimized buffer composition to develop a simple and highly pure purification method for heparin synthase PmHS.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0008] A method for purifying heparin synthase PmHS2 includes the following steps:
[0009] S1. Add buffer A to the fermentation broth of heparin synthase PmHS to be purified, mix well, and then perform ultrafiltration once to remove metal ions and impurities with a molecular weight of less than 10kDa.
[0010] S2. Pump the ultrafiltrate obtained in step S1 into a nickel column that has been equilibrated with buffer A, then reequilibrate with buffer A, and then elute with buffer B, and collect the eluent.
[0011] S3. Pump the eluent obtained in step S2 into the cation exchange column that has been equilibrated with buffer A, then reequilibrate with buffer A, and then elute with buffer C, and collect the eluent.
[0012] S4. Add buffer A to the eluent obtained in step S3, and perform ultrafiltration concentration to obtain high-purity heparin synthase PmHS2; wherein,
[0013] Buffer A: 15-25 mM Tris-HCl, pH = 7.5-8.5;
[0014] Buffer B: 15-25mM Tris-HCl, 1±0.5M imidazole, pH=7.5-8.5;
[0015] Buffer C: 15-25mM Tris-HCl, 1±0.2M NaCl, pH=7.5~8.5.
[0016] In a further preferred embodiment of the present invention, the heparin synthase PmHS fermentation broth to be purified has been centrifuged and filtered to remove cell debris and other impurities before purification, and stored at low temperature.
[0017] A further preferred embodiment of the present invention is that the storage temperature is 2 to 10°C, preferably 4 ± 0.5°C.
[0018] In a further preferred embodiment of the present invention, in step S1, the total volume of the added buffer solution A is 0.8 to 2 times that of the heparin synthase PmHS fermentation broth.
[0019] In a further preferred embodiment of the present invention, in step S2, the flow rate of the buffer solution B is controlled to be 10-20 mL / min.
[0020] In a further preferred embodiment of the present invention, in step S3, the flow rate of the buffer solution C is controlled to be 10-20 mL / min.
[0021] In a further preferred embodiment of the present invention, in step S4, the volume of the added buffer A is 2-4 times that of the elution solution.
[0022] In a further preferred embodiment of the present invention, the number of times the secondary ultrafiltration is performed after adding buffer A in step S4 is 3.
[0023] In a further preferred embodiment of the present invention, the cation exchange medium of the ion exchange column is selected from SP cation exchange column packing material.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) The purification method of heparin synthase PmHS2 provided by the present invention only requires two purification processes to meet product requirements, which is very suitable for industrial production.
[0026] (2) The present invention found that the purity and yield of the sample are highest when the pH is 8.0.
[0027] (3) The purification method for heparin synthase PmHS2 provided by the present invention uses reagents with low raw material cost and the obtained protein has an SDS-PAGE purity of over 95%. Attached Figure Description
[0028] Figure 1 This is a 12% agarose gel electrophoresis image obtained in Example 1. Detailed Implementation
[0029] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.
[0030] In the following embodiments, unless otherwise specified, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased.
[0031] Unless otherwise specified, the reagents and materials involved in the following embodiments are all commercially available products well known to those skilled in the art. Unless otherwise specified, the technical means used in the following embodiments are all technical means well known to those skilled in the art.
[0032] In this embodiment of the invention, the information on the raw materials and related equipment used is shown in Table 1 below:
[0033] Table 1. Information related to raw materials and equipment
[0034]
[0035]
[0036] In this embodiment of the invention, the supernatant of the heparin synthase PmHS fermentation broth was collected after homogenization and centrifugation and stored at 4°C for later use. Before purification, the cryogenically stored heparin synthase PmHS fermentation broth was filtered using a 0.2 μm ultrafiltration tube to obtain a further purified heparin synthase PmHS fermentation broth with cell debris and other impurities removed, which was used for purification in the following embodiments.
[0037] Example 1
[0038] This embodiment provides a method for purifying heparin synthase PmHS, which specifically includes the following steps:
[0039] (1) Liquid exchange and concentration: The fermentation broth of heparin synthase PmHS was mixed with buffer A at a volume ratio of 1:1. The liquid was then exchanged and concentrated using an ultrafiltration membrane with a 10kDa cutoff to remove metal ions and impurities with a molecular weight of less than 10kDa, thus obtaining the ultrafiltrate.
[0040] (2) Chromatographic purification: Take nickel column packing material, pack the column, regenerate the nickel column with 2 mol / L imidazole, rinse repeatedly with purified water, and equilibrate with buffer A for 2-3 column volumes; pump the ultrafiltrate obtained in step (1) into the equilibrated nickel column, then reequilibrate with buffer A, and then perform gradient elution with buffer B, and collect the eluent; the flow rate of the elution buffer B is a linear flow rate of 15 mL / min; wherein, buffer A: 20 mM Tris-HCl, pH = 8.0; buffer B: 20 mM Tris-HCl, 1 M imidazole, pH = 8.0.
[0041] (3) Chromatographic purification: Take SP cation chromatography packing material, pack it into a column, regenerate the SP column with 2 mol / L NaCl, rinse repeatedly with purified water, equilibrate with buffer A for 2-3 column volumes, pump the eluent obtained in step (2) into the equilibrated SP column, perform gradient elution with buffer C, and collect the eluent; the flow rate of the elution buffer C is a linear flow rate of 15 mL / min; wherein, buffer A: 20 mM Tris-HCl, pH = 8.0; buffer C: 20 mM Tris-HCl, 1 M NaCl, pH = 8.0.
[0042] (4) Collect the eluent obtained from the chromatography purification in step (3), add buffer A, and perform ultrafiltration concentration three times to obtain high-purity heparin synthase PmHS2.
[0043] Finally, the eluent was analyzed by electrophoresis, and the results are as follows: Figure 1 As shown in Table 2, the above steps yield electrophoretically purified heparin synthase PmHS2. The activity of the purified enzyme was measured, and the results are shown in Table 3. The yield of heparin synthase PmHS2 obtained in the above steps is calculated and shown in Table 3.
[0044] Example 2
[0045] Compared with Example 1, the difference is that: buffer C: 20mM Tris-HCl, 1.2M NaCl, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0046] Example 3
[0047] Compared with Example 1, the difference is that: buffer C: 20mM Tris-HCl, 0.8M NaCl, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0048] Example 4
[0049] Compared with Example 1, the difference is that: buffer C: 20mM Tris-HCl, 1.5M NaCl, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0050] Example 5
[0051] Compared with Example 1, the difference is that: Buffer B: 20mM Tris-HCl, 0.5M imidazole, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS purity value and yield are shown in Table 3.
[0052] Example 6
[0053] Compared with Example 1, the difference is that: Buffer B: 20mM Tris-HCl, 2.0M imidazole, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0054] Example 7
[0055] Compared with Example 1, the difference is that: Buffer B: 20mM Tris-HCl, 1.5M imidazole, pH=8.0. The obtained heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0056] Comparative Example 1
[0057] Compared with Example 1, the difference is that cation column purification was not performed; only the concentrate obtained in step (1) was purified by nickel column chromatography. The results of the obtained heparin synthase activity are shown in Table 2, and the purity value and yield of PmHS2 are shown in Table 3.
[0058] Comparative Example 2
[0059] Compared with Example 1, the difference is that nickel column purification was not performed; instead, the concentrate obtained in step (1) was directly purified by cation chromatography. The heparin synthase activity results are shown in Table 2, and the PmHS2 purity value and yield are shown in Table 3.
[0060] Table 2. Enzyme activities of purified heparin synthase PmHS2 in each example and comparative example.
[0061]
[0062] Table 3. Yields and purities of purified heparin synthase PmHS2 in each example and comparative example.
[0063]
[0064]
[0065] 12% agarose gel electrophoresis image as shown Figure 1 As shown in the figure; lane 1 is the crude supernatant of heparin synthase PmHS2, lane 2 is the SP flow-through of heparin synthase PmHS2, lane 3 is the SP elution peak of heparin synthase PmHS2, and lane 4 is the marker. It can be seen from the figure that the purity of the heparin synthase PmHS2 sample obtained after purification using the method of Example 1 can reach ≥95%.
[0066] The enzyme activity of the purified products from the examples and comparative examples was measured. The results showed that the enzyme activity of the examples was higher than that of the comparative examples. Therefore, the purification method for heparin synthase PmHS2 provided in this invention not only ensures the total protein recovery rate but also exhibits superior enzyme activity.
[0067] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.
Claims
1. A method for purifying heparin synthase PmHS2, characterized in that, Includes the following steps: S1. Add buffer A to the fermentation broth of heparin synthase PmHS to be purified, mix well, and then perform ultrafiltration once to remove metal ions and impurities with a molecular weight of less than 10kDa. S2. Pump the ultrafiltrate obtained in step S1 into a nickel column that has been equilibrated with buffer A, then reequilibrate with buffer A, and then elute with buffer B, and collect the eluent. S3. Pump the eluent obtained in step S2 into the cation exchange column that has been equilibrated with buffer A, then reequilibrate with buffer A, and then elute with buffer C, and collect the eluent. S4. Add buffer A to the eluent obtained in step S3, and perform ultrafiltration concentration to obtain high-purity heparin synthase PmHS2; wherein, Buffer A: 15-25mM Tris-HCl, pH = 7.5-8.5; Buffer B: 15-25mM Tris-HCl, 1±0.5M imidazole, pH = 7.5-8.5; Buffer C: 15-25mM Tris-HCl, 1±0.2M NaCl, pH = 7.5-8.
5.
2. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, In step S1, the heparin synthase PmHS fermentation broth to be purified has been centrifuged and filtered to remove cell debris and other impurities before purification, and stored at low temperature.
3. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, In step S1, the total volume of the added buffer A is 0.8 to 2 times that of the heparin synthase PmHS fermentation broth.
4. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, In step S2, the flow rate of the buffer solution B is controlled at 10-20 mL / min.
5. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, In step S3, the flow rate of the buffer solution C is controlled at 10-20 mL / min.
6. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, In step S4, the total volume of the added buffer A is 2-4 times that of the elution solution.
7. The purification method for heparin synthase PmHS2 according to claim 6, characterized in that, The number of times the secondary ultrafiltration is performed after adding buffer A is 3.
8. The purification method for heparin synthase PmHS2 according to claim 1, characterized in that, The cation exchange medium of the ion exchange column is selected from SP cation column packing material.