A method for purifying defibrase from snake venom

Abstract

The invention belongs to the technical field of protein separation and purification, and specifically relates to a method for purifying defibrase from snake venom. The method comprises the following steps: S1: dissolving a crude snake venom product with a balanced buffer, filtering and removing precipitates to obtain a clarified liquid; S2: balancing an anion chromatography column with the balanced buffer, applying the clarified liquid to the column, eluting it with an eluent, and collecting an eluted component containing a defibrase target protein; S3: balancing an ionic mixed mode chromatography column with a low-pH phosphate buffer, applying the eluted component obtained in S2 to the column after conductivity treatment, eluting it with a high-pH phosphate buffer, collecting the eluted component of the defibrase target protein, and obtaining pure defibrase. The purification method of the invention combines anionic chromatography and ionic mixed mode chromatography, realizes the separation and purification of high-purity defibrase, has low cost, is convenient to operate, and is suitable for large-scale promotion.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and purification, and particularly relates to a method for purifying defibrase from snake venom. Background Art

[0002] As a type of thrombin-like enzyme (TLE) in snake venom hemorrhagic toxins, defibrase is the most studied and most mature snake venom biochemical drug in clinical application.

[0003] Defibrase is a proteolytic enzyme extracted from the venom of the white-browed agkistrodon acutus in Changbai Mountain [National Drug Standard - Defibrase (WSI-XG-031-2000)]. It is a thrombin-like (TLE) proteolytic enzyme, a glycoprotein, and has both arginine esterase and thrombin-like activities. Defibrase is a protease extracted from a specific snake venom. It has clear pharmacological effects such as thrombolysis, antithrombosis, viscosity reduction, and fiber reduction. It is widely used in various embolic diseases such as cerebral thrombosis, transient ischemic attack, myocardial infarction, femoral artery embolism, thromboangiitis obliterans, pulmonary embolism, etc. The anticoagulant effect of defibrase is mainly to act directly on plasma fibrinogen, and has no obvious effect on other coagulation factors in the body such as II, VI, VI X, etc., so defibrase is not easy to cause bleeding. Defibrase also has trypsin-like fibrinase activity, which can promote the release of tissue plasminogen activator from the vascular endothelium, increase the activity of tissue plasminogen activator (t-pA) and urokinase in human plasma, and jointly participate in activating plasminogen in blood clots and plasma to convert it into plasmin. Therefore, defibrase has the effects of resisting blood coagulation, dissolving thrombus, removing fibers, reducing blood viscosity, opening blood vessels to improve microcirculation, and scavenging free radicals (Tang Shengxi, 1999).

[0004] At present, there are many methods for preparing defibrase disclosed. For example, in "Isolation and Purification of Defibrase from Agkistrodon acutus Venom and Experimental Study on Its Rectal Administration", it is recorded that Benzamidine Bestarose 4FF affinity chromatography and Spehadex G-75 molecular sieve chromatography were combined to achieve the separation and purification of defibrase from Agkistrodon acutus venom; or in "Purification and Characterization Analysis of Defibrase from Agkistrodon acutus", Benzamidine Sepharose 6B 150mL was loaded into column A and Sephadex G 25200mL was loaded into column B, and two chromatography columns were used for purification in series to obtain high-purity defibrase from Agkistrodon acutus.

[0005] In order to purify high-purity snake venom defibrase, the existing preparation methods often need to be purified by affinity chromatography, and the high specificity of affinity chromatography is used to purify the defibrase, but the cost of affinity chromatography is high, which is not conducive to large-scale industrial production. Therefore, the present invention develops a purification process method for obtaining high-purity defibrase using ion chromatography technology. Summary of the invention

[0006] The main purpose of the present invention is to provide a method for extracting high-purity defibrase from snake venom using ion chromatography technology to solve the problem of high cost of affinity chromatography, which is mainly used in the extraction and purification of defibrase. Using the purification method disclosed in the present invention, high-purity defibrase target protein can be purified by anion chromatography and ion mixed mode chromatography.

[0007] The first aspect of the present invention discloses a method for purifying defibrase from snake venom, the method comprising the following steps:

[0008] S1: Dissolve the crude snake venom in the equilibrium buffer and filter out the precipitate to obtain a clear solution;

[0009] S2: After balancing the anion chromatography column with the balancing buffer, the clarified liquid obtained in S1 is loaded onto the column, eluted with an elution solution, and the eluted component containing the defibrase target protein is collected;

[0010] S3: After balancing the ionic mixed mode chromatography column with a low pH phosphate buffer, the eluted component obtained in S2 is loaded onto the column after conductivity treatment, eluted with a high pH phosphate buffer, and the eluted component of the defibrase target protein is collected to obtain a pure defibrase;

[0011] Wherein, the pH value of the low pH phosphate buffer is 6.5-7.5, and the pH value of the high pH phosphate buffer is 8.5-9.5;

[0012] The stationary phase of the anion chromatography column used in S2 is DEAE Purose 6HP; the stationary phase of the ionic mixed-mode chromatography column used in S3 is Rigose MMC HiRes.

[0013] Preferably, in S3, the conductivity treatment is: adjusting the conductivity of the eluted component containing the defibrase target protein obtained in S2 to 2-4 mS / cm, and adjusting the pH to 6.5-7.5.

[0014] Preferably, in S3, the high pH phosphate buffer is a high pH phosphate buffer containing NaCl, and the concentration of NaCl is 0.25-0.35 mol / L.

[0015] Preferably, in S3, the pH value of the high pH phosphate buffer is 9, and the concentration of NaCl is 0.3mol / L.

[0016] Preferably, the equilibration buffer in S1 and S2 is a phosphate buffer with a pH value of 7-8;

[0017] The eluent in S2 is a phosphate buffer containing NaCl, with a pH value of 7-8 and a NaCl concentration of 0.1-0.3 mol / L.

[0018] Preferably, in S3, the eluted component obtained in S2 is subjected to conductivity treatment and filtered through a 0.45 μm filter membrane before being loaded onto the column.

[0019] Preferably, in S1, the conductivity and pH value of the sample solution obtained after dissolution are adjusted to be consistent with the equilibrium buffer solution, and then filtered through a 0.45 μm filter membrane to obtain the clarified solution.

[0020] The second aspect of the present invention discloses a high-purity snake venom defibrase prepared by the above method, wherein the molecular weight of the snake venom defibrase is 31-41 kDa.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention adopts chromatographic separation based on different principles, selects the specific ion type mixed mode chromatography of Rigose MMC HiRes chromatography medium, cooperates with the anion chromatography in the first step, and designs and adjusts the parameter conditions such as pH and buffer. Only two steps of chromatography are required to achieve high purity results. Under grayscale analysis, the purity of the purified sample is significantly higher than the existing technical level.

[0023] Among them, during the purification process, the crude snake venom is first dissolved with an equilibrium buffer. Its function is that, on the one hand, the equilibrium buffer can provide a stable environment to avoid sudden changes in pH value and irreversible effects on the folding state, solubility and activity of the protein; on the other hand, the equilibrium buffer can reduce the adsorption of impurities in the sample, simplify the subsequent purification process, and improve the purity of the purified protein.

[0024] In the first step of anion chromatography, the different proteins in the snake venom are initially separated by the difference in reversible binding force between the different proteins in the snake venom and the charged groups on the ion exchange resin. In the present invention, the separation effect of different proteins in the snake venom in the anion chromatography column is further optimized by adjusting parameters such as phosphate buffer and salt concentration.

[0025] The Rigose MMC HiRes chromatography medium selected in the present invention is designed by optimizing the structure of the functional ligand, combining cationic and hydrophobic interaction modes, enhancing the affinity and selectivity for the target protein, thereby improving the purification efficiency.

[0026] The purification method developed by the present invention combines anion chromatography and ion-type mixed mode chromatography, realizes the separation and purification of high-purity defibrase, has low cost, convenient operation, and is suitable for large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 , the chromatogram of DEAE Purose 6HP in Example 1;

[0028] Figure 2 , Rigose MMC HiRes chromatogram in Example 1;

[0029] Figure 3 , SDS-PAGE gel image of the purified sample of Example 1;

[0030] Figure 4 , the DEAE Purose 6HP chromatogram in Example 2;

[0031] Figure 5 , Rigose MMC HiRes chromatogram in Example 2;

[0032] Figure 6 , SDS-PAGE gel image of the purified sample of Example 2;

[0033] Figure 7 , the DEAE Purose 6HP chromatogram in Example 3;

[0034] Figure 8 , Rigose MMC HiRes chromatogram in Example 3;

[0035] Fig. 9 , SDS-PAGE gel image of the purified sample in Example 3. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The process parameters not specified in the embodiments of the present application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0037] Example 1

[0038] (S1) accurately weighing 0.3 g of freeze-dried powder of Agkistrodon acutus venom from Changbai Mountain, dissolving it in 15 mL of equilibrium buffer (phosphate buffer with a pH of 7.5) at room temperature to a final concentration of 20 mg / mL, and adjusting the pH and conductivity to be consistent with the equilibrium buffer, filtering with a 0.45 um filter membrane to obtain a clear solution;

[0039] (S2) After balancing the DEAE Purose 6HP anion chromatography column with a balancing buffer (phosphate buffer with a pH of 7.5), the clarified liquid obtained in S1 was loaded onto the column at a flow rate of 120 cm / h. During loading, air bubbles were not allowed to enter the pipe and column. After the sample was loaded, the column was rinsed with a phosphate buffer with a pH of 7.5, and the flow-through liquid was collected. After the UV light stabilized, it was eluted with 0.15 M NaCl and a phosphate buffer with a pH of 7.5. The collection began when the UV peak appeared, and the collection of the eluate stopped when the UV peak ended.

[0040] (S3) conducting conductivity treatment on the eluate collected in S2: adjusting the conductivity and pH thereof to be consistent with the conductivity and pH of a phosphate buffer solution with a pH of 7, and filtering through a 0.45 μm filter membrane for later use;

[0041] After balancing the Rigose MMC HiRes ionic mixed mode chromatography column with a phosphate buffer having a pH of 7, the eluate treated with conductivity and filtration was loaded onto the column at a flow rate of 120 cm / h, and eluted with a phosphate buffer having a pH of 9 and 0.3 M NaCl. Collection began when the ultraviolet peak appeared and stopped when the ultraviolet peak ended, thereby obtaining pure defibrase.

[0042] The final product was analyzed by grayscale analysis (Qinxiang GenoSens 2000 series gel imaging system). The target band at 31-41KDa accounted for 90% of the whole lane band, that is, the purity of the purified defibrase was 90%. Figure 3 .

[0043] Example 2

[0044] (S1) accurately weighing 0.3 g of freeze-dried powder of Agkistrodon acutus venom from Changbai Mountain, dissolving it in 15 mL of equilibrium buffer (phosphate buffer with a pH of 7.5) at room temperature to a final concentration of 20 mg / mL, and adjusting the pH and conductivity to be consistent with the equilibrium buffer, filtering with a 0.45 um filter membrane to obtain a clear solution;

[0045] (S2) After balancing the DEAE Purose 6HP anion chromatography column with a balancing buffer (phosphate buffer with a pH of 7.5), the clarified liquid obtained in S1 was loaded onto the column at a flow rate of 120 cm / h. During loading, air bubbles were not allowed to enter the pipe and column. After the sample was loaded, the column was rinsed with a phosphate buffer with a pH of 7.5, and the flow-through liquid was collected. After the UV light stabilized, it was eluted with 0.15 M NaCl and a phosphate buffer with a pH of 7.5. The collection began when the UV peak appeared, and the collection of the eluate stopped when the UV peak ended.

[0046] (S3) conducting conductivity treatment on the eluate collected in S2: adjusting the conductivity and pH thereof to be consistent with the conductivity and pH of a phosphate buffer solution with a pH of 7.5, and filtering through a 0.45 μm filter membrane for later use;

[0047] After balancing the Rigose MMC HiRes ion-type mixed mode chromatography column with a phosphate buffer of pH 7.5, the eluate treated with conductivity and filtration was loaded onto the column at a flow rate of 120 cm / h, and eluted with a phosphate buffer of 0.3 M NaCl and pH 9. Collection began when the ultraviolet peak appeared and stopped when the ultraviolet peak ended, thereby obtaining pure defibrase.

[0048] The final product was analyzed by grayscale analysis (Qinxiang GenoSens 2000 series gel imaging system). The target band at 31-41KDa accounted for 95% of the whole lane band, that is, the purity of the purified defibrase was 95%. Figure 6 .

[0049] Example 3

[0050] (S1) accurately weighing 0.3 g of freeze-dried powder of Agkistrodon acutus venom from Changbai Mountain, dissolving it in 15 mL of equilibrium buffer (phosphate buffer with a pH of 7.5) at room temperature to a final concentration of 20 mg / mL, and adjusting the pH and conductivity to be consistent with the equilibrium buffer, filtering with a 0.45 um filter membrane to obtain a clear solution;

[0051] (S2) After balancing the DEAE Purose 6HP anion chromatography column with a balancing buffer (phosphate buffer with a pH of 7.5), the clarified liquid obtained in S1 was loaded onto the column at a flow rate of 120 cm / h. During loading, air bubbles were not allowed to enter the pipe and column. After the sample was loaded, the column was rinsed with a phosphate buffer with a pH of 7.5, and the flow-through liquid was collected. After the UV light stabilized, it was eluted with 0.15 M NaCl and a phosphate buffer with a pH of 7.5. The collection began when the UV peak appeared, and the collection of the eluate stopped when the UV peak ended.

[0052] (S3) adjusting the conductivity and pH of the eluate eluted with the 0.15 M NaCl, pH 7.5 phosphate buffer according to the next step of pH 7.5 phosphate buffer so that its conductivity and pH are consistent with the next step of balancing buffer, and filtering through a 0.45 μm filter membrane for later use;

[0053] After balancing the Rigose MMC HiRes ionic mixed mode chromatography column with a phosphate buffer having a pH of 7.5, the eluate treated with conductivity and filtration was loaded onto the column at a flow rate of 120 cm / h, and eluted with a phosphate buffer having a pH of 9 and 0.25 M NaCl. Collection began when the ultraviolet peak appeared and stopped when the ultraviolet peak ended, thereby obtaining pure defibrase.

[0054] The final product was analyzed by grayscale analysis (Qinxiang GenoSens 2000 series gel imaging system). The target band at 31-41KDa accounted for 97% of the whole lane band, that is, the purity of the purified defibrase was 97%. Fig. 9 .

[0055] The present invention is not limited to the above-mentioned embodiments. The protection scope of the present invention is defined by the attached claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principle and essence of the present invention, which should all be equivalent replacement methods and fall within the protection scope of the present invention.

Claims

1. A method for purifying defibrase from snake venom, characterized in that: The method is to purify defibrase from snake venom, which comprises the following steps: S1: Dissolve the crude snake venom in the equilibrium buffer and filter out the precipitate to obtain a clear solution; S2: After balancing the anion chromatography column with the balancing buffer, the clarified liquid obtained in S1 is loaded onto the column, eluted with an elution solution, and the eluted component containing the defibrase target protein is collected; S3: After balancing the ionic mixed mode chromatography column with a low pH phosphate buffer, the eluted component obtained in S2 is loaded onto the column after conductivity treatment, eluted with a high pH phosphate buffer, and the eluted component of the defibrase target protein is collected to obtain a pure defibrase; Wherein, the pH value of the low pH phosphate buffer is 6.5-7.5, and the pH value of the high pH phosphate buffer is 8.5-9.5; The stationary phase of the anion chromatography column used in S2 is DEAE Purose 6HP; the stationary phase of the ionic mixed-mode chromatography column used in S3 is Rigose MMC HiRes.

2. A method for purifying defibrase from snake venom according to claim 1, characterized in that: In S3, the conductivity treatment is: adjusting the conductivity of the eluted component containing the defibrase target protein obtained in S2 to 2-4 mS / cm, and adjusting the pH to 6.5-7.

5.

3. A method for purifying defibrase from snake venom according to claim 1, characterized in that: In S3, the high pH phosphate buffer is a high pH phosphate buffer containing NaCl, and the concentration of NaCl is 0.25-0.35 mol / L.

4. A method for purifying defibrase from snake venom according to claim 3, characterized in that: In S3, the pH value of the high pH phosphate buffer is 9, and the concentration of NaCl is 0.3 mol / L.

5. A method for purifying defibrase from snake venom according to claim 1, characterized in that: The equilibration buffer in S1 and S2 is a phosphate buffer with a pH value of 7-8; The eluent in S2 is a phosphate buffer containing NaCl, with a pH value of 7-8 and a NaCl concentration of 0.1-0.3 mol / L.

6. A method for purifying defibrase from snake venom according to claim 1, characterized in that: In S3, the eluted component obtained in S2 is subjected to conductivity treatment and filtered through a 0.45 μm filter membrane before being loaded onto the column.

7. A method for purifying defibrase from snake venom according to claim 1, characterized in that: In S1, the conductivity and pH value of the sample solution obtained after dissolution are adjusted to be consistent with the equilibrium buffer solution, and then filtered through a 0.45 μm filter membrane to obtain the clarified solution.

8. A high-purity snake venom defibrase, characterized in that: The snake venom defibrase is prepared by the method according to any one of claims 1 to 7, and the molecular weight of the snake venom defibrase is 31-41 kDa.

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