Method for purifying and expressing batroxobin and its applications

Abstract

This method provides a recombinant batroxobin expression purification method. By adjusting and optimizing the pH and conductivity of the supernatant of the culture medium, the target protein can bind better to the chromatographic filler, while most impurity proteins pass through without binding to the filler. This improves the purity of the collected solution, suppresses the activity of hydrolytic enzymes in the fermentation supernatant, and ensures that the target protein does not pass through, thus guaranteeing a high yield. Furthermore, by using a combination of cationic chromatography, anionic chromatography, single-pass tangential flow filtration concentration, and gel filtration chromatography, highly pure and active batroxobin protein can be purified. The elution method is changed from a simple salt gradient to a combined salt gradient and pH gradient elution method, significantly improving protein purity. This purification method employs a continuous flow purification process, eliminating the need for operations such as pH and electrical conductivity adjustment, dilution, and solution exchange for intermediates. This reduces process time, improves enzyme activity, and offers advantages such as reducing storage tanks, personnel, and factory area. In vitro and in vivo verification reveals that batroxobin purified by this method has a similar coagulation effect to commercially available natural batroxobin and has broad applicability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the expression, purification method and application of batroxobin.

Background Art

[0002] Batroxobin is a serine protein hydrolase (thrombin-like) first isolated from the venom of Bothrops atrox in Brazil by the Austrian scholar Von Klobusitzky in 1963. The batroxobin protein has only one peptide chain, but there are 12 cysteines in the batroxobin molecule, forming 6 intramolecular disulfide bonds, and there is also glycosylation modification. There are two N-glycosylation sites, Asn146-Asn17-Thr1 and Asn225-Lys226-Thr227, in its molecule. Although researchers have grasped many properties of batroxobin, the molecular biology research of such snake venom components has been progressing slowly.

[0003] Batroxobin is a serine protease, and its specific action substrate is fibrinogen. Different from thrombin, it only cleaves the A chain of fibrinogen without acting on the B chain. When it hydrolyzes the peptide bond at the Arg16-Gly17 site in the plasma fibrinogen A chain, it can release fibrinogen peptide A, thereby rapidly converting fibrinogen in the blood into fibrin. These fibrin aggregates into a soft thrombus that is easily hydrolyzed by plasmin to close the wound and achieve a rapid hemostatic effect. Also, in vivo, it does not activate blood coagulation factor XIII, so the side chains of the fibrin clot generated by hydrolysis cannot crosslink and are easily decomposed by plasmin, thus not causing blood system embolism.

[0004] These soft clots are easily broken down by hydrolytic enzymes, leading to a decrease in blood fibrinogen concentration, improving blood viscosity and fluid dynamics, and acting as a defibrinogenase. Based on these biochemical properties, batroxobin has been successfully developed as a hemostatic and fibrin-removing agent. In Europe, batroxobin is used as a hemostatic agent instead of human thrombin. The high production cost of extracting batroxobin from snake venom, the single source of supply, and the low yield and high cost have always been factors limiting the broad application of batroxobin.

[0005] The production of proteins rich in disulfide bonds and glycosylation modifications using genetic engineering methods, particularly serine protein hydrolase molecules with multiple pairs of disulfide bonds, has always been a technical challenge. This is because the mispair rate of disulfide bonds is very high, resulting in very little thrombin-like expression in E. coli, with most of the resulting proteins being inclusion bodies.

[0006] Eukaryotic cell expression systems (yeast, CHO, and insect cells, etc.) can guarantee a high accuracy in disulfide bond pairing. Eukaryotic expression systems can guarantee the accuracy of disulfide bond pairing, and protein folding and organization occur in the endoplasmic reticulum, producing proteins with good biological activity under the synergistic effect of molecular chaperones. Yeast possesses prokaryotic characteristics such as rapid cell proliferation, ease of culture, and simple genetic manipulation, while also possessing eukaryotic functions such as precise processing and modification of expressed proteins and rational spatial folding. Its properties are more stable than prokaryotic expressed proteins, making it highly advantageous for eukaryotic gene expression. It can effectively overcome the deficiencies of the E. coli system, such as the lack of post-translational processing and modification, and the difficulty in regenerating subsequent proteins. Therefore, yeast expression systems are attracting increasing attention and utilization. In 2004, You Weon-Kyoo from South Korea reported that when batroxobin is expressed in Pichia, the expression level reaches 3.431 NIH / mL, and 7 mg can be purified per liter of fermentation broth.

[0007] Due to the characteristics of the enzyme structure of batroxobin itself, the activity of batroxobin tends to decrease during the fermentation production process using genetic engineering. The main reason for this is that proteases in the yeast host system exert a biodegradative effect on batroxobin accumulated in the fermentation liquid, which may affect the yield and enzyme activity of batroxobin.

[0008] Therefore, conventional technology has the problem that the activity of batroxobin produced by fermentation is low, and enzymatic degradation is insufficient. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a method for purifying batroxobin expression, addressing the problems of conventional techniques, such as low yield and low enzyme activity of purified batroxobin. [Means for solving the problem]

[0010] To solve the above technical problems, the present invention employs the following technical solutions.

[0011] The present invention 1) A step of taking the supernatant of the batroxobin yeast fermentation liquid and adjusting the pH and conductivity, 2) A step of performing chromatography by passing the cation resin through the resin, 3) A step of performing chromatography by passing the anionic resin through the resin, 4) A process of concentration using single-pass tangential flow filtration, 5) A step of performing gel filtration chromatography on the concentrated protein solution, 6) The process of eluting and collecting the target protein peak to obtain purified batroxobin, A method for purifying batroxobin expression, including the method described herein, is disclosed.

[0012] Preferably, in step 1), the pH is adjusted to 5.8 and the conductivity is adjusted to 6 mS / cm.

[0013] Preferably, in step 2), the chromatographic elution process involves first removing impurity proteins with a low-salt buffer, then using PB with the buffer pH adjusted to 8.0 to elute and remove the dye, and finally using Tris-HCl with a buffer pH of 9.0 for elution.

[0014] Preferably, the low-salt buffer is a 0.15 M NaCl buffer, which is adjusted to pH 6.0 with NaAc / HAc.

[0015] Preferably, in step 3), the elution operation of the chromatography is performed using an elution buffer of 50 mM Tris-HCl + 0.5 M NaCl with a pH of 9.0.

[0016] Preferably, in step 5), the elution operation in chromatography is performed using an elution buffer of 20 mM PB + 0.15 M NaCl with a pH of 6.0.

[0017] Preferably, the cationic resin used for chromatography in step 2) is SP Sepharose FF.

[0018] Preferably, the anionic resin used for chromatography in step 3) is Q Sepharose FF.

[0019] Preferably, the gel filter filler used for chromatography in step 5) is Superdex 75 PG.

[0020] Preferably, the method further includes measuring the protein concentration of the purified batroxobin protein.

[0021] In the present invention, during the purification process of batroxobin, by adjusting and optimizing the pH and conductivity of the supernatant of the culture medium, the target protein can better bind to the chromatography filler, and most of the impurity proteins flow through without binding to the filler, thereby improving the purity of the collected solution, suppressing the activity of the hydrolase in the fermentation supernatant, ensuring that the target protein does not flow through, and guaranteeing a high yield. The elution method is changed from a simple salt gradient to a combined elution method of salt gradient and pH gradient, significantly improving the purity of the protein. Furthermore, by combining cation chromatography, anion chromatography, single-pass tangential flow filtration concentration, and gel filtration chromatography, a highly pure and active batroxobin protein is purified. The purification method of the present invention adopts a continuous flow purification process, does not require operations such as adjusting pH and electrical conductivity, dilution, and solution exchange for the intermediate, has the advantages of reducing the process time, improving the enzyme activity, and reducing the storage tank, workers, and factory area. Through in vitro and in vivo verification, it is revealed that the batroxobin purified by the method of the present invention has a blood coagulation effect similar to that of commercially available natural batroxobin and has broad applicability.

Brief Description of the Drawings

[0022] [Figure 1] It is a diagram for screening and identifying transformed yeast colonies by PCR. [Figure 2] It is a diagram of the blood coagulation effect of batroxobin purified by culture solutions under different pH conditions. [Figure 3] It is a diagram of the blood coagulation effect of batroxobin purified by culture solutions with different conductivities. [Figure 4] It is a diagram of the blood coagulation effect of batroxobin purified by adding an anion resin and single-pass tangential flow filtration. [Figure 5] It is a diagram of SDS electrophoresis measurement of the purified batroxobin protein. [Figure 6]This is the relative molecular mass spectrometry spectrum of an intact protein. [Figure 7] This is the relative molecular mass spectrometry spectrum after de-N-linked sugar reduction. [Figure 8] This diagram illustrates the coagulation effect of purified recombinant batroxobin on mouse bleeding. [Modes for carrying out the invention]

[0023] Specific embodiments of the present invention will be described in detail below with reference to the drawings. It should be understood that the specific embodiments described herein are for illustrative and interpretive purposes only and do not limit the present invention.

[0024] Example 1: Construction and transformation of a yeast expression vector containing the batroxobin gene 1) Cloning of batroxobin-coding genes First, the batroxobin gene sequence was artificially synthesized using the nucleic acid sequence of batroxobin (J02684.1) disclosed in GeneBank. Based on the information of the gene and the enzyme cleavage sites within the gene, a primer sequence for amplifying the batroxobin coding gene was designed, and the enzyme cleavage sites of XhoI and SacII were added to both ends of the primer. The designed amplification primer sequence is as follows:

[0025] Sense primer: 5'-aactcgaggtcattgga ggtgatgaat-3' (SEQ ID NO: 1), Antisense primer: 5'-aaccgcggcgggcaagt cgcagttt-3' (SEQ ID NO: 2).

[0026] Using the amplification primers described above, PCR amplification was performed on the synthesized batroxobin gene J02684.1.

[0027] The conditions for the PCR amplification reaction are: The cycles were 95°C for 3 minutes, 95°C for 40 seconds, 53°C for 30 seconds, 72°C for 1 minute, 35 cycles, and 72°C for 15 minutes.

[0028] The resulting amplification product was stored at 4°C in preparation for use.

[0029] 2) Construction and transformation of yeast expression vectors After purifying and recovering the PCR amplification product from step 1), the yeast expression vector pPICZαA was also subjected to double enzymatic digestion with XhoI and SacII. After recovering the respective products of the enzymatic digestion, they were ligated with T4 ligase, and the ligated products were then transformed into E. coli BL21 to identify recombinant bacteria, extract the recombinant plasmid, and perform double enzymatic digestion and PCR amplification on the target gene to select positive clones. The recombinant plasmid was named pPICZαA-Batr.

[0030] The pPICZαA-Batr plasmid was extracted, linearized with SacI, and then transformed into Pichia X-33 using electrotransformation. All transformed cells were spread in YPD + Zeocin (100 μg / ml) medium and cultured at 30°C for 4-5 days. Colonies that grew rapidly and burst were selected and identified by colony PCR, and further identification was performed using the amplification primers described above. See Figure 1 for a diagram of yeast colony identification by PCR. The identified positive colony clones were saved for use.

[0031] Example 2: Fermentation expression of yeast process bacterium X-33 / pPICZαA-Batr 1) Initial screening of expression levels in positive colonies Positive clones are selected, first inoculated into glycerol complex medium (BMGY), and cultured overnight with shaking. The culture medium is then removed by centrifugation, and the precipitated cells are resuspended in BMMY and OD. 600Induced expression was performed by diluting the solution to 1, sampling was taken approximately every 12 hours, and methanol was added until the final concentration reached 1%. After 72 hours, the supernatant was collected by centrifugation. After each sampling, the supernatant was retained by centrifugation and the thrombin activity was measured for the first time. The measurement results are shown in Table 1. Colonies with high thrombin activity were obtained by initial selection and cloned for fermentation in a fermenter. The method for measuring thrombin activity is as follows.

[0032] 0.2 ml of human citrate-coagulated plasma was taken, added to a 96-well plate, and incubated at 37°C for 3 minutes. 0.2 ml of preheated sample solution (also at 37°C) was added, and the mixture was immediately shaken to ensure uniform mixing. The time was then counted, and the plasma coagulation status was examined starting at 40 seconds. The initial coagulation time was recorded, and three samples were measured simultaneously, with an error of <20 seconds. If the initial coagulation time was <40 seconds, the sample solution was appropriately diluted multiple times using the dilution formula, and the concentration of the test sample solution that coagulated within (60 ± 20) seconds was recorded. Under the above conditions, the amount of enzyme capable of coagulating 0.2 ml of human citrate-coagulated plasma in 60 seconds was defined as one enzyme activity unit.

[0033] [Table 1]

[0034] Based on the measurement results, the batroxobin activity expressed in colony 2 was the highest, and the coagulation acceleration time was the shortest. Therefore, subsequent fermentation and purification operations were carried out using colony 2.

[0035] 2) Fermentation of yeast X-33 / pPICZαA-Batr-2 The preserved Colony No. 2 was inoculated into a test tube containing 5 mL of BMGY yeast culture solution and incubated overnight. The culture solution in the test tube was transferred to a shaking flask containing 180 mL of BMGY culture solution to be propagated as a seed solution. This was then inoculated into a biological fermenter containing 5 L of BMMY fermentation solution, and fermentation was carried out on a pilot scale using a general methanol yeast fermentation method. The supernatant was collected, purified, and prepared for use.

[0036] Example 3: Purification of yeast X-33 / pPICZαA-Batr-2 fermentation broth 3-1) Initial purification of the fermentation supernatant: Adjustment of the fermentation liquid to different pH levels Following a general method for purifying yeast fermentation broth proteins, the fermentation broth was first adjusted to different pH levels, and the activity of the purified enzymes was observed.

[0037] i) 200 mL of the fermentation supernatant was taken from each sample, and the pH was adjusted to 5.8, 6.5, and 7.2, respectively.

[0038] ii) The supernatant liquid was passed through a cationic filler and chromatographed. The cationic filler was SP Sepharose FF. First, impurity proteins were removed with a low-salt solution (NaAc / HAc, 0.15M NaCl, pH 6.0). The pH was then adjusted to 8.0 with buffer (PB) to elute and remove the dye. Finally, the pH was adjusted to 9.0 with buffer (Tris-HCl) to elute the target protein.

[0039] iii) The protein eluent was concentrated using an ultrafiltration tube (10 kd) and prepared for use.

[0040] iv) Gel filtration chromatography was performed on the concentrated protein solution. Superdex 75 PG was used as the gel filtration filler. The chromatography column was first washed with equilibration buffer (20 mM PB + 0.15 M NaCl, pH 6.0) and then equilibrated. Single-pass tangential flow filtration collection solution was then loaded, and the loading volume was set to ≤5% of the column volume to collect the target protein peak.

[0041] v) The protein concentration in the eluent was measured and adjusted to match the concentration, and the enzyme activity of the collected batroxobin was measured using the method of Example 2. The measurement results are shown in Figure 2.

[0042] Measurement results indicate that raising the pH of the loading sample to around 5.8 allows the target protein to bind better to the chromatographic filler, while most impurity proteins pass through without binding to the filler, thereby improving the purity and yield of the collected solution and enhancing enzyme activity.

[0043] 3-2) Initial purification of the fermentation supernatant: Adjustment of the conductivity of the fermentation liquid The pH of the fermentation liquid was adjusted to approximately 5.8, and the conductivity was further adjusted to observe the activity of the purified enzymes.

[0044] i) 200 mL of the fermentation supernatant was taken from each sample, and the pH was adjusted to 5.8 for each sample. Then, the conductivity of the culture medium was adjusted to 4 mS / cm, 6 mS / cm, and 8 mS / cm, respectively.

[0045] ii) The supernatant liquid was passed through a cationic filler and chromatographed. The cationic filler was SP Sepharose FF. First, impurity proteins were removed with a low-salt solution (NaAc / HAc, 0.15M NaCl, pH 6.0). The pH was then adjusted to 8.0 with buffer (PB) to elute and remove the dye. Finally, the pH was adjusted to 9.0 with buffer (Tris-HCl) to elute the target protein.

[0046] iii) The protein eluent was concentrated using an ultrafiltration tube (10 kd) and prepared for use.

[0047] iv) Gel filtration chromatography was performed on the concentrated protein solution. Superdex 75 PG was used as the gel filtration filler. The chromatography column was first washed with equilibration buffer (20 mM PB + 0.15 M NaCl, pH 6.0) and equilibrated. The concentrated solution was then loaded, and the loading volume was set to ≤5% column volume. The target protein peak was collected.

[0048] v) The protein concentration in the eluent was measured and adjusted to match the concentration, and the enzyme activity of the collected batroxobin was measured using the method of Example 2. The measurement results are shown in Figure 3.

[0049] Measurement results indicate that adjusting the conductivity of the loading sample to approximately 6 mS / cm suppresses the activity of hydrolytic enzymes in the fermentation supernatant and ensures that the target protein does not penetrate, thereby guaranteeing high yield and improving enzyme activity.

[0050] 3-3) Initial purification of the fermentation supernatant: Purification by adding an anion exchange chromatography step. The anions readily bind to the dyes in the culture medium, and by employing a linear elution method based on a salt gradient, the purity of the target protein was increased to approximately 95%.

[0051] i) 200 mL of the supernatant of the fermentation broth was taken from each sample, the pH was adjusted to 5.8, and then the conductivity of the culture medium was adjusted to 6 mS / cm.

[0052] ii) The supernatant liquid was passed through a cationic filler and chromatographed. The cationic filler was SP Sepharose FF. First, impurity proteins were removed with a low-salt solution (NaAc / HAc, 0.15M NaCl, pH 6.0). The pH was then adjusted to 8.0 with buffer (PB) to elute and remove the dye. Finally, the pH was adjusted to 9.0 with buffer (Tris-HCl) to elute the target protein.

[0053] iii) The cation eluent was loaded onto the anionic resin, and the anionic resin was made into Q Sepharose FF. After completion, the chromatography column was washed with equilibration buffer, and then 0-100% elution was performed at 15 CV with elution buffer (50 mM Tris-HCl + 0.5 M NaCl, pH 9.0), and the elution peak of the target protein was collected.

[0054] After chromatography was completed, the chromatography filler was washed and disinfected. Specifically, the anionic chromatography filler still had a noticeable pigment attached after elution, and the pigment could not be completely removed even with 0.5M NaOH and 1M NaCl. With multiple uses, the pigment accumulated, causing the collected solution to become darker in color, reducing the filler yield, lowering the column efficiency, and making packing difficult. Therefore, the pigment remaining on the filler was first eluted with 0.5M H3PO4, with a retention time of 8-10 minutes, and then the filler was regenerated and washed and disinfected with 0.5M NaOH + 1M NaCl to return the filler to its original milky white color.

[0055] The manufacturing process is explained as follows:

[0056] The pH of the supernatant obtained by centrifugation was adjusted to 5.8-6.0 and the conductivity to 5-6 mS / cm using sterile water for injection and 10% glacial acetic acid.

[0057] The cation chromatography column was first equilibrated by washing with equilibration buffer (20mM NaAc / HAc, pH 6.0), and then the sample, whose pH and conductivity had been adjusted, was loaded. After loading was complete, the chromatography column was washed with equilibration buffer, and the elution flow was started. Specifically, the column was first spray-washed with buffer 1 (20mM NaAc / HAc + 0.15M NaCl, pH 6.0) to elute impurity proteins, then spray-washed again with buffer 2 (20mM PB, pH 8.0) to elute dyes and other impurities, and finally eluted with elution buffer (50mM Tris-HCl, pH 9.0), and the elution peak was collected.

[0058] The anion chromatography column was first washed with equilibration buffer (50 mM Tris-HCl, pH 9.0), and then loaded with cation exchange chromatography collection solution. After loading was complete, the chromatography column was washed with equilibration buffer, and then 0-100% elution was performed at 15 CV with elution buffer (50 mM Tris-HCl + 0.5 M NaCl, pH 9.0), and the elution peak of the target protein was collected.

[0059] The chromatographic filler was equilibrated to pH 6.0 using equilibration buffer (20 mM NaAc / HAc, pH 6.0), followed by washing the chromatography column with 0.5 M H3PO4 for 8-10 minutes. After equilibration with the same equilibration buffer, the filler was regenerated and disinfected with 0.5 M NaOH + 1 M NaCl for 30 minutes.

[0060] iv) The protein eluent was concentrated using an ultrafiltration tube (10 kD) and prepared for use.

[0061] v) Gel filtration chromatography was performed on the concentrated protein solution. Superdex 75 PG was used as the gel filtration filler. The chromatography column was first washed with equilibration buffer (20 mM PB + 0.15 M NaCl, pH 6.0) and equilibrated. Then the concentrated solution was loaded, and the loading volume was set to ≤5% column volume. The target protein peak was collected.

[0062] vi) The protein concentration in the eluent was measured and adjusted to match the concentration, and the enzyme activity of the collected batroxobin was measured using the method of Example 2. The measurement results are shown in Figure 4.

[0063] 3-4) Initial purification of the fermentation supernatant: Addition of a single-pass tangential flow filtration step Single-pass tangential flow filtration is easy to operate, suitable for expansion, allows for rapid enrichment of target proteins, and enables convenient subsequent operations by controlling the loading volume as needed, thereby reducing the number of cycles and filler volume required for gel filtration chromatography.

[0064] i) 200 mL of the supernatant of the fermentation broth was taken from each sample, the pH was adjusted to 5.8, and then the conductivity of the culture medium was adjusted to 6 mS / cm.

[0065] ii) The supernatant liquid was passed through a cationic filler and chromatographed. The cationic filler was SP Sepharose FF. First, impurity proteins were removed with a low-salt solution (NaAc / HAc, 0.15M NaCl, pH 6.0). The pH was then adjusted to 8.0 with buffer (PB) to elute and remove the dye. Finally, the pH was adjusted to 9.0 with buffer (Tris-HCl) to elute the target protein.

[0066] iii) The cation eluent was loaded onto the anionic resin, and the anionic resin was made into Q Sepharose FF. After completion, the chromatography column was washed with equilibration buffer, and then 0-100% elution was performed at 15 CV with elution buffer (50 mM Tris-HCl + 0.5 M NaCl, pH 9.0), and the elution peak of the target protein was collected.

[0067] The anion chromatography filler was equilibrated to pH 6.0 using equilibration buffer (20 mM NaAc / HAc, pH 6.0), followed by washing the chromatography column with 0.5 M H3PO4 for 8-10 minutes. After equilibration with the same equilibration buffer, the filler was regenerated and disinfected with 0.5 M NaOH + 1 M NaCl for 30 minutes.

[0068] iv) Perform single-pass tangential flow filtration on the module (0.11m 2 The 10Kd modules were first washed with equilibration buffer (50mM Tris-HCl, pH 9.0). A series connection was used between each subsequent module, and a parallel connection was used for modules of the same order. After equilibration, the anion exchange chromatography collection solution was loaded, the permeate end became waste liquid, and the final sample was collected at the reflux end. The modules were then washed with sterile water for injection and 0.5M NaOH.

[0069] v) Gel filtration chromatography was performed on the concentrated protein solution. Superdex 75 PG was used as the gel filtration filler. The chromatography column was first washed with equilibration buffer (20 mM PB + 0.15 M NaCl, pH 6.0) and then equilibrated. After that, the single-pass tangential flow filtration collection solution was loaded, the loading volume was set to ≤5% of the column volume, and the target protein peak was collected.

[0070] vi) The protein concentration in the eluent was measured and adjusted to match the concentration, and the enzyme activity of the collected batroxobin was measured using the method of Example 2. The measurement results are shown in Figure 4.

[0071] As can be seen from the measurement results in Figure 4, the activity of purified batroxobin can be further improved by adding anionic resin adsorption elution and single-pass tangential flow filtration during the purification process. Analysis suggests that the possible reasons for this are that the purification process reduces the content of impurity proteins, further increases the concentration of batroxobin, and to some extent reduces the interfering effect of impurity proteins on batroxobin.

[0072] Example 4: SDS electrophoresis measurement of purified batroxobin protein Electrophoretic measurements are performed on batroxobin prepared by the methods described in 3-4 of Example 3, mainly as follows.

[0073] The purified sample was taken and electrophoretic measurement was performed on a 15% polyacrylamide gel. The protein sample and loading buffer were mixed in a 4:1 ratio, boiled for 5 minutes to denature the protein, cooled to room temperature, and then loaded using a microwell loader. The electrophoretic voltage was 80-100V for the concentrated gel and 100-120V for the separated gel. The electrophoretic measurement results are shown in Figure 5.

[0074] Here, band 1 is the negative control, and band 2 is batroxobin prepared by the method 3-4 in Example 3. As can be seen from the above, the protein prepared by this purification method is free of other impurity proteins and has high purity.

[0075] Example 5: Relative molecular mass measurement of purified batroxobin protein The calculated theoretical molecular weight of batroxobin is 25.6 kD, but the molecular weight measured by reductive electrophoresis of batroxobin protein expressed by the secretion of constructed methanol yeast process bacteria was approximately 33 kD, which did not match the calculated theoretical value. This is because the batroxobin molecule has two N-glycosylation sites, and based on the general rules for studying most glycosylated proteins, each glycosylation can increase the molecular weight of the protein by approximately 0.3 to 0.5 kD. Therefore, this difference in molecular weight should be the result of glycosylation modification. Specifically, please refer to the mass spectrum measurement results below.

[0076] 1) De-N-linked glycoprotein treatment 40 μg of each purified sample was taken, added to DTT, reduced at 55°C for 60 min, cooled to room temperature, 1 μL PNGase F was added and mixed uniformly, and incubated overnight at 37°C.

[0077] 2) Intact protein treatment A 40 μg sample was treated with 6 M guanidinium chloride to denature it, then DTT was added until the final concentration reached 50 mM. The sample was reduced and denatured at 100°C for 10 minutes, and after dilution, the liquid quality was measured.

[0078] 3) High-performance liquid chromatography parameters The separation was performed using a UPLC (Acquity UPLC I-Class, Waters) liquid-phase system. Phase A was a 0.1% FA aqueous solution, and Phase B was a 0.1% FA acetonitrile solution. 1 μL of sample was loaded from the autosampler, and further separation was performed using a chromatography column (BioResolve RP Column, 450 Å, 2.7 μm, 2.1 × 50 mm, Waters) with a flow rate of 0.3 ml / min, UV measurement wavelength of 280 nm, column temperature of 80°C, and analysis time of 10 min. As for the separation gradient, the linear gradient of liquid B was maintained at 15% from 0 to 1 minute, increased from 15% to 60% from 1 to 7 minutes, increased from 60% to 90% from 7 to 7.5 minutes, maintained at 90% from 7.5 to 8 minutes, and maintained at 15% from 8 to 10 minutes.

[0079] 4) Mass spectral parameters Mass spectral analysis was performed using a Waters XevoG2~XS Q-Tof mass spectrometer. The analysis time was 10 min, the measurement method was set to positive ions, and the parent ion scan range was 500~4000 m / z.

[0080] 5) Data Analysis The raw data was processed using UNIFI (1.8.2, Waters) software, and the relative molecular mass spectrometry spectrum of the intact protein is shown in Figure 6, while the relative molecular mass spectrometry spectrum after de-N-linked sugar reduction is shown in Figure 7.

[0081] The relative molecular weight of the intact protein in the purified sample was 30195 Da. Here, the N-glycosylation modification was mainly high-mannitol (M10-M15, etc.) with differing data, and phosphorylation modification (Phosphate) occurred on some of the mannitol. The relative molecular weight after de-N-linked sugar reduction was 25505 Da in all cases.

[0082] Example 6: Measurement of enzyme activity of purified recombinant batroxobin and commercially available natural batroxobin. Commercially available batroxobin was taken, dissolved in buffer (20 mM PB + 0.15 M NaCl, pH 6.0), and the protein concentration was measured using a general method to match that of purified recombinant batroxobin prepared by method 3-4 in Example 3.

[0083] Refer to the method in Example 2 for measuring enzyme activity. The method for measuring thrombin activity is as follows:

[0084] 0.2 ml of human citrate-controlled plasma was taken, added to a 96-well plate, and incubated at 37°C for 3 minutes. 0.2 ml of preheated sample solution (also at 37°C) was added, and the mixture was immediately shaken to ensure uniform mixing. The time was then counted, and the plasma coagulation status was examined starting at 40 seconds. The initial coagulation time was recorded, and three tubes were measured simultaneously, with an error of <20 seconds. If the initial coagulation time was <40 seconds, the sample solution was diluted multiple times using the formula, and the concentration of the test sample solution that coagulated within (60 ± 20) seconds was recorded. Under the above conditions, the amount of enzyme capable of coagulating 0.2 ml of human citrate-controlled plasma in 60 seconds was defined as one enzyme activity unit. The measurement results are shown in Table 2.

[0085] [Table 2]

[0086] Example 7 Effects of purified recombinant batroxobin and commercially available batroxobin on mouse hemorrhage To identify the in vivo activity of the recombinant batroxobin produced, it was specifically measured by the blood clotting time after the tail of a mouse was cut off, as described below.

[0087] 1) Nine healthy mice were randomly and uniformly divided into three groups, with three mice per group. 2) The corresponding buffer solution was intravenously injected into the mice. One group was injected with PB buffer to serve as a negative control. Another group was injected with commercially available batroxobin (dissolved in buffer solution (20 mM PB + 0.15 M NaCl, pH 6.0)). The last group was injected with purified recombinant batroxobin prepared by method 3-4 in Example 3. 3) Half an hour after injection, the tail of each mouse was cut 0.5 cm from the tip with scissors. Blood spontaneously flowed out, and the time was immediately counted. The average clotting time for each group of mice was calculated. The measurement results are shown in Figure 8.

[0088] Based on the measurement results, recombinant purified batroxobin produced by the present invention already achieves or comes close to achieving the coagulation effect of natural batroxobin, and exhibits a certain level of coagulation effect in vivo.

[0089] In this invention, the process method of the present invention is described by the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention cannot be carried out without relying on the above process steps. As those skilled in the art will see, any improvements to the present invention, equivalent substitutions of raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. 1) A step of taking the supernatant of the recombinant batroxobin yeast fermentation liquid and adjusting the pH and conductivity, 2) A step of performing chromatography by passing the cation resin through the resin, 3) A step of performing chromatography by passing the anionic resin through the resin, 4) A process of concentration by single-pass tangential flow filtration, 5) A step of performing gel filtration chromatography on the concentrated protein solution, 6) The process of eluting and collecting the target protein peak to obtain purified batroxobin, A method for purifying recombinant batroxobin expression, characterized by including the following:

2. The purification method according to claim 1, characterized in that step 1) adjusts the pH to 5.8 and the conductivity to 6 mS / cm.

3. The purification method according to claim 1, characterized in that, in step 2), the chromatographic elution process involves first removing impurity proteins with a low-salt buffer, then using PB with a buffer adjusted to pH 8.0 to elute and remove the dye, and finally using Tris-HCl with a buffer at pH 9.0 for elution.

4. The purification method according to claim 3, characterized in that the low-salt buffer is a 0.15 M NaCl buffer, and its pH is adjusted to 6.0 using NaAc / HAc.

5. The purification method according to claim 1, characterized in that the chromatographic elution operation in step 3) uses an elution buffer of 50 mM Tris-HCl + 0.5 M NaCl with a pH of 9.

0.

6. The purification method according to claim 1, characterized in that in step 5) the chromatographic elution operation uses an elution buffer of 20 mM PB + 0.15 M NaCl with a pH of 6.

0.

7. The purification method according to claim 1, characterized in that the cationic resin used for chromatography in step 2) is SP Sepharose FF.

8. The purification method according to claim 1, characterized in that the anionic resin used for chromatography in step 3) is Q Sepharose FF.

9. The purification method according to claim 1, characterized in that the gel filtration filler used in chromatography in step 5) is Superdex 75 PG.

10. The method according to any one of claims 1 to 9, further comprising the step of performing a protein concentration measurement operation on the recombinant batroxobin protein obtained by purification.

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