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WO2024212736A1 - 一种巴曲酶的表达纯化方法及应用 - Google Patents

一种巴曲酶的表达纯化方法及应用 Download PDF

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Publication number
WO2024212736A1
WO2024212736A1 PCT/CN2024/080242 CN2024080242W WO2024212736A1 WO 2024212736 A1 WO2024212736 A1 WO 2024212736A1 CN 2024080242 W CN2024080242 W CN 2024080242W WO 2024212736 A1 WO2024212736 A1 WO 2024212736A1
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batroxobin
chromatography
buffer
protein
purification method
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PCT/CN2024/080242
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English (en)
French (fr)
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张燕
李晴
唐芳
郭森林
关录凡
李郑武
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上海腾瑞制药股份有限公司
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Publication of WO2024212736A1 publication Critical patent/WO2024212736A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21074—Venombin A (3.4.21.74)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of bioengineering, and specifically to an expression and purification method and application of batroxobin.
  • Batroxobin is a serine protease (thrombin-like enzyme) that was first isolated from the venom of the Brazilian lancehead pit viper (Bothrops atrox) by Austrian scholar Von Klobusitzky in 1963.
  • batroxobin protein has only one peptide chain, there are 12 cysteines in the batroxobin molecule, forming six intramolecular disulfide bonds; it is also glycosylated, with two N-glycosylation sites in the molecule: ASnl46-ASn''17-Thr'" and Asn225-Lys226-Thr227.
  • Batroxobin is a serine protease. Its specific substrate is fibrinogen. Unlike thrombin, it only cuts the A chain of fibrinogen and does not act on the B chain. When it hydrolyzes the Arg16-Gly17 peptide bond in the A chain of plasma fibrinogen, it can release fibrinopeptide A, thereby quickly converting the fibrinogen in the blood into fibrin. These fibrins can then aggregate into loose thrombi that are easily hydrolyzed by fibrinolysin to seal the wound and achieve rapid hemostasis.
  • batroxobin has been successfully developed into a hemostatic and defibrinolytic drug.
  • batroxobin is replacing human thrombin as a hemostatic drug.
  • the production cost of extracting batroxobin from snake venom is too high.
  • the single source, low output and high price have always been factors restricting the widespread application of batroxobin.
  • a high disulfide bond pairing accuracy rate can be guaranteed in eukaryotic cell expression systems (yeast, CHO and insect cells, etc.).
  • the eukaryotic expression system can ensure the positive pairing rate of disulfide bonds.
  • the folding and assembly of proteins are carried out on the endoplasmic reticulum. Under the synergistic action of molecular chaperones, proteins with good biological activity can be produced.
  • Yeast has the characteristics of prokaryotes, such as fast cell growth, easy cultivation, and simple genetic manipulation; it also has the functions of eukaryotic organisms to correctly process, modify, and reasonably fold the expressed proteins. Its properties are more stable than those of prokaryotic expressed proteins, which is very conducive to the expression of eukaryotic genes.
  • batroxobin Due to the characteristics of the enzyme structure of batroxobin itself, the activity of batroxobin tends to decrease during the genetic engineering fermentation production process. The main reason is that the protease in the yeast host system may degrade the batroxobin accumulated in the fermentation broth, thereby affecting the production and enzyme activity of batroxobin.
  • the existing technology has the problems of low activity of batroxobin obtained by fermentation and insufficient enzyme degradation.
  • the purpose of the present invention is to provide a method for expressing and purifying batroxobin to address the problems of low yield and low enzyme activity of purified batroxobin in the prior art.
  • the present invention/utility model adopts the following technical solutions:
  • the present invention discloses a method for expressing and purifying batroxobin, comprising the following steps:
  • step 1) the pH value is adjusted to 5.8 and the conductivity is adjusted to 6 mS/cm.
  • the elution operation of the chromatography in step 2) is to first remove impurities with a low-salt buffer, then use PB with the buffer pH adjusted to 8.0 to elute the pigment, and finally use Tris-HCl with a buffer pH of 9.0 for elution.
  • the low-salt buffer is 0.15M NaCl buffer, and the pH value is adjusted to 6.0 by NaAc/HAc.
  • the elution operation in the chromatography in step 5 uses an elution buffer of 20 mM PB + 0.15 M NaCl, pH 6.0.
  • the cationic resin used for chromatography in step 2) is SP Sepharose FF.
  • the anion resin used for chromatography in step 3 is Q Sepharose FF.
  • the gel filtration filler used in chromatography in step 5 is Superdex 75 PG.
  • the method further comprises the operation of measuring the protein concentration of the purified batroxobin protein.
  • the present invention optimizes the pH value and conductivity of the culture medium supernatant so that the target protein can be well bound to the chromatographic filler, while most of the impurities cannot bind to the filler and flow through, thereby improving the purity of the collected liquid, inhibiting the hydrolase activity in the fermentation supernatant, and ensuring that the target protein does not flow through, thereby ensuring a high yield.
  • the elution mode is changed from a simple salt gradient to a salt gradient and pH gradient composite elution mode, which greatly improves the purity of the protein.
  • cation chromatography, anion chromatography, unidirectional tangential flow filtration concentration and gel filtration chromatography are used in conjunction to purify and obtain a high-purity and active batroxobin protein.
  • the purification method of the present invention adopts a continuous flow purification process, and the intermediate does not need to adjust the pH value, conductivity, dilution, liquid change and other operations, which has the advantages of reducing the process time, improving the enzyme activity, and reducing the storage tanks, personnel, and plant area.
  • In vitro and in vivo verifications show that the batroxobin purified by the method of the present invention has a coagulation effect similar to that of commercially available natural batroxobin, and has broad application prospects.
  • Figure 1 PCR screening and identification of transformed yeast colonies.
  • Figure 2 Graph showing the coagulation effect of batroxobin purified from culture medium under different pH conditions.
  • Figure 4 The coagulation effect of adding anion resin and unidirectional tangential flow filtration on purified batroxobin.
  • Figure 8 Effect of purified recombinant batroxobin on bleeding and coagulation in mice.
  • the gene sequence was first artificially synthesized. Based on the information of the gene and the information of the restriction sites in the gene, the primer sequence for amplifying the batroxobin encoding gene was designed, and the restriction site sequences of XhoI and SacII were added to both ends of the primers.
  • the designed amplification primer sequence is as follows:
  • Sense primer 5′-aactcgaggtcattgga ggtgatgaat -3′;
  • Antisense primer 5’-aaccgcggcgggcaagt cgcagttt-3’.
  • the above-mentioned amplification primers were used to perform PCR amplification with the synthetic batroxobin gene J02684.1.
  • the obtained amplification product was stored at 4°C for future use.
  • the PCR amplification product in step 1) is purified and recovered, it is double-digested with XhoI and SacII.
  • the yeast expression vector pPICZ ⁇ A is also double-digested with XhoI and SacII.
  • the digested products are recovered, they are ligated with T4 ligase and transformed into Escherichia coli BL21.
  • the recombinant bacteria are identified, the recombinant plasmid is extracted, and the positive clones are screened by double digestion and PCR amplification of the target gene.
  • the recombinant plasmid is named pPICZ ⁇ A-Batr.
  • the error should be ⁇ 20 s. If the initial coagulation time is ⁇ 40 s, then when the sample solution is diluted several times, record the concentration of the test sample solution that coagulates within (60 ⁇ 20) s. Under the above conditions, the amount of enzyme that can make 0.2 ml of human-citrate anticoagulated plasma coagulate in 60 s is defined as one enzyme activity unit.
  • Table 1 Preliminary thrombin activity assay of different positive clones engineered yeast X-33/pPICZ ⁇ A-Batr induced expression for 36 hours
  • colony 2 is used in the subsequent fermentation and purification operations.
  • the mixture was transferred to a biofermentation tank containing 5 L of BMMY fermentation broth and a pilot-scale fermentation was carried out according to the conventional method of methanol yeast fermentation. The supernatant was collected for purification and later use.
  • the different pH values of the fermentation broth are first adjusted to observe the activity of the purified enzyme.
  • the concentrated protein solution was subjected to gel filtration chromatography.
  • the gel filtration filler used was Superdex 75 PG.
  • the chromatography column was first rinsed with equilibrium buffer (20mM PB+0.15M NaCl, pH6.0). After equilibrium, the unidirectional tangential flow filtration collection liquid was loaded, and the loading volume was ⁇ 5% of the column volume to collect the target protein peak.
  • Example 5 Determine the protein concentration of the eluate and adjust it to a consistent concentration. Use the method in Example 2 to determine the enzyme activity of the collected batroxobin. See Figure 2 for the determination results.
  • the pH value of the fermentation broth was adjusted to about 5.8, and the conductivity was further adjusted to observe the activity of the purified enzyme.
  • the concentrated protein solution was subjected to gel filtration chromatography.
  • the gel filtration filler used was Superdex 75 PG.
  • the chromatography column was first rinsed with equilibrium buffer (20mM PB+0.15M NaCl, pH6.0). After equilibrium, the concentrated solution was loaded with the sample. The loading volume was ⁇ 5% of the column volume, and the target protein peak was collected.
  • Example 5 Determine the protein concentration of the eluate and adjust it to a consistent concentration. Use the method in Example 2 to determine the enzyme activity of the collected batroxobin. See Figure 3 for the determination results.
  • adjusting the conductivity of the sample to about 6 can not only inhibit the activity of hydrolases in the fermentation supernatant, but also ensure that the target protein does not flow through, ensuring a high yield, thereby improving enzyme activity.
  • Anions easily bind to pigments in the culture medium, and the purity of the target protein is further increased to about 95% by using the salt gradient linear elution mode.
  • the pH of the centrifuged supernatant was adjusted to 5.8-6.0 and the conductivity to 5-6 mS/cm with water for injection and 10% glacial acetic acid.
  • the cationic chromatography column is first flushed and balanced with equilibrium buffer (20mM NaAc/HAc, pH6.0), and then the sample with adjusted pH and conductivity is loaded. After loading, the chromatography column is flushed with equilibrium buffer and the elution process begins: first, the impurities are eluted with elution buffer 1 (20mM NaAc/HAc +0.15M NaCl, pH6.0), and then impurities such as pigments are eluted with elution buffer 2 (20mM PB, pH8.0), and finally, elution is performed with elution buffer (50mM Tris-HCl, pH9.0), and the elution peak is collected.
  • equilibrium buffer 20mM NaAc/HAc, pH6.0
  • the anion chromatography column was first rinsed with equilibrium buffer (50mM Tris-HCl, pH9.0), and the cation exchange chromatography collection solution was loaded. After loading, the chromatography column was rinsed with equilibrium buffer, and then eluted with elution buffer (50mM Tris-HCl+0.5M NaCl, pH9.0) for 0 ⁇ 100% for 15CV, and the elution peak of the target protein was collected.
  • equilibrium buffer 50mM Tris-HCl, pH9.0
  • elution buffer 50mM Tris-HCl+0.5M NaCl, pH9.0
  • the chromatography filler was first balanced to pH 6.0 with a balance buffer (20 mM NaAc/HAc, pH 6.0), and then the column was cleaned with 0.5 M H 3 PO 4 with a retention time of 8 to 10 minutes. After balancing with the above balance buffer, the filler was regenerated and disinfected with 0.5 M NaOH + 1 M NaCl with a retention time of 30 minutes.
  • the concentrated protein solution was subjected to gel filtration chromatography.
  • the gel filtration filler used was Superdex 75 PG.
  • the chromatography column was first rinsed with equilibrium buffer (20mM PB+0.15M NaCl, pH6.0). After equilibrium, the concentrated solution was loaded with the sample. The loading volume was ⁇ 5% of the column volume, and the target protein peak was collected.
  • Example 6 Determine the protein concentration of the eluate and adjust it to a consistent concentration. Use the method in Example 2 to determine the enzyme activity of the collected batroxobin. See Figure 4 for the determination results.
  • Single-pass tangential flow filtration is easy to operate and suitable for scale-up. It can quickly concentrate the target protein, control the loading volume as needed for subsequent operations, and reduce the number of cycles and filler volume of gel filtration chromatography.
  • the anion chromatography filler was first balanced to pH 6.0 with a balance buffer (20 mM NaAc/HAc, pH 6.0), and then the column was cleaned with 0.5 M H 3 PO 4 with a retention time of 8 to 10 minutes. After balancing with the above balance buffer, the filler was regenerated and disinfected with 0.5 M NaOH + 1 M NaCl with a retention time of 30 minutes.
  • the gel filtration filler used is Superdex 75 PG.
  • the chromatography column is first rinsed with equilibrium buffer (20mM PB+0.15M NaCl, pH6.0). After equilibrium, the collected liquid by unidirectional tangential flow filtration is loaded. The loading volume is ⁇ 5% of the column volume, and the target protein peak is collected.
  • Example 6 Determine the protein concentration of the eluate and adjust it to a consistent concentration. Use the method in Example 2 to determine the enzyme activity of the collected batroxobin. See Figure 4 for the determination results.
  • 40ug of the purified sample was denatured by adding 6M guanidine hydrochloride, and DTT was added to a final concentration of 50mM.
  • the sample was reduced and denatured at 100°C for 10 min, and then diluted for liquid quality detection.
  • UPLC Acquity UPLC I-Class, Waters liquid phase system
  • Mobile phase A was 0.1% FA aqueous solution
  • mobile phase B was 0.1% FA acetonitrile solution.
  • 1 ⁇ L of the sample was loaded by an autosampler and then separated by a chromatographic column (BioResolve RP Column, 450 ⁇ , 2.7 ⁇ m, 2.1X50mm, Waters) at a flow rate of 0.3ml/min, UV detection wavelength of 280nm, column temperature of 80°C, and analysis time of 10min.
  • the separation gradient was: 0min-1min, liquid B linear gradient maintained at 15%, 1min-7min, liquid B linear gradient from 15% to 60%; 7min-7.5min, liquid B linear gradient from 60% to 90%; 7.5min-8min, liquid B maintained at 90%; 8min-10min, liquid B maintained at 15%.
  • Mass spectrometry analysis was performed using a XevoG2-XS Q-Tof mass spectrometer (Waters). Analysis time: 10 min, detection mode: positive ion, parent ion scanning range: 500-4000 m/z.
  • the original data were processed by UNIFI (1.8.2, Waters) software.
  • the relative molecular mass analysis spectrum of the intact protein is shown in FIG6
  • the relative molecular mass analysis spectrum after reduction and de-N-glycanization is shown in FIG7 .
  • the relative molecular mass of the purified sample intact protein is 30195Da, among which the N-glycosylation modification is mainly high mannose with different data (M10-M15, etc.) and phosphorylation modification (Phosphate) occurs on some mannose; the relative molecular mass after reduction and de-N-glycosylation is 25505 Da.
  • batroxobin was taken and dissolved in a buffer solution (20 mM PB + 0.15 M NaCl, pH 6.0). The protein concentration was determined by conventional methods and was consistent with the purified recombinant batroxobin prepared by method 3-4 in Example 3.
  • the method for determining enzyme activity is as follows:
  • Example 7 Effects of purified recombinant batroxobin and commercially available batroxobin on bleeding in mice
  • the in vivo activity of recombinant batroxobin was determined by the coagulation time of mice after tail amputation.
  • the specific operation is as follows:
  • mice Nine healthy mice were randomly divided into three groups, with 3 mice in each group;
  • the recombinant purified batroxobin prepared by the method of the present invention has reached or is close to the coagulation effect of natural batroxobin, and also has a certain coagulation effect in vivo.
  • the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the raw materials selected by the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

提供了一种重组巴曲酶的表达纯化方法，通过对培养基上清的pH值以及电导率的调整优化，使目的蛋白能很好地结合在层析填料上，而大部分杂蛋白无法和填料结合而流穿，提高收集液纯度，既能抑制发酵上清液中的水解酶活性，又能保证目的蛋白不流穿，保证高收率。进一步将阳离子层析、阴离子层析以及单向切向流过滤浓缩和凝胶过滤层析配套使用，纯化获得了高纯度有活性的巴曲酶蛋白。洗脱模式由简单的盐梯度改为盐梯度和pH梯度复合洗脱方式，极大提高了蛋白的纯度。该纯化方法采用连续流纯化工艺，中间体无需调节pH值、电导、稀释、换液等操作，降低工艺时间，提高酶活性，减少储罐、人员、厂房面积等优点。体外以及体内的验证表明，采用该方法纯化的巴曲酶具有与市售的天然巴曲酶类似的凝血效果，具有广泛的应用前景。

Description

一种巴曲酶的表达纯化方法及应用 技术领域
 本发明涉及生物工程领域，具体涉及一种巴曲酶的表达纯化方法及应用。
 背景技术
 巴曲酶是1963年奥地利学者Von Klobusitzky 首次从巴西矛头蝮蛇（Bothrops atrox）毒液中分离得到一种丝氨酸蛋白水解酶（类凝血酶）。巴曲酶蛋白虽然只有一条肽链，但是巴曲酶分子中有12个半胱氨酸，形成六种分子内 二硫键；还有糖基化修饰，其分子内有两个N-糖基化位点：ASnl46-ASn''17-Thr'"和 Asn225-Lys226-Thr227 。虽然研究者对巴曲酶的性质有了比较多的了解，但是，对这种蛇毒成分的分子生物学研究一直进展缓慢。
 巴曲酶是一种丝氨酸蛋白酶类，巴曲酶的特异性作用底物是纤维蛋白原，与凝血酶不同的是，它只切割纤维蛋白原的A链，而不作用B链。当它水解血浆纤维蛋白原A链中的Arg16-Gly17位肽键时，能够释放出纤维蛋白肽A，从而快速地将血液中的纤维蛋白原转变成纤维蛋白，接着这些纤维蛋白就能聚集成疏松的易于被纤维蛋白酶水解的血栓来封闭伤口，实现快速止血的功效。同时,在体内它并不激活凝血因子XIII，由其水解产生的纤维蛋白凝块的侧链不能交联‚易被纤维蛋白溶酶降解‚所以不会造成血液系统的栓塞。
 这种软凝块易被水解酶降解，导致血中纤维蛋白原浓度降低，从而改善血液黏度和血液的流体力学特性‚起到降纤酶的功效。基于这些生物化学特性‚巴曲酶已经被成功地开发成止血药和降纤药。在欧洲巴曲酶正在替代人凝血酶作为止血药物。从蛇的毒液中提取巴曲酶的生产成本太高。来源单一,并且产量低价格高一直是制约巴曲酶广泛应用的因素。
 采用基因工程的手段生产富含二硫键和糖基化修饰的蛋白一直都是一个技术难题，尤其是生产有多对二硫键的丝氨酸蛋白水解酶类分子。这是因为二疏键配对错误率很高，在E．coli 中表达蛇毒类凝血酶的量很少，几乎全是包涵体。
 真核细胞表达系统(酵母、CHO和昆虫细胞等）中能保证较高二硫键配对正确率。真核生物表达系统可以保证二硫键的正配率,蛋白的折叠和组装是在内质网上进行的,在分子伴侣的协同作用下,就可以产生具有良好的生物活性的蛋白。酵母具有原核表达系统细胞生长快,易于培养,遗传操作简单等原核生物的特点;又具有真核生物对表达的蛋白进行正确加工、修饰、合理的空间折叠等功能，性质较原核表达的蛋白质更加稳定，非常有利于真核基因的表达，能有效克服大肠杆菌系统缺乏蛋白翻泽后加工、修饰的不足，后期蛋白复性难的缺点。所以,酵母表达系统受到越来越多的重视和利用。韩国的You Weon-Kyoo 于2004年报道了巴曲酶在毕氏酵母中进行表达，表达量达到3.431NIH／mL，从每升发酵液中可纯化得到7mg。
 由于巴曲酶自身酶结构的特性，在采用基因工程发酵生产的过程中，巴曲酶的活性有下降趋势，主要原因是由于酵母宿主系统中蛋白酶可能对发酵液中累积的巴曲酶产生降解作用，从而影响巴曲酶的产量以及酶活性。
 因此，现有技术中存在发酵制备获得的巴曲酶的活性偏低，以及酶降解的不足。
 发明内容
 本发明的目的在于针对现有技术中纯化的巴曲酶产量的偏低，酶活性不高的问题，提供一种巴曲酶的表达纯化方法。
 为解决上述技术问题，本发明/实用新型采用如下技术方案：
本发明公开了一种巴曲酶的表达纯化方法，包括如下步骤：
1）取巴曲酶酵母发酵液上清，调节pH值和电导率；
2）过阳离子树脂进行层析；
3）过阴离子树脂层析；
4）单向切向流过滤浓缩；
5）将浓缩的蛋白液进行凝胶过滤层析。
 6）洗脱收集目的蛋白峰，获得纯化的巴曲酶。
 优选的，步骤1）中调节pH值为5.8，调节电导率为6mS/cm。
 优选的，步骤2）中层析的洗脱操作为先用低盐缓冲液将杂蛋白去除，使用缓冲液pH调整为8.0的PB，将色素洗脱去除，最后采用缓冲液pH为9.0的Tris-HCl洗脱。
 优选的，所述低盐缓冲液为0.15M NaCl缓冲液，NaAc/HAc调节pH值6.0。
 优选的，步骤3）中层析的洗脱操作采用洗脱缓冲液为50mM Tris-HCl+0.5M NaCl，pH9.0。
 优选的，步骤5）中层析的洗脱操作采用洗脱缓冲液为20mM PB+0.15M NaCl，pH6.0。
 优选的，步骤2）中层析采用的阳离子树脂为SP Sepharose FF。
 优选的，步骤3）中层析采用的阴离子树脂为Q Sepharose FF。
 优选的，步骤5）中层析采用的凝胶过滤填料为Superdex 75 PG。
 优选的，所述的方法，进一步包括对纯化获得的巴曲酶蛋白进行蛋白浓度测定的操作。
 本发明在对巴曲酶纯化的过程中，通过对培养基上清的pH值以及电导率的调整优化，使目的蛋白能很好地结合在层析填料上，而大部分杂蛋白无法和填料结合而流穿，提高收集液纯度，既能抑制发酵上清液中的水解酶活性，又能保证目的蛋白不流穿，保证高收率。洗脱模式由简单的盐梯度改为盐梯度和pH梯度复合洗脱方式，极大提高了蛋白的纯度。进一步将阳离子层析、阴离子层析以及单向切向流过滤浓缩和凝胶过滤层析配套使用，纯化获得了高纯度有活性的巴曲酶蛋白。本发明的纯化方法采用连续流纯化工艺，中间体无需调节pH值、电导、稀释、换液等操作，降低工艺时间，提高酶活性，减少储罐、人员、厂房面积等优点。体外以及体内的验证表明，采用本发明方法纯化的巴曲酶具有与市售的天然巴曲酶类似的凝血效果，具有广泛的应用前景。
 附图说明
 图1. PCR对转化的酵母菌落的筛选鉴定图。
 图2.不同pH值条件下的培养液纯化的巴曲酶的凝血效果图。
 图3.不同电导率的培养液纯化的巴曲酶的凝血效果图。
 图4. 增加阴离子树脂以及单向切向流过滤对于纯化的巴曲酶的凝血效果图。
 图5.纯化的巴曲酶蛋白的SDS电泳检测图。
 图6. 完整蛋白相对分子质量分析图谱。
 图7. 还原脱N糖后相对分子质量分析图谱。
 图8. 纯化的重组巴曲酶对小鼠出血凝血效果图。
 具体实施方式
 以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
 实施例1含有巴曲酶基因的酵母表达载体的构建及转化
1）巴曲酶编码基因的克隆
采用GeneBank 中公开的巴曲酶（J02684.1）的核酸序列，首先人工合成出该基因序列。根据该基因的信息以及基因中的酶切位点信息等，设计扩增巴曲酶编码基因的引物序列，并在引物两端添加XhoI以及 SacII的酶切位点序列。设计的扩增引物序列如下：
正义引物：5’-aactcgaggtcattgga ggtgatgaat -3’；
反义引物：5’-aaccgcggcgggcaagt cgcagttt-3’。
 采用上述扩增引物以合成的巴曲酶基因J02684.1进行PCR扩增。
 PCR扩增反应条件如下：
95℃3 min；95℃ 40s,53℃ 30s,72 ℃ 1min,35个循环；72 ℃ 15min。
 获得的扩增产物4℃保存备用。
 2）酵母表达载体的构建及转化
将步骤1）中的PCR扩增产物纯化回收后采用XhoI以及 SacII进行双酶切，同时对酵母表达载体pPICZαA也采用XhoI以及 SacII进行双酶切。分别对酶切的产物进行回收后，再用T4连接酶进行连接后，将连接产物转化大肠杆菌BL21。鉴定重组菌，提取重组质粒，经双酶切及PCR 扩增目的基因筛选阳性克隆，重组质粒命名为pPICZαA-Batr。
 提取pPICZαA-Batr质粒，用Sac I 线性化后，采用电转化法转化毕赤酵母X-33所有转化子涂布在YPD＋Zeocin（100μg／ml）培养基上‚30℃培养4-5天后挑选长得快且饱满的菌落进行菌落PCR 鉴定，采用上述扩增引物进行PCR鉴定。其中，PCR对酵母菌落的鉴定图参见图1。对于鉴定的阳性菌落克隆进行保存备用。
 实施例2 工程酵母菌X-33/pPICZαA-Batr的发酵表达
1）阳性菌落酶表达量的初步筛选
挑取阳性克隆，首先接种于甘油复合培养基（BMGY）中振荡培养过夜，离心去除培养液，沉淀菌体重悬于BMMY 中并稀释至OD 600为1进行诱导表达，每约12小时取样并补加甲醇至终浓度为1％，72h 后离心收集上清。每次取样经离心保留上清并初步测定凝血酶活性，测定结果参见表1。初步筛选获得凝血酶活性较高的菌落克隆以用于发酵罐发酵。其中，凝血酶活性测定的方法如下：
取人－枸橼酸凝血血浆0.2 ｍｌ，加入96孔板中，置于37℃保温3 min，加入37℃预热的样品溶液0.2 ｍｌ，立即振荡混匀计时，于40 s开始检查血浆凝固情况，记录初凝时间，同时测定３管，误差应＜２０ｓ。若初凝时间＜４０ｓ，则是当稀释公式样品溶液若干倍，记录（６０±２０）ｓ内凝固的供试样品溶液浓度。在上述条件下，能使0.2 ｍｌ人－枸橼酸抗凝血浆在６０ｓ凝固的酶量，定义为一个酶活单位。
 表1：不同阳性克隆工程酵母菌X-33/pPICZαA-Batr诱导表达36小时的初步凝血酶活性测定
从测定结果来看，菌落2中表达的巴曲酶的活性最高，促凝固的时间最短，在后期的发酵以及纯化的操作中采用2号菌落来进行。
 2）酵母菌X-33/pPICZαA-Batr-2的发酵
将保存的2号菌落接种到5mL BMGY酵母培养液的试管中，培养过夜。将试管中的培养液转入含有180mL BMGY培养液的摇瓶中增殖作为种子液，再转
接至有5L BMMY发酵液的生物发酵罐中，按甲醇酵母发酵的常规方法进行中试规模的发酵，收集上清以纯化备用。
     实施例3 酵母菌X-33/pPICZαA-Batr-2发酵液的纯化
3-1)对发酵上清液的初步纯化: 对发酵液的不同pH值进行调整
按照常规的对酵母发酵液蛋白的纯化方法，首先对发酵液的不同pH值进行调整，看纯化的酶的活性。
 ①分别取发酵液上清200mL，调节pH值分别为5.8、6.5以及7.2；
②将上述上清液过阳离子填料进行层析，所述阳离子填料为SP Sepharose FF，先用低盐（NaAc/HAc，0.15M NaCl、pH值6.0）将杂蛋白去除，使用缓冲液（PB）pH调整为8.0，将色素洗脱去除，最后采用缓冲液pH（Tris-HCl）至9.0，目的蛋白被洗脱；
③将蛋白洗脱液采用超滤管（10Kd）进行浓缩备用；
④将浓缩的蛋白液进行凝胶过滤层析，采用的凝胶过滤填料为Superdex 75 PG，层析柱先用平衡缓冲液（20mM PB+0.15M NaCl，pH6.0）冲洗，平衡后将单向切向流过滤收集液进行上样，上样体积≤5%柱体积，收集目的蛋白峰。
 ⑤测定洗脱液的蛋白的浓度，并调整至浓度一致，采用实施例2中的方法对收集的巴曲酶进行酶活性测定。测定结果参见图2。
 从检测结果来看，提高上样样品的pH至5.8左右，使目的蛋白能很好地结合在层析填料上，而大部分杂蛋白无法和填料结合而流穿，提高收集液纯度和收率，从而提高酶活性。
 3-2) 对发酵上清液的初步纯化: 对发酵液的电导率进行调整
对发酵液的pH值进行调整至5.8左右，进一步分别调整电导率，看纯化的酶的活性。
 ①分别取发酵液上清200mL，调节pH值分别为5.8后，分别调整培养基电导率为4mS/cm、 6mS/cm以及8mS/cm；
②将上述上清液过阳离子填料进行层析，所述阳离子填料为SP Sepharose FF，先用低盐（NaAc/HAc，0.15M NaCl、pH值6.0）将杂蛋白去除，使用缓冲液（PB）pH调整为8.0，将色素洗脱去除，最后采用缓冲液pH（Tris-HCl）至9.0，目的蛋白被洗脱；
③将蛋白洗脱液采用超滤管（10Kd）进行浓缩备用；
④将浓缩的蛋白液进行凝胶过滤层析，采用的凝胶过滤填料为Superdex 75 PG，层析柱先用平衡缓冲液（20mM PB+0.15M NaCl，pH6.0）冲洗，平衡后将浓缩液进行上样，上样体积≤5%柱体积，收集目的蛋白峰。
 ⑤测定洗脱液的蛋白的浓度，并调整至浓度一致，采用实施例2中的方法对收集的巴曲酶进行酶活性测定。测定结果参见图3。
 从检测结果来看，调整上样样品的电导率至6左右，既能抑制发酵上清液中的水解酶活性，又能保证目的蛋白不流穿，保证高收率，从而提高酶活性。
 3-3）对发酵上清液的初步纯化: 增加阴离子交换层析步骤纯化
阴离子容易结合培养基中的色素，进一步利用盐梯度线性洗脱模式，将目的蛋白纯度提高至95%左右。
 ①分别取发酵液上清200mL，调节pH值分别为5.8后，调整培养基电导率为6mS/cm；
②将上述上清液过阳离子填料进行层析，所述阳离子填料为SP Sepharose FF，先用低盐（NaAc/HAc，0.15M NaCl，pH6.0）将杂蛋白去除，使用缓冲液（PB）pH调整为8.0，将色素洗脱去除，最后采用缓冲液pH（Tris-HCl）至9.0，目的蛋白被洗脱；
③将阳离子洗脱液上样阴离子树脂，所述阴离子树脂为Q Sepharose FF，结束后用平衡缓冲液冲洗层析柱，再用洗脱缓冲液（50mM Tris-HCl+0.5M NaCl，pH9.0）进行0~100%洗脱15CV，收集目的蛋白洗脱峰。
 在层析结束后对层析填料进行清洁与消毒：经过洗脱后的阴离子层析填料仍带有明显的色素，用0.5M NaOH和1M NaCl也无法将色素完全去除，使用次数多了以后色素也将累积，会导致收集液颜色变深、填料收率降低、柱效变差、不容易装填等问题。所以先用0.5M H 3PO 4将填料上残留的色素洗脱，保留时间为8~10分钟，再用0.5M NaOH+1M NaCl对填料进行再生与清洁消毒，使填料回到初始的乳白色。
 工艺描述：
用注射用水及10%冰醋酸调节离心上清液的pH至5.8~6.0，电导率至5~6mS/cm。
 阳离子层析柱先用平衡缓冲液（20mM NaAc/HAc，pH6.0）冲洗平衡，然后将调节好pH和电导率的样品进行上样。上样结束后用平衡缓冲液冲洗层析柱，开始洗脱流程：先用淋洗1缓冲液（20mM NaAc/HAc +0.15M NaCl，pH6.0）洗脱杂蛋白，再用淋洗2缓冲液（20mM PB，pH8.0）洗脱色素等杂质，最后用洗脱缓冲液（50mM Tris-HCl，pH9.0）进行洗脱，收集洗脱峰。
 阴离子层析柱先用平衡缓冲液（50mM Tris-HCl，pH9.0）冲洗，将阳离子交换层析收集液进行上样。上样结束后用平衡缓冲液冲洗层析柱，再用洗脱缓冲液（50mM Tris-HCl+0.5M NaCl，pH9.0）进行0~100%洗脱15CV，收集目的蛋白洗脱峰。
 层析填料先用平衡缓冲液（20mM NaAc/HAc，pH6.0）将pH值平衡至6.0，然后先用0.5M H 3PO 4清洁层析柱，保留时间为8~10分钟，再用上述平衡缓冲液平衡后，用0.5M NaOH+1M NaCl对填料进行再生与消毒，保留时间为30分钟。
 ④将蛋白洗脱液采用超滤管（10Kd）进行浓缩备用；
⑤将浓缩的蛋白液进行凝胶过滤层析，采用的凝胶过滤填料为Superdex 75 PG，层析柱先用平衡缓冲液（20mM PB+0.15M NaCl，pH6.0）冲洗，平衡后将浓缩液进行上样，上样体积≤5%柱体积，收集目的蛋白峰。
 ⑥测定洗脱液的蛋白的浓度，并调整至浓度一致，采用实施例2中的方法对收集的巴曲酶进行酶活性测定。测定结果参见图4。
 3-4）对发酵上清液的初步纯化: 增加单项切向流过滤步骤
单项切向流过滤，操作简便适合放大，可以快速浓缩目的蛋白，可根据需求控制上样体积以便后续操作，并减少凝胶过滤层析的循环数和填料体积。
 ①分别取发酵液上清200mL，调节pH值分别为5.8后，调整培养基电导率为6mS/cm；
②将上述上清液过阳离子填料进行层析，所述阳离子填料为SP Sepharose FF，先用低盐（NaAc/HAc，0.15M NaCl，pH6.0）将杂蛋白去除，使用缓冲液（PB）pH调整为8.0，将色素洗脱去除，最后采用缓冲液pH（Tris-HCl）至9.0，目的蛋白被洗脱；
③将阳离子洗脱液上样阴离子树脂，所述阴离子树脂为Q Sepharose FF，结束后用平衡缓冲液冲洗层析柱，再用洗脱缓冲液（50mM Tris-HCl+0.5M NaCl，pH9.0）进行0~100%洗脱15CV，收集目的蛋白洗脱峰。
 阴离子层析填料先用平衡缓冲液（20mM NaAc/HAc，pH6.0）将pH值平衡至6.0，然后先用0.5M H 3PO 4清洁层析柱，保留时间为8~10分钟，再用上述平衡缓冲液平衡后，用0.5M NaOH+1M NaCl对填料进行再生与消毒，保留时间为30分钟。
 ④单向切向流过滤，膜包（0.11m 2,10Kd）先用平衡缓冲液（50mM Tris-HCl，pH9.0）冲洗，每级膜包之间采用串联方式，同级膜包采用并联方式，平衡后将阴离子交换层析收集液进行上样，透过端为废液，收集最终的回流端样品；膜包清洗，用注射用水和0.5M NaOH清洗膜包。
 ⑤将浓缩的蛋白液进行凝胶过滤层析，采用的凝胶过滤填料为Superdex 75 PG，层析柱先用平衡缓冲液（20mM PB+0.15M NaCl，pH6.0）冲洗，平衡后将单向切向流过滤收集液进行上样，上样体积≤5%柱体积，收集目的蛋白峰。
 ⑥测定洗脱液的蛋白的浓度，并调整至浓度一致，采用实施例2中的方法对收集的巴曲酶进行酶活性测定。测定结果参见图4。
 从图4的检测结果来看，在纯化的操作过程中进一步增加阴离子树脂吸附洗脱以及单向切向流过滤能够进一步增加纯化获得的巴曲酶的活性，分析原因可能是纯化的步骤降低了杂蛋白的含量，进一步提高了巴曲酶的浓度，并在一定程度上减轻了杂蛋白对巴曲酶的干扰作用。
 实施例4 纯化的巴曲酶蛋白的SDS电泳检测
对实施例3中3-4的方法纯化制备的巴曲酶进行电泳检测，主要操作如下。
 取纯化的样品,在15%浓度的聚丙稀酰胺凝胶中进行电泳检测。按照蛋白样品与上样缓冲液4: 1的比例混合,浴煮沸5min,使蛋白变性,室温冷却后,使用微孔上样器上样。电泳电压为:浓缩胶80-100V,分离胶100-120V。电泳检测结果参见图5。其中条带1是阴性对照，条带2是实施例3中3-4的方法纯化制备的巴曲酶。可以看出，该纯化方法制备的蛋白无其他杂蛋白，纯度较高。
 实施例5 纯化的巴曲酶蛋白的相对分子质量检测
巴曲酶理论计算分子量为25.6KD，但是我们所构建甲醇酵母工程菌分泌表达出的巴曲酶蛋白还原电泳测得分子量约33KD，这同理论计算值不相符。出现这种情况的原因是巴曲酶分子中有两个N-糖基化位点，根据对大部分糖基化蛋白研究的一般规律，每个糖基化可使蛋白的分子量增加约0.3~0.5KD，所以这种分子量的差别应是糖基化修饰的结果，具体可见下述质谱的检测结果。
 1)脱N糖蛋白处理
纯化的样品各取40ug，加入DTT，55℃还原60 min，降至室温加入1 uL PNGase F混匀，37℃孵育过夜。
 2)完整蛋白处理
纯化的样品40ug，加入6M盐酸胍进行变性处理，加入DTT至终浓度50mM，100℃还原变性10 min，稀释后进行液质检测。
 3)高效液相色谱参数
采用UPLC（Acquity UPLC I-Class，Waters）液相系统进行分离。流动相A为0.1% FA水溶液，B为0.1% FA乙腈溶液。样品由自动进样器上样1µL，再经色谱柱（BioResolve RP Column，450Å，2.7µm，2.1X50mm，Waters）分离，流速为0.3ml/min，紫外检测波长280nm，柱温80℃，分析时长10min。分离梯度为：0分钟-1分钟，B液线性梯度维持15%，1分钟-7分钟，B液线性梯度从15%到60%；7分钟-7.5分钟，B液线性梯度从60%到90%；7.5分钟-8分钟，B液维持在90%；8分钟-10分钟，B液维持在15%。
 4)质谱参数
采用XevoG2-XS Q-Tof 质谱仪（Waters）进行质谱分析。分析时长：10min，检测方式：正离子，母离子扫描范围：500-4000m/z。
 5)数据分析
原始数据由UNIFI(1.8.2，Waters)软件处理，完整蛋白相对分子质量分析图谱见图6，还原脱N糖后相对分子质量分析图谱见图7。
 纯化的样品完整蛋白的相对分子质量为30195Da，其中，N糖基化修饰主要为不同数据的高甘露糖（M10-M15等）且部分甘露糖上发生磷酸化修饰（Phosphate）；还原脱 N糖后的相对分子质量均为25505 Da。
 实施例 6 纯化的重组巴曲酶与市售的天然巴曲酶的酶活力的测定
取市售巴曲酶，溶于缓冲液（20mM PB+0.15M NaCl，pH6.0），并常规方法测定蛋白浓度与实施例3中3-4方法制备的纯化重组巴曲酶一致。
 酶活力的测定方法参考实施例2中的方法：凝血酶活性测定的方法如下：
取人－枸橼酸凝血质控血浆0.2 ｍｌ，加入96孔板中，置于37℃保温3 min，加入37℃预热的样品溶液0.2 ｍｌ，立即振荡混匀计时，于40 s开始检查血浆凝固情况，记录初凝时间，同时测定３管，误差应＜２０ｓ。若初凝时间＜４０ｓ，则是当稀释公式样品溶液若干倍，记录（６０±２０）ｓ内凝固的供试样品溶液浓度。在上述条件下，能使0.2 ｍｌ人－枸橼酸抗凝血浆在６０ｓ凝固的酶量，定义为一个酶活单位。测定结果参见表2。
 表2：重组巴曲酶的酶活力测定
实施例 7纯化的重组巴曲酶与市售巴曲酶对小鼠出血的影响
对于重组制备的巴曲酶的体内活性鉴定采用小鼠断尾后的凝血时间来测定。具体操作如下：
1）取健康小鼠9只，随机平均分成三组，每组3只；
2）于小鼠静脉注射相应的缓冲液，其中1组注射PB缓冲液作为阴性对照，其中1组注射市售巴曲酶（溶于缓冲液（20mM PB+0.15M NaCl，pH6.0）），最后一组注射实施例3中3-4方法制备的纯化重组巴曲酶；
3）注射后半小时后，用剪刀在距小鼠尾尖0.5cm处断尾，血液自行流出后立即计时。计算各组小鼠的平均凝血时间。测定结果参见图8。
 从测定结果来看，采用本发明方法制备的重组纯化巴曲酶已经达到或接近天然巴曲酶的凝血效果，在体内也具有一定的凝血效果。
 本发明通过上述实施例来说明本发明的工艺方法，但本发明并不局限于上述工艺步骤，即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1.  一种重组巴曲酶的表达纯化方法，其特征在于，包括如下步骤：
    1）取重组巴曲酶酵母发酵液上清，调节pH值和电导率；
    2）过阳离子树脂进行层析；
    3）过阴离子树脂层析；
    4）单向切向流过滤浓缩；
    5）将浓缩的蛋白液进行凝胶过滤层析；
    6）洗脱收集目的蛋白峰，获得纯化的巴曲酶。
  2.  根据权利要求1所述的纯化方法，其特征在于，步骤1）中调节pH值为5.8，调节电导率为6mS/cm。
  3.  根据权利要求1所述的纯化方法，其特征在于，步骤2）中层析的洗脱操作为先用低盐缓冲液将杂蛋白去除，使用缓冲液pH调整为8.0的PB，将色素洗脱去除，最后采用缓冲液pH为9.0的Tris-HCl洗脱。
  4.  根据权利要求3所述的纯化方法，其特征在于，所述低盐缓冲液为0.15M  NaCl缓冲液，NaAc/HAc调节pH值6.0。
  5.  根据权利要求1所述的纯化方法，其特征在于，步骤3）中层析的洗脱操作采用洗脱缓冲液为50mM Tris-HCl+0.5M NaCl，pH9.0。
  6.  根据权利要求1所述的纯化方法，其特征在于，步骤5）中层析的洗脱操作采用洗脱缓冲液为20mM PB+0.15M NaCl，pH6.0。
  7.  根据权利要求1所述的纯化方法，其特征在于，步骤2）中层析采用的阳离子树脂为SP Sepharose FF。
  8.  根据权利要求1所述的纯化方法，其特征在于，步骤3）中层析采用的阴离子树脂为Q Sepharose FF。
  9.  根据权利要求1所述的纯化方法，其特征在于，步骤5）中层析采用的凝胶过滤填料为Superdex 75 PG。
  10.  根据权利要求1-9任一权利要求所述的方法，进一步包括对纯化获得的重组巴曲酶蛋白进行蛋白浓度测定的操作。
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