CN1212013A - 从蛇毒中纯化凝血酶样蛋白酶 - Google Patents
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Abstract
本文描述了一种用于蛇毒中纯化凝血酶样蛋白酶的方法,该方法包括通过三步层析从混合物中分离出所说的蛋白酶。
Description
本发明涉及一种用于从蛇毒中纯化凝血酶样蛋白酶的方法。
上述蛋白酶的例子有batroxobin,crotalase，并且更具体地讲为das Ancrod。后者为一种从Agkistrodon rhodostoma蛇的毒液中分离出来的抗凝剂(Merck索引1989,No.664)。已经描述了多种用于从蛇毒中制备das Ancrod的方法(英国专利1,094,301、英国专利1,177,506、英国专利1,293,793、美国专利3,743,722、美国专利3,879,369、德国公开说明书2,428,955和德国公开说明书2,734,427)。上述方法基本上以层析步骤为基础并且生成了不同收率和纯度的das Ancrod。
迄今为止，从蛇毒中制备高纯度的das Ancrod仍未获得成功。人们总是分离出以das Ancrod作为主要组分的酶的混合物，而其中取决于制备过程，所说的产物总是或多或少地受到异种蛋白的污染。
现在已经发现了一种从蛇毒中制备高纯度的凝血酶样蛋白酶的方法。
本发明涉及一种用于从蛇毒中纯化凝血酶样蛋白酶的方法，该方法包括：
a．通过亲和层析或者在碱性离子交换剂上进行层析使蛋白酶的粗产物进行预纯化，
b．使含有如上获得的凝血酶样蛋白酶的组分在弱阳离子交换剂上进行层析或者在碱性范围内通过在玻璃上吸附来分离上述组分，以及
c．使来自步骤2的主要组分进行凝胶层析或者在酸性范围内通过在玻璃上进行层析来纯化该组分，
但是其中步骤2和3中的至少一步包括通过在玻璃上吸附或层析而进行的分离。
本发明还涉及来自蛇毒的纯度为95至100％的凝血酶样蛋白酶。
值得推荐的是步骤b通过在玻璃上吸附来加以纯化，而步骤c则借助于凝胶层析加以纯化。
在本发明的一个特别优选的实例中，在步骤b和步骤c中的纯化都是通过在玻璃上进行吸附或层析来实现的。
精胺-、精氨酸-或肝素-Sepharose特别适于通过亲和层析进行的预纯化。
适于预纯化的碱性离子交换剂具体有DEAE纤维素和DEAE-Sepharose。
可以提及的用于离子交换层析的缓冲液具体为tris-磷酸盐和磷酸钠缓冲液。
离子交换层析在pH5-9、优选6-8.5时进行。
在预纯化的过程中，大约有70-80％的异种蛋白和其它的成分从粗酶中被除去。
如果纯化的第二步采用阳离子交换剂来实现的话，那么随之应考虑以下弱酸性交换剂：CM-SEPHAROSE,pH5-9，和AMBERLITE CG50,pH5-9。
在玻璃上进行层析是指在pH为7.5-9.0、其中优选8.0-8.5时将das Ancrod和性质近似的凝血酶样酶以及强碱性的蛋白酶结合到玻璃基质上。用平衡缓冲液(优选tris-磷酸盐或磷酸钠缓冲液)从柱中洗下大约60％的呈未结合形式的酸性极强的异种蛋白。通过加入氯化钠将缓冲液的离子强度增加到0.3-1.0M，从而从玻璃中分级洗脱出纯度大于90％的das Ancrod。
在第二步纯化步骤中，把所说的酶浓缩到大约90％。
对于作为纯化步骤c的凝胶层析而言，合适的凝胶具体为SEPHACRYLS-100HR、SUPERDEX、SEPHADEX、ULTROGEL和SUPEROSE。
如果上述纯化步骤c选择了在玻璃上进行层析的话，那么在4-6的酸性pH范围内，碱性的异种蛋白将从das Ancrod溶液中被吸附在玻璃的表面上，同时可以用平衡缓冲液从柱中直接洗脱出纯度远大于95％的das Ancrod。通过加入如氯化钠等的盐可以调节缓冲液所需的离子强度。
这种新的方法特别适于制备纯的das Ancrod，而根据该纯化方法所得的das Ancrod的纯度显然高于95％。
实施例1a．预纯化
将3g干燥的马来腹蛇(der malaiischen Grubenotter)毒液(dasGift)溶解在pH为8.5的50ml Tris(羟甲基)氨基甲烷(TRIS)-磷酸盐缓冲液中，离心除去蛇毒中不溶的细胞部分，并且将澄清的黄色溶液加入到直径为1.6cm、用DEAE-SEPHAROSE-FF(Pharmacia)装填至高度为30cm的层析柱中。把毒液中的凝血酶样酶以及具有酸性的蛋白质结合到基质上，在150-200ml/h的流速下进行层析。通过在室温下用大约300ml的平衡缓冲液(10mM TRIS-磷酸盐缓冲液，pH 8.5)洗涤该柱直至洗脱物的A280-值下降至小于0.5以及再用pH为6.0的400ml 35mM的TRIS-磷酸盐缓冲液洗涤至洗脱物的A280-值＜0.4时，便洗脱出了大约70-80％的异种蛋白(以在280nm下起始溶液的光密度为基准)。用pH为6.0的150-200ml的150mM TRIS-磷酸盐缓冲液洗脱出含有das Ancrod的主要部分，其收率为85％。b．主要的纯化
通过在具有截断阈为10000道尔顿的膜上进行超滤来把含有dasAncrod的洗脱物浓缩至20ml，并用pH为8.0的100mM TRIS-磷酸盐缓冲液重新缓冲。将上述溶液加入到直径为1.6cm并用BIORAN-CPG玻璃(Schott，孔径：25-35nm，颗粒尺寸：30-60μm)装填到高度为15cm的柱中。通过在室温下用pH为8.0的300ml 100mM的TRIS-磷酸盐缓冲液并在每小时250ml的流速下洗涤柱子，从柱中洗脱出大约60％的异种蛋白(以在280nm下所加物质的光密度为基准)。
为了进行洗脱，使用了以100mM的TRIS-磷酸盐缓冲至pH为8.0的0.5M氯化钠溶液。以大约10ml的部分自动收集洗脱物。在一个具有随后的拖尾区的峰处洗脱出凝血酶样酶。为了获得其中最多含5％的凝血酶样次要组分的主要组分(对应于das Ancrod)，在从主峰向脱尾范围的单个部分过渡的过程中通过反相HPLC研究了其纤维蛋白原酶的比活性及其组成。仅把比活性大于1700U/OD280nm并在HPLC中含有少于10％的次要组分的部分与主峰合并(约80ml)。从BIORAN柱中获得了纯度为96％的主要组分，其收率为72％。c．精制的纯化
通过在YM 10膜(Amicon)上超滤来把含有主要组分das Ancrod的洗脱物浓缩至2ml，并且把得到的浓缩物加入直径为1.6cm并用SEPHLARYL S-100 HR装填到高度为85cm的柱中。该柱事先已用pH为6.9的100mM氯化钠和100mM磷酸钠的缓冲液平衡。通过上述的凝胶层析从das Ancrod中分离出残余的蛋白酶和TRIS。该步骤的收率约为90％。实施例2a．预纯化
将2.1g干燥的马来腹蛇的毒液溶解在pH为8.5的50ml 35mM的TRIS-磷酸盐缓冲液中，离心除去蛇毒中不溶的细胞部分，并且将澄清的黄色溶液加入到直径为1.6cm、用DEAE-SEPHAROSE-FF(Pharmacia)装填至高度为30cm并用上述缓冲液加以平衡的层析柱中。通过在室温下用600ml的平衡缓冲液洗涤柱直至洗脱物的A280值＜0.2时，便洗脱出了大约70％的异种蛋白(以在280nm下起始溶液的光密度为基准)。用pH为6.0的200-250ml 150mM的TRIS-磷酸盐缓冲液洗脱主要的部分，其收率为90-100％。b．主要的纯化
与实施例1一样，将上述洗脱物浓缩至20ml，并用pH为8.5的50mM磷酸钠缓冲液重新缓冲。将该溶液加样到BIORAN-CPG玻璃柱中(直径：1.6cm，高度：15cm，孔径：25-35nm，颗粒尺寸：30-60μm)。通过在室温下用pH为8.5的300ml 50mM的磷酸钠缓冲液并在每小时250ml的流速下洗涤柱子，便从柱中洗脱出大约60％的异种蛋白(以在280nm下进行的光密度为基准)。
为了洗脱凝血酶样酶，使用了已用50mM磷酸钠缓冲至pH为8.0的1M氯化钠溶液。采用150ml的缓冲液洗脱出大约80％的所加酶的组分。c．精制的纯化
如上所述将洗脱物浓缩至20ml，并用pH为5.0的50mM磷酸钠缓冲液重新缓冲。将该溶液加入直径也为1.6cm、用BIORAN-CPG玻璃装填至高度为15cm、但孔径为90-110nm、颗粒尺寸为30-60μm的柱中。当pH为5.0时，仅有碱性蛋白质和次要组分结合到BIORAN玻璃上，而纯度约为100％的主要组分das Ancrod则从柱中用平衡缓冲液洗脱出来，其收率约为80-90％(以所加组分为基准)。实施例3a,b．预纯化以及主要的纯化
以类似于实施例2的方法将2.1g干燥的马来腹蛇的毒液在DEAE-SEPHAROSE上进行预分离，以类似于实施例1的方法将洗脱物浓缩至20ml，并用pH为6.2的40mM TRIS-磷酸盐缓冲液重新进行缓冲。将该溶液加入直径为1.6cm、用CM-SEPHAROSE-FF(Pharmacia)装填至高度为20cm并且用上述缓冲液平衡的层析柱中。通过在室温下用300m1的平衡缓冲液并在250ml/h的流速下洗涤柱子，从柱中洗脱出大约60％的异种蛋白(以在280nm下进行的光密度为基准)。
为了洗脱出凝血酶样酶，与实施例2相仿，使用了以50mM磷酸钠缓冲至pH为8.0的1M氯化钠溶液。使用150ml的该缓冲液便洗脱出大约80％的所加凝血酶样酶的组分。c．精制的纯化
与实施例2相仿，使用pH为5.0的50mM磷酸盐缓冲液在BIORAN-CPG玻璃(孔径约为100nm；颗粒尺寸为30-60μm)上进行das Ancrod的精制的纯化。从柱中洗脱出纯度高于95％的das Ancrod，其收率约为85％(以所加组分为基准)。
Claims (4)
1．一种用于从蛇毒中纯化凝血酶样蛋白酶的方法，其特征在于：
a．通过亲和层析或在碱性离子交换剂上进行层析使蛋白酶的粗产物进行预纯化，
b．使含有如上获得的凝血酶样蛋白酶的组分在弱阳离子交换剂上进行层析或者在碱性范围内通过在玻璃上吸附来分离上述组分，以及
c．使来自步骤2的主要组分进行凝胶层析或者在酸性范围内通过在玻璃上进行层析来纯化该组分，
但是其中步骤2和3中的至少一步包括通过在玻璃上层析而进行的分离。
2．一种如权利要求1所要求保护的方法，其特征在于，在步骤b中通过吸附的纯化是在玻璃上进行的，步骤c是通过凝胶层析来实现的。
3．一种如权利要求1所要求保护的方法，其特征在于，在步骤b和c中通过层析的纯化是在玻璃上进行的。
4．纯度高于95％的，天然来源的das Ancrod。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19607210A DE19607210A1 (de) | 1996-02-26 | 1996-02-26 | Verfahren zur Reinigung von thrombinähnlichen Proteasen aus Schlangengiften |
| DE19607210.7 | 1996-02-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1212013A true CN1212013A (zh) | 1999-03-24 |
| CN1188517C CN1188517C (zh) | 2005-02-09 |
Family
ID=7786490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB971925852A Expired - Lifetime CN1188517C (zh) | 1996-02-26 | 1997-02-18 | 从蛇毒中纯化凝血酶样蛋白酶 |
Country Status (32)
| Country | Link |
|---|---|
| US (1) | US6200791B1 (zh) |
| EP (1) | EP0883684B1 (zh) |
| JP (1) | JP3817269B2 (zh) |
| KR (1) | KR100493835B1 (zh) |
| CN (1) | CN1188517C (zh) |
| AR (1) | AR005995A1 (zh) |
| AT (1) | ATE283350T1 (zh) |
| AU (1) | AU711650B2 (zh) |
| BG (1) | BG64875B1 (zh) |
| BR (1) | BR9707738A (zh) |
| CO (1) | CO4771158A1 (zh) |
| CZ (1) | CZ292482B6 (zh) |
| DE (2) | DE19607210A1 (zh) |
| ES (1) | ES2233997T3 (zh) |
| HK (1) | HK1017380A1 (zh) |
| HR (1) | HRP970108B1 (zh) |
| HU (1) | HU225489B1 (zh) |
| ID (1) | ID16046A (zh) |
| IL (1) | IL125395A (zh) |
| MX (1) | MX9806304A (zh) |
| MY (1) | MY130458A (zh) |
| NO (1) | NO319561B1 (zh) |
| NZ (1) | NZ331120A (zh) |
| PL (1) | PL186211B1 (zh) |
| PT (1) | PT883684E (zh) |
| RU (1) | RU2186849C2 (zh) |
| SK (1) | SK284874B6 (zh) |
| TR (1) | TR199801668T2 (zh) |
| TW (1) | TW505696B (zh) |
| UA (1) | UA46831C2 (zh) |
| WO (1) | WO1997032015A1 (zh) |
| ZA (1) | ZA971597B (zh) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1102173C (zh) * | 1999-03-26 | 2003-02-26 | 福建医科大学 | 蕲蛇酶及其生产工艺 |
| CN103160486A (zh) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | 一种猪凝血酶的制备方法 |
| CN108559740A (zh) * | 2018-05-12 | 2018-09-21 | 北京博康宁生物医药科技有限公司 | 重组安克洛酶及工业规模制备方法和治疗急性脑梗的应用 |
| CN109652398A (zh) * | 2018-12-29 | 2019-04-19 | 上海太阳生物技术有限公司 | 凝血因子ⅹ激活剂rvv-ⅹ的制备方法及制得的rvv-ⅹ |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219431A1 (en) * | 2002-05-24 | 2003-11-27 | Empire Pharmaceuticals, Inc. | Treatment of neuronal and neurological damage associated with spinal cord injury |
| US9375499B2 (en) * | 2006-07-14 | 2016-06-28 | Wisconsin Alumni Research Foundation | Adsorptive membranes for trapping viruses |
| CN109929020A (zh) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | 一种眼镜蛇毒的纯化方法及其产品 |
| CN110016471B (zh) * | 2019-04-10 | 2020-10-02 | 北京博康宁生物医药科技有限公司 | 重组安克洛酶及工业规模制备和纯化方法及其组合物 |
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| GB1094301A (en) * | 1964-02-21 | 1967-12-06 | Nat Res Dev | Improvements relating to anticoagulants |
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| GB1293793A (en) | 1969-04-28 | 1972-10-25 | Chrysler United Kingdom Ltd Fo | Improvements in or relating to valves |
| US3743722A (en) | 1971-07-14 | 1973-07-03 | Abbott Lab | Anti-coagulant isolation |
| US3879369A (en) | 1972-05-12 | 1975-04-22 | Abbott Lab | Anti-coagulant isolation from malayan pit viper using affinity chromatography |
| GB1472574A (en) | 1973-09-03 | 1977-05-04 | Biochimici Fargal Pharmasint S | Process for the extraction of components having anticoagulant activity -in vivo- from snake venoms and related products |
| NL7605805A (nl) * | 1976-05-28 | 1977-11-30 | Stichting Rega V Z W | Werkwijze voor het zuiveren van interferon. |
| CH626917A5 (zh) | 1976-08-17 | 1981-12-15 | Pentapharm Ag | |
| JPS5569595A (en) * | 1978-11-20 | 1980-05-26 | Suguru Abe | Novel substance having fibrinolytic activity, remedy for thrombotic embolic disease comprising it, and its preparation |
| EP0328256A1 (en) * | 1988-01-21 | 1989-08-16 | Owens-Corning Fiberglas Corporation | Glass fibers coated with agarose for use as column packing or chromatographic media for bioseparations |
| US5712117A (en) * | 1995-02-08 | 1998-01-27 | Zymogenetics, Inc. | Cytoplasmic antiproteinase-2 and coding sequences |
-
1996
- 1996-02-26 DE DE19607210A patent/DE19607210A1/de not_active Withdrawn
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1997
- 1997-02-18 DE DE59712093T patent/DE59712093D1/de not_active Expired - Lifetime
- 1997-02-18 UA UA98094992A patent/UA46831C2/uk unknown
- 1997-02-18 HU HU9900505A patent/HU225489B1/hu not_active IP Right Cessation
- 1997-02-18 PT PT97903312T patent/PT883684E/pt unknown
- 1997-02-18 WO PCT/EP1997/000755 patent/WO1997032015A1/de active IP Right Grant
- 1997-02-18 ES ES97903312T patent/ES2233997T3/es not_active Expired - Lifetime
- 1997-02-18 US US09/125,374 patent/US6200791B1/en not_active Expired - Lifetime
- 1997-02-18 NZ NZ331120A patent/NZ331120A/xx not_active IP Right Cessation
- 1997-02-18 IL IL12539597A patent/IL125395A/en not_active IP Right Cessation
- 1997-02-18 JP JP53055397A patent/JP3817269B2/ja not_active Expired - Lifetime
- 1997-02-18 CZ CZ19982697A patent/CZ292482B6/cs not_active IP Right Cessation
- 1997-02-18 PL PL97328568A patent/PL186211B1/pl not_active IP Right Cessation
- 1997-02-18 CN CNB971925852A patent/CN1188517C/zh not_active Expired - Lifetime
- 1997-02-18 EP EP97903312A patent/EP0883684B1/de not_active Expired - Lifetime
- 1997-02-18 SK SK1132-98A patent/SK284874B6/sk not_active IP Right Cessation
- 1997-02-18 TR TR1998/01668T patent/TR199801668T2/xx unknown
- 1997-02-18 MX MX9806304A patent/MX9806304A/es unknown
- 1997-02-18 AU AU17913/97A patent/AU711650B2/en not_active Ceased
- 1997-02-18 RU RU98117870/13A patent/RU2186849C2/ru active
- 1997-02-18 AT AT97903312T patent/ATE283350T1/de active
- 1997-02-18 BR BR9707738A patent/BR9707738A/pt not_active Application Discontinuation
- 1997-02-18 KR KR10-1998-0706648A patent/KR100493835B1/ko not_active IP Right Cessation
- 1997-02-21 TW TW086102111A patent/TW505696B/zh not_active IP Right Cessation
- 1997-02-24 MY MYPI97000700A patent/MY130458A/en unknown
- 1997-02-24 ID IDP970561A patent/ID16046A/id unknown
- 1997-02-25 AR ARP970100755A patent/AR005995A1/es active IP Right Grant
- 1997-02-25 ZA ZA971597A patent/ZA971597B/xx unknown
- 1997-02-25 CO CO97010013A patent/CO4771158A1/es unknown
- 1997-02-26 HR HR970108A patent/HRP970108B1/xx not_active IP Right Cessation
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1998
- 1998-07-31 BG BG102663A patent/BG64875B1/bg unknown
- 1998-08-25 NO NO19983893A patent/NO319561B1/no not_active IP Right Cessation
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1102173C (zh) * | 1999-03-26 | 2003-02-26 | 福建医科大学 | 蕲蛇酶及其生产工艺 |
| CN103160486A (zh) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | 一种猪凝血酶的制备方法 |
| CN108559740A (zh) * | 2018-05-12 | 2018-09-21 | 北京博康宁生物医药科技有限公司 | 重组安克洛酶及工业规模制备方法和治疗急性脑梗的应用 |
| CN108559740B (zh) * | 2018-05-12 | 2021-05-18 | 吉林天衡康泰生物技术有限公司 | 重组安克洛酶及工业规模制备方法和治疗急性脑梗的应用 |
| CN109652398A (zh) * | 2018-12-29 | 2019-04-19 | 上海太阳生物技术有限公司 | 凝血因子ⅹ激活剂rvv-ⅹ的制备方法及制得的rvv-ⅹ |
| CN109652398B (zh) * | 2018-12-29 | 2021-07-20 | 上海太阳生物技术有限公司 | 凝血因子ⅹ激活剂rvv-ⅹ的制备方法及制得的rvv-ⅹ |
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