CN110387365A - 基因工程组织型纤溶酶原激活剂重组异构体（nt-PA）包涵体的复性方法 - Google Patents
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Abstract
本发明提供了一种大肠杆菌表达组织型纤溶酶原激活剂重组异构体（nt‑PA）形成的包涵体正确折叠复性实现产业化的新型工艺方法。该方法是通过开发的独特的变性及复性体系使nt‑PA包涵体中大量错配的二硫键重新打开并形成正确的二硫键，从而提高复性率，最终增加nt‑PA的产率，降低成本，可应用于工业化生产。
Description
技术领域
本发明属生物医药领域，具体涉及一种大肠杆菌表达组织型纤溶酶原激活剂重组异构体(nt-PA)形成的包涵体正确折叠复性实现产业化的的新型工艺方法。
背景技术
组织纤溶酶原激活剂t-PA能高效特异地溶解血栓而不易出现系统性纤溶状态，但t-PA进入血浆后易与纤溶酶原激活剂抑制物(PAI) 形成复合物而迅速失去活性。同时t-PA的血浆半衰期短，治疗所需剂量高，颅内出血的发生率比其他溶栓药物高，重组t-PA的价格也较昂贵。组织型纤溶酶原激活剂(t-PA)的39KD重组异构体(简称 nt-PA)是t-Pa的一种分子改造衍生物。它包含有野生型t-PA的Kringle 2结构域和蛋白酶结构域，不含糖基化位点。nt-PA具有t-PA的溶血栓活性，但半衰期延长，并且出血副作用小，比t-PA具有使用方便、安全、经济的优势，是取代t-PA的理想溶血栓药物。
因为nt-PA不含糖基化位点，可以用大肠杆菌系统进行表达。但由于nt-PA含有十对二硫键，用大肠杆菌系统表达的nt-PA往往互相交联在一起，形成不溶性的聚积物，形成包涵体。存在于包涵体内的重组蛋白通常无生物活性，这是由于大肠杆菌系统缺少二硫键异构酶，因此表达的重组蛋白分子内及分子间二硫键的错配，而产生无活性的蛋白质。
发明内容
为了使大肠杆菌系统表达的nt-PA包涵体中错配的二硫键实现正确折叠，生产出有活性的溶栓药物，本发明采用独特的变性和复性体系，使错配的二硫键打开重新形成正确的二硫键，从而提高复性率，降低异构体比例，简化后续纯化步骤，最终提高nt-PA的产率，并可应用于工业化生产。
本发明是通过以下技术方案实现的：
用工程菌表达nt-PA→菌体破碎→包涵体分离→变性变性溶解→在复性体系中按一定比例加入重组人PDI以催化nt-PA的复性→脱盐→亲和层析→阳离子交换层析，经上述步骤，即可制备得到纯度大于 95％，比活性达到6×105IU/mg的nt-PA。
具体实施方式
nt-PA变性体系如下：3-8M盐酸胍，25-100mM Tris，pH7-10， 20-120mM DTT。在此变性体系中加入大肠杆菌表达的nt-PA包涵体进行变性，其中DTT的浓度是变性的关键，变性后的蛋白需经纯化水浓缩，去除变形体系中的其他物质以免干扰复性体系。
nt-PA复性体系如下：50mM Tris，pH7-10，氧化型谷胱甘肽 2-12mM，还原型谷胱甘肽0.1-2mM，nt-PA蛋白浓度0.05-2mg/ml。在此复性体系中加入重组人二硫键异构酶PDI，使nt-PA与PDI的摩尔比为100:1-10:1。在加入PDI的复性体系中进行复性，nt-PA的复性率比不加PDI时有显著提高，二硫键错配造成的异构体比例大大降低，nt-PA的终产率也相应提高。
nt-PA制备方法如下：将表达后的nt-PA工程菌菌体离心收集，悬浮于50mM Tris，pH 7.5，超声破碎并10000×g，30分钟后离心制得nt-PA包涵体。包涵体用6.5M盐酸胍，50mMTris，100mM DTT 充分溶解后，12000×g，20分钟离心，取上清，即为变性的nt-PA。
将变性的nt-PA逐滴滴入加入PDI的复性体系中，使得nt-PA终浓度达到0.5mg/ml，室温下放置过夜。将上述复性液超滤脱盐后，用亲和层析柱纯化，再经过CM-Sepharose柱层析(平衡缓冲：30mM PBS， pH6.5，0-1M NaCl线性梯度洗脱)，得到纯度＞95％，比活性＞6×105IU/mg的nt-PA。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围。
Claims (7)
1.一种组织型纤溶酶原激活剂重组异构体(nt-PA)包涵体的复性方法。其特征在于通过一种独特的变性、复性体系使nt-PA包涵体中大量错配的二硫键重新打开并形成正确的二硫键，提高nt-PA的复性率。
2.根据权利要求1所述一种组织型纤溶酶原激活剂重组异构体(nt-PA)包涵体的复性方法，其特征在于包括两种体系：变性体系和复性体系。
3.根据权利要求2所述nt-PA包涵体的变性体系，其特征在于包括：nt-PA包涵体、盐酸胍、三羟甲基氨基甲烷(Tris)、二硫苏糖醇(DTT)，采用盐酸胍的浓度范围为3-8M，Tris的浓度范围为25-100mM，DTT的浓度范围为20-120mM。
4.根据权利要求2所述nt-PA包涵体的复性体系，其特征在于包括：变性nt-PA、二硫键异构酶(PDI)、Tris、氧化型谷胱甘肽(GSSG)、还原型谷胱甘肽(GSH)，采用Tris的浓度范围为25-100mM，氧化型谷胱甘肽的浓度范围为2-12mM，还原型谷胱甘肽的浓度范围为0.1-2mM。
5.如权利要求4所述的nt-PA包涵体的复性体系，其特征在于PDI为重组人二硫键异构酶(rhPDI)或其它来源的PDI。
6.如权利要求4所述的nt-PA包涵体的复性体系，其特征在于PDI催化复性体系中，变性nt-PA与PDI的摩尔比为100:1-10:1，变性nt-PA的蛋白浓度为0.01～2mg/ml，其中最优的浓度为0.5mg/ml。
7.权利要求1-6任意一种组织型纤溶酶原激活剂重组异构体(nt-PA)包涵体的复性方法。
步骤：nt-PA工程菌表达→菌体破碎→包涵体分离→变性→PDI催化复性→脱盐→亲和层析→离子交换层析等步骤完成。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1299822A (zh) * | 1999-12-13 | 2001-06-20 | 王革 | 用新的复性方法制备组织型纤溶酶原激活剂重组异构体 |
| CN1654643A (zh) * | 2004-02-11 | 2005-08-17 | 王革 | 用辅助复性的方法制备重组瑞替普酶 |
| CN103509772A (zh) * | 2012-06-18 | 2014-01-15 | 王革 | 组织型纤溶酶原激活剂的新型组合突变体 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1299822A (zh) * | 1999-12-13 | 2001-06-20 | 王革 | 用新的复性方法制备组织型纤溶酶原激活剂重组异构体 |
| CN1654643A (zh) * | 2004-02-11 | 2005-08-17 | 王革 | 用辅助复性的方法制备重组瑞替普酶 |
| CN103509772A (zh) * | 2012-06-18 | 2014-01-15 | 王革 | 组织型纤溶酶原激活剂的新型组合突变体 |
Non-Patent Citations (3)
| Title |
|---|
| 孔扬: "重组组织型纤溶酶原激活剂变异体（RETEPLASE）工程菌的培养与体外复性的研究", 中国博士学位论文全文数据库 基础科学辑, no. 3, pages 006 - 90 * |
| 赵友春: "重组组织型纤溶酶原激活剂变异体（RETEPLASE）及复性研究", 万方数据知识服务平台, pages 1 - 159 * |
| 陈望化: "重组组织型纤溶酶原激活剂变异体（RETEPLASE）复性研究", 中国优秀博硕士学位论文全文数据库（硕士）基础科学辑, no. 12, pages 006 - 137 * |
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