Refolding and purification of a urokinase plasminogen activator fragment by chromatography
Refolding and purification of a urokinase plasminogen activator fragment by chromatography
复制标题
DOI：
10.1016/s0378-4347(99)00360-6
复制
发表时间：
2000-01-14
影响因子：
3
通讯作者：
Binding, P
中科院分区：
文献类型：
--
作者：
Fahey, EM;Chaudhuri, JB;Binding, P
重组尿激酶纤溶酶原激活剂（u-PA）片段在大肠杆菌中以包涵体形式表达。纯化和复性通过三阶段过程实现。通过将 Triton X-100 和尿素中的洗涤步骤与离心相结合来捕获包涵体。然后通过尺寸排阻色谱法（SEPROS）进行缓冲液交换来复性溶解的包涵体。使用具有较高分级范围的尺寸排阻介质导致 u-PA 活性的回收率增加，最大分级范围达到 M-r 10 000-1 500 000，之后由于重折叠的 u-PA 和变性剂之间的分辨率较低，回收率降低。使用阳离子交换色谱法浓缩重折叠的 u-PA 级分，该色谱法选择性地结合正确折叠的 u-PA。结果是浓缩的、活性的、均质的 u-PA。 (C) 2000 Elsevier Science B.V. 保留所有权利。
A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of M-r 10 000-1 500 000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA. (C) 2000 Elsevier Science B.V. All rights reserved.