Non-fusion expression in Escherichia coli:: Single-step purification of recombinant human annexin A5 for detection of apoptosis
Non-fusion expression in Escherichia coli:: Single-step purification of recombinant human annexin A5 for detection of apoptosis
复制标题
大肠杆菌中的非融合表达：重组人膜联蛋白 A5 的一步纯化用于检测细胞凋亡。
DOI：
10.1016/j.pep.2005.05.017
复制
发表时间：
2006-01-01
影响因子：
1.6
通讯作者：
Sun, SH
中科院分区：
文献类型：
--
作者：
Wang, F;He, XW;Sun, SH
重组人膜联蛋白A5 （rh-膜联蛋白A5）最初用于体外检测早期细胞凋亡。随着放射性标记和成像技术的发展，放射性标记物标记的膜联蛋白A5在体内监测凋亡细胞中发挥着越来越重要的作用。为了获得高纯度的rh-膜联蛋白A5，我们构建了pjla503 -膜联蛋白A5表达质粒，该质粒可以在大肠杆菌中以可溶性形式过表达人膜联蛋白A5。建立了一种基于Ca2+依赖性磷脂酰丝氨酸（PS）结合活性的纯化方法，使rh-膜联蛋白A5的纯度提高到98%。为了证实该纯化方案产生的rh-膜联蛋白A5的PS亲和力，我们制备了一个简单可靠的脂膜模型并用于结合试验。作为检测凋亡的探针，异硫氰酸荧光素标记的rh-膜联蛋白A5与凋亡细胞孵育。结果表明，标记的rh-膜联蛋白A5对PS分子具有高亲和力，并能指示不同的凋亡状态。(C) 2005爱思唯尔公司版权所有。
Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states. (C) 2005 Elsevier Inc. All rights reserved.