Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells
Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells
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DOI：
10.1016/j.ymeth.2007.11.010
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发表时间：
2008-03-01
期刊：
影响因子：
4.8
通讯作者：
Martin, Seamus J.
中科院分区：
文献类型：
--
作者：
Brumatti, Gabriela;Sheridan, Clare;Martin, Seamus J.
细胞凋亡是一种细胞死亡模式，伴随着质膜的特异性改变，促进吞噬细胞对这些细胞的识别和吞噬。虽然已经定义了几种这样的膜改变，磷脂酰丝氨酸从内到外质膜小叶的再分布已成为哺乳动物中凋亡细胞最广泛使用的标志物之一。这在很大程度上是由于一个敏感的和特异性的探针的磷脂酰丝氨酸结合蛋白，膜联蛋白V的形式，这一事件的可用性。在这里，我们描述了重组多组氨酸标记的膜联蛋白V从大肠杆菌的表达和纯化的方法。重组膜联蛋白V是高度可溶的，因此易于表达和纯化至高产率;通常在每ml细菌培养物4 μ g蛋白质的范围内。我们还描述了这种蛋白质的FITC荧光团的共轭方法，其用于检测凋亡细胞的流式细胞术或荧光显微镜。(C)2007年爱思唯尔公司All rights reserved.
Apoptosis is a mode of cell death that is accompanied by specific alterations to the plasma membrane that promote the recognition and engulfment of these cells by phagocytes. Although several such membrane alterations have been defined, redistribution of phosphatidylserine from the inner to the outer plasma membrane leaflet has become one of the most widely used markers for apoptotic cells in mammals. This is largely due to the availability of a sensitive and specific probe for this event in the form of the phosphatidylserine-binding protein, annexin V. Here, we describe methods for the expression and purification of recombinant polyhistidine-tagged annexin V from Escherichia coli. Recombinant annexin V is highly soluble and is thus readily expressed and purified to high yields; typically in the region of 4 mu g of protein per ml of bacterial culture. We also describe methods for conjugation of this protein to the FITC fluorophore and for its use for the detection of apoptotic cells by flow cytometry or fluorescence microscopy. (C) 2007 Elsevier Inc. All rights reserved.