Burger A, Berendes R, Voges D, Huber R, Demange P
Max-Planck-Institut für Biochemie, Martinsried, Germany.
FEBS Lett. 1993 Aug 23;329(1-2):25-8. doi: 10.1016/0014-5793(93)80185-w.
Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.
膜联蛋白V以钙依赖的方式与酸性磷脂结合，并在体外表现出离子通道活性。我们正在通过单通道测量、X射线晶体学和电子显微镜研究膜联蛋白V的突变体，以了解离子通道活性的结构-功能关系。我们在此描述一种获得此类研究所需的非常纯的重组膜联蛋白V的方法。第一步是通过渗透休克温和地打开细菌细胞。在纯化过程中，利用膜联蛋白V与脂质体的可逆钙介导结合。在最后的纯化步骤中，蛋白质进行离子交换色谱，并以单一峰洗脱，不含任何可检测到的污染物。
