Pouny Y, Weitzman C, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.
Biochemistry. 1998 Nov 10;37(45):15713-9. doi: 10.1021/bi981519z.
Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin affinity purification. Although a high degree of purity is obtained, only about 15-20% of the permease is recovered due to incomplete biotinylation. In this communication, a simple modification is described that allows quantitative recovery of highly purified permease. Membranes containing permease with the biotin acceptor domain from the Klebsiella pneumoniae oxaloacetate decarboxylase are extracted with 5 M urea or treated with dicyclohexylcarbodiimide to inactivate F1/Fo ATPase and biotinylated in vitro with biotin ligase, ATP and d-biotin. Subsequently, the membranes are harvested, washed to remove free biotin and solubilized with 2% n-dodecyl-beta-D-maltopyranoside. Biotinylated permease is then purified in one step by affinity chromatography on monomeric avidin-Sepharose. The purified material is homogeneous and exhibits full activity with respect to ligand binding and counterflow.
康斯勒等人[康斯勒，T.G.，佩尔松，B.L.等（1993年）《美国国家科学院院刊》90，6934 - 6938]描述了一种从大肠杆菌中一步纯化乳糖通透酶（一种疏水性膜转运蛋白）的方法。含有生物素受体结构域的通透酶构建体在体内进行生物素化，然后进行溶解和抗生物素蛋白亲和纯化。尽管获得了高度的纯度，但由于生物素化不完全，仅回收了约15 - 20%的通透酶。在本通讯中，描述了一种简单的改进方法，可实现高度纯化的通透酶的定量回收。用5 M尿素提取含有来自肺炎克雷伯菌草酰乙酸脱羧酶的带有生物素受体结构域的通透酶的膜，或用二环己基碳二亚胺处理以灭活F1/Fo ATP酶，并在体外使用生物素连接酶、ATP和d - 生物素进行生物素化。随后，收获膜，洗涤以去除游离生物素，并用2%的n - 十二烷基 - β - D - 麦芽糖苷溶解。然后通过在单体抗生物素蛋白 - 琼脂糖上进行亲和色谱一步纯化生物素化的通透酶。纯化后的物质是均一的，并且在配体结合和逆流方面表现出完全活性。
