Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1F0 ATP synthase
Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1F0 ATP synthase
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DOI：
10.1016/j.bbabio.2004.09.012
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发表时间：
2005-01-07
影响因子：
4.3
通讯作者：
Vik, SB
中科院分区：
文献类型：
--
作者：
Ishmukhametov, RR;Galkin, MA;Vik, SB
用Ni-NTA亲和层析纯化了组氨酸标记的大肠杆菌无半胱氨酸F1F0ATP合成酶。在纯化过程中，总ATPase活性损失不超过50%，纯化程度约为80倍。纯化后的酶基本不含其他蛋白质，对三磷酸腺苷具有较高的催化活性(pH=8，37℃时为75U/mg)，对N，N‘-二环己基碳二亚胺敏感(70%)。将F1F0掺入大豆脂质体中，可得到偶联良好、活性高的蛋白脂质体。从用法国压力机破碎细胞到制备蛋白脂质体的整个过程仅用了约8h。本文介绍了对ATP水解率和依赖于ATP的9-氨基-6-氯-2-甲氧基吖啶(ACMA)荧光猝灭速率的一些改进方法。(C)2004爱思唯尔B.V.保留所有权利。
His-tagged cysteine-less F1F0 ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degreesC), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1F0 into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described. (C) 2004 Elsevier B.V. All rights reserved.