The dnaA protein of Escherichia coli. Abundance, improved purification, and membrane binding.
The dnaA protein of Escherichia coli. Abundance, improved purification, and membrane binding.
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DOI：
10.1016/s0021-9258(18)68616-8
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发表时间：
1988-05
期刊：
影响因子：
--
通讯作者：
K. Sekimizu;B. Y. Yung;A. Kornberg
中科院分区：
文献类型：
--
作者：
K. Sekimizu;B. Y. Yung;A. Kornberg
5株菌株提取液中dna蛋白的免疫分析显示，相对于细胞质量，dna蛋白的丰度相当稳定，在800 ~ 2100分子/细胞之间变化；过量生产的细胞含有这个数字的100倍。野生型细胞中大约一半的dna蛋白通过裂解过程被溶解。在不溶性组分中，dna蛋白通过其与ATP的高亲和力结合而被鉴定出来。从过量生产的细胞中纯化dna蛋白的一种改进的、快速的方法似乎依赖于其与磷脂的共沉淀，并依赖于胍盐酸的溶解。该过程的产量提高了5倍，也消除了一种有效的atp酶污染物。纯化后的dnaA蛋白与dnaB和dnaC蛋白不同，通过蔗糖梯度离心分析判断，纯化后的dnaA蛋白与磷脂囊泡结合。
Immunoassays of dnaA protein in extracts from five strains showed a rather constant abundance relative to cell mass, with a variation of 800-2100 molecules/cell; overproducing cells contained 100-fold that number. About half of the dnaA protein in wild type cells was solubilized by a lysis procedure. Within the insoluble fractions, dnaA protein was identified by its characteristic high-affinity binding of ATP. An improved, rapid procedure for purifying dnaA protein from overproducing cells appears to depend on its coprecipitation with phospholipids and depends on solubilization by guanidine HCl. The procedure, with a 5-fold increased yield, also eliminates a potent ATPase contaminant. Purified dnaA protein, unlike dnaB and dnaC proteins, binds to phospholipid vesicles as judged by analysis on sucrose gradient centrifugation.