The grpE protein of Escherichia coli. Purification and properties.
The grpE protein of Escherichia coli. Purification and properties.
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DOI：
10.1016/s0021-9258(18)45398-7
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发表时间：
1987-12
期刊：
影响因子：
--
通讯作者：
M. Żylicz;D. Ang;Costa Georgopoulos
中科院分区：
文献类型：
--
作者：
M. Żylicz;D. Ang;Costa Georgopoulos
大肠杆菌的grpE基因首先被发现，因为它的突变，grpE 280，阻止了噬菌体λ DNA在体内的复制。随后的工作导致了grpE蛋白在二维凝胶中的鉴定及其作为热休克蛋白的分类。在这里，我们报告的grpE蛋白的纯化。我们表明，过量生产grpE发生在dnaK 103细菌不产生功能性的Mr 72，000 dnaK蛋白。grpE蛋白主要通过其在dnaK亲和柱上的特异性保留从该菌株中纯化。这两种蛋白质之间的相互作用在2 M KCl存在下是稳定的，允许从该柱中洗涤其他蛋白质。然后用ATP洗脱grpE，这破坏了相互作用。在纯化过程中，grpE活性通过其补充依赖于λ O和λ P蛋白的体外λ dv DNA复制系统的能力来监测。ATP对dnaK-grpE复合物的影响也观察到在甘油梯度的两种蛋白质的沉降过程中。纯化的grpE蛋白在变性和天然条件下的Mr约为23，000，分别通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和沉降测定。然而，在天然条件下，在dnaK的存在下，grpE与dnaK共沉积。当ATP加入到梯度中时，复合物被破坏，两种蛋白质作为单体独立沉淀。
The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.