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首页> 外文期刊>Protein Expression and Purification >Construction of Escherichia coli dnaK-deletion mutant infected by lambda DE3 for overexpression and purification of recombinant GrpE proteins
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Construction of Escherichia coli dnaK-deletion mutant infected by lambda DE3 for overexpression and purification of recombinant GrpE proteins

机译：λDE3感染的大肠杆菌dnaK缺失突变体的构建，用于重组GrpE蛋白的过表达和纯化

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Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E coli dnaK-deletion mutant. The E. coli Delta dnaK52 mutant was infected by lambda DE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants. (c) 2008 Elsevier Inc. All rights reserved.
机译：大肠埃希氏菌被广泛用于产生重组蛋白，因为该微生物易于操作，培养成本低并且持续时间短以产生重组蛋白。然而，在重组蛋白的纯化过程中，分子伴侣DnaK的污染有时是一个问题，因为DnaK有时对随后的实验具有负面影响。以前，已经进行了一些努力来通过几次顺序色谱法或用一些昂贵的化学药品（例如ATP）洗涤来去除DnaK污染物。在这里，我们开发了一种简单且便宜的方法来表达和纯化基于大肠杆菌dnaK缺失突变体的重组蛋白。大肠杆菌Delta dnaK52突变体被λDE3噬菌体感染，以在T7启动子的控制下过表达所需的重组蛋白。使用该宿主细胞，通过一步镍亲和色谱法过表达并纯化了重组的六组氨酸标签融合的GrpE，它是DnaK的陪伴分子，并与DnaK强烈相互作用。结果，无需使用ATP洗涤就获得了高度纯化的重组GrpE。纯化的重组GrpE显示出折叠的二级结构和二聚体结构，如先前的发现。体外ATPase活性测定和萤光素酶复性活性测定证明重组GrpE与DnaK协同工作。因此，该开发的方法将是快速的并且对于表达和纯化难以去除DnaK污染物的重组蛋白有用。 （c）2008 Elsevier Inc.保留所有权利。

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