On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli
On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli
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DOI：
10.1016/s1570-0232(02)00745-6
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发表时间：
2003-03-25
影响因子：
3
通讯作者：
Santarelli, X
中科院分区：
文献类型：
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作者：
Lemercier, G;Bakalara, N;Santarelli, X
利用PCR技术从布氏锥虫中克隆了一个外切多聚磷酸酶基因，并在大肠杆菌中高效表达了相应的蛋白。大肠杆菌中表达，以表征其生化活性，并产生抗体以确定其定位。由于这种蛋白在细菌中的过度表达导致形成无活性的包涵体，这些结构首先在变性条件下溶解（6 M尿素）。其次，在使用固定化金属亲和层析（IMAC）的捕获步骤之后，在1%Triton X-100的存在下，在柱上从6 M至0 M尿素进行蛋白质的逐渐重折叠。Triton X-100用于消除复性步骤中的蛋白质聚集。纯化的酶是有活性的，表明至少部分蛋白质被正确地重折叠。(C)2002 Elsevier Science B. V.保留所有权利。
An exopolyphosphatase gene has been cloned by polymerase chain reaction (PCR) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine tag) exopolyphosphatase in E. coli in order to characterize its biochemical activity and to produce antibody to determine its localization. Because overexpression of this protein in bacteria resulted in the formation of inactive inclusion bodies, these structures were first solubilized in denaturant condition (6 M urea). Secondly, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein Was performed on-column from 6 M to 0 Murea in the presence of 1% Triton X-100. Triton X-100 was used to abolish protein aggregation during the refolding step. The purified enzyme was active, demonstrating that at least part of the proteins was properly refolded. (C) 2002 Elsevier Science B.V. All rights reserved.