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首页> 外文期刊>Protein Expression and Purification >Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B
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Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

机译：分子克隆，在大肠杆菌中的过表达以及艰难梭菌毒素A和B的6x his标记的C末端结构域的纯化

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Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 28]
机译：来自核糖型01和-17艰难梭菌菌株的基因组DNA被用于扩增编码毒素A（TcdA）和B（TcdB）的羧基末端结构域的序列。推导的C末端TcdB核糖型01和-17结构域具有99.5％的氨基酸序列同一性，而与TcdA-01相比，TcdA核糖型17包含607个氨基酸的缺失。当与先前测序的艰难梭菌毒素进行比较时，在菌株VPI10643的TcdA-01和TcdA之间发现了99.3％的氨基酸同一性，而在菌株F-1470的TcdA-17和TcdA之间发现了98.8％的同一性。将获得的序列在3'端与编码六组氨酸标签的序列融合，并克隆到大肠杆菌表达载体中。重组蛋白在大肠杆菌中表达，并使用一步金属螯合物色谱法纯化。 TcdA-01的重组羧基末端结构域是从可溶性大肠杆菌裂解物级分中纯化的，而TcdA-17和TcdB-17羧基末端结构域是从包涵体中纯化的。每升细菌培养物中至少要纯化40mg的每种蛋白质。通过Western印迹和ELISA用针对天然艰难梭菌毒素的抗体特异性检测重组毒素结构域。这项研究表明，可以使用大肠杆菌表达系统生产TcdA和TcdB的羧基末端结构域，并易于纯化。这些重组，稳定且无毒的蛋白质提供了一种方便的来源，可用于诊断艰难梭菌感染，代替天然毒素，作为免疫测定试剂盒中的对照和校准物，并获得特异性单克隆抗体。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：28]

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