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首页> 外文期刊>Protein Expression and Purification >Purification and characterization of YihA, an essential GTP-binding protein from Escherichia coli
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Purification and characterization of YihA, an essential GTP-binding protein from Escherichia coli

机译：大肠杆菌的必需GTP结合蛋白YihA的纯化和表征

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摘要

YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis. It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics. YihA encodes a putative GTP-binding protein. We have cloned and overexpressed the gene encoding E coli YihA and initiated biochemical studies as a first step towards understanding its biological function. We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins. It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA. The K-d values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 muM, respectively. Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 22]
机译：YihA先前已被表征为大肠杆菌和枯草芽孢杆菌中未知功能的必需基因。它在细菌中是保守的，并且是发现新抗生素的有吸引力的靶标。 YihA编码一个假定的GTP结合蛋白。我们已经克隆并过表达编码大肠杆菌YihA的基因，并开始进行生化研究，作为了解其生物学功能的第一步。我们通过圆二色性表明，纯化的蛋白具有大多数GTP结合蛋白的典型二级结构。它特异性地结合鸟嘌呤核苷酸，如2'-（或3'）-O-（N-甲基蒽基）核苷酸（薄荷核苷酸）和YihA色氨酸之间的荧光共振能量转移所证明的。 GDP和GTP的K-d值是通过与2'-（或3'）-O-（N-甲基蒽基）GDP竞争确定的，分别为3和27μM。使用YihA的突变体，我们表明核苷酸结合发生在从一级序列预测的假定的GTP结合域上。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：22]

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