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首页> 外文期刊>Protein Expression and Purification >Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa
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Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

机译：耐生小球藻重组谷胱甘肽-S-转移酶的克隆，表达，纯化及鉴定

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摘要

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.
机译：枯草杆菌是由木质部取食的叶蝉传播的重要病原细菌，它定植在植物的木质部中，并在包括橙和酸橙树的柑橘杂色萎黄病（CVC）在内的几种重要农作物上引起疾病。谷胱甘肽-S-转移酶（GST）形成了一组多功能同工酶，可催化谷胱甘肽（GSH）依赖性的共轭反应和异源生物和内源性化合物的细胞解毒反应中的还原反应。 GST是在细胞内空间中发现的主要解毒酶，主要存在于从原核生物到哺乳动物的胞质溶胶中，并且可能通过抑制凋亡信号调节激酶1参与应激激活信号的调节。在本研究中，我们描述了克隆毕赤酵母中的谷胱甘肽-S-转移酶到pET-28a（+）载体中的表达，在大肠杆菌中的表达，纯化和初步结构表征。重组xfGST（rxfGST）的纯化接近同质是使用亲和色谱和尺寸排阻色谱（SEC）实现的。 SEC证明rxfGST是溶液中的同型二聚体。重组蛋白的二级和三级结构分别通过圆二色性和荧光光谱分析。测定了该酶的活性，结果相加表明rxfGST是稳定的分子，正确折叠并且具有高活性。 GST家族的几个成员已被广泛研究。但是，xfGST是研究较少的亚家族的一部分，但尚未在结构和生化特征上进行描述。此外，这些研究应为rxfGST的未来研究和生物技术方法提供有用的基础。 （C）2008 Elsevier Inc.保留所有权利。

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