Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 μM equivalent to Trolox in a 100 μg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd.

中文翻译：

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白虾凡纳滨对虾重组金属硫蛋白的克隆，表达，纯化和生化特性。

金属硫蛋白（MTs）是具有抗氧化能力的富含半胱氨酸的蛋白质，参与金属的稳态和解毒以及其他细胞过程，并有助于抵消活性氧（ROS）产生的氧化应激。在几种压力条件下，包括金属中毒和缺氧（缺氧），ROS的产生都会增加。在缺氧期间，虾对虾南美白对虾中会诱导MT基因的表达。然而，该基因编码的MT蛋白尚未纯化或鉴定。在这项工作中，南美白对虾MT的编码序列被克隆并在大肠杆菌中以融合蛋白的形式过度表达，该融合蛋白包含内含蛋白和几丁质结合结构域（CBD）。MT通过几丁质亲和色谱纯化，并评估其抗氧化能力以及与镉（Cd）和铜（Cu）的结合能力。该MT在100μg/ mL溶液中的抗氧化能力为Trolox的27.23μM。向培养基中添加CdCl2可增加273倍的Cd含量，而添加CuCl2可将纯化MT中的Cu含量增加569倍。因此，虾MT基因编码具有抗氧化能力并结合Cu和Cd的功能蛋白。