The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNA^UTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNA^UTu and seven chimeric tRNAs that were constructed on the basis of tRNA^UTu. We found that chimeric tRNA^UTu2, which substitutes the acceptor stem and T-stem of tRNA^UTu with those from tRNA^Sec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNA^UTuT6, which has a single base change (A59C) compared to tRNA^UTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNA^UTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

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嵌合tRNA在缺乏琥珀色的大肠杆菌中高效表达谷胱甘肽过氧化物酶

含硒的谷胱甘肽过氧化物酶（GPx）的活性中心是硒代半胱氨酸（Sec），它是在生物体的tRNA上生物合成的。Sec的解码取决于特定的延伸因子和Sec插入序列（SECIS）以抑制UGA密码子。利用传统的重组DNA技术，哺乳动物GPx的表达极为困难。最近，与延伸因子Tu（EF-Tu）兼容的嵌合tRNA（tRNA ^UTu）使硒蛋白表达更加容易。在这项研究中，人类谷胱甘肽过氧化物酶（hGPx）在无琥珀色的大肠杆菌C321.ΔA.exp中表达，使用的是tRNA ^UTu和在tRNA ^UTu的基础上构建的七个嵌合tRNA 。我们发现嵌合的tRNA ^UTu2用来自tRNA ^Sec的受体干和替代tRNA ^UTu的受体茎和T茎，能够以高产量表达反应性hGPx。我们还发现，与tRNA ^UTu相比，具有单一碱基变化（A59C）的嵌合tRNA ^UTuT6介导了^hGPx1的最高反应性表达。所表达的hGPx1以四聚体形式存在，并以正协同性反应。通过^添加或不添加亚硒酸钠的tRNA UTuT6产生的hGPx2的SDS-PAGE分析表明，Sec的掺入率接近90％。我们的方法可在无琥珀色的大肠杆菌中高效表达硒蛋白，并应进一步表征硒蛋白体外。