Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli host strain
Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli host strain
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DOI：
10.1016/bs.mie.2021.10.004
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发表时间：
2022-01-01
期刊：
影响因子：
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通讯作者：
Arner, Elias S. J.
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文献类型：
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作者：
Cheng, Qing;Arner, Elias S. J.
含有稀有氨基酸硒代半胱氨酸（Sec）的硒蛋白，通常是利用Sec的硒原子促进氧化还原反应的催化的酶，具有挑战性地获得高量的纯形式。限制硒蛋白供应的技术挑战来自其翻译的复杂性，需要将UGA终止密码子重新编码为Sec的有义密码子。这反过来又涉及到一个SEC专用的延伸因子，直接或间接地与硒蛋白编码mRNA中的一个称为SECIS元件（硒半胱氨酸插入序列）的结构，一个SEC专用的tRNA种类与UGA的反密码子，以及几个参与硒蛋白合成的辅助酶和蛋白质的相互作用。在这里，我们描述了一种替代的方法，用于重组硒蛋白生产使用UAG作为Sec密码子在一个特定的E。大肠杆菌缺乏其他UAG密码子和缺乏释放因子RF1，通常终止在UAG翻译。我们还描述了如何重组硒蛋白可以被纯化，并进一步分析最终Sec的内容。该方法可用于生产重组形式的天然硒蛋白，以及用于生产合成硒蛋白，其可被设计为使用Sec的独特生物物理特性用于各种生物技术应用。
Selenoproteins containing the rare amino acid selenocysteine (Sec), typically being enzymes utilizing the selenium atom of Sec for promoted catalysis of redox reactions, are challenging to obtain at high amounts in pure form. The technical challenges limiting selenoprotein supply derive from intricacies in their translation, necessitating the recoding of a UGA stop codon to a sense codon for Sec. This, in turn, involves the interactions of a Sec-dedicated elongation factor, either directly or indirectly, with a structure in the selenoprotein-encoding mRNA called a SECIS element (Selenocysteine Insertion Sequence), a dedicated tRNA species for Sec with an anticodon for the UGA, and several accessory enzymes and proteins involved in the selenoprotein synthesis. Here, we describe an alternative method for recombinant selenoprotein production using UAG as the Sec codon in a specific strain of E. coli lacking other UAG codons and lacking the release factor RF1 that normally terminates translation at UAG. We also describe how such recombinant selenoproteins can be purified and further analyzed for final Sec contents. The methodology can be used for production of natural selenoproteins in recombinant form as well as for production of synthetic selenoproteins that may be designed to use the unique biophysical properties of Sec for diverse biotechnological applications.