谷胱甘肽过氧化物酶的新型表达方法与机制研究

Selenium-containing glutathione peroxidase (GPX), a critical enzyme in antioxidant defense system of living organisms, can protect cell and tissues against oxidative damage by catalyzing the reduction of H2O2, hydroxyl radical and other harmful peroxides using glutathione (GSH) as the reducer and so it can treat many human diseases caused by oxidative stress by removing reactive oxygen species (ROS). However, GPX has limited availability and poor stability, because the catalytic group selenocysteine (Sec) is encoded by UGA, usually as a stop codon, which need complicated mechanism with participation of a variety of cis-element and trans-acting factor to translate into Sec and read through the rate less than 3% in eukaryotic and 5% in prokaryotic. Therefore, the preparation of artificial GPX and their mimics have attracted more and more attentions. In this study, we try to obtain the special translation system suited for Sec by redesigning the Sec specific tRNA and the directed evolution of E.coli extension factor (EF) based on sequence alignment, computer simulation, molecular docking, mutant library construction and screening technology. Finally, in the UAG engineering bacterium, Sec involved in protein synthesis through the same as conventional amino acid translation mechanism, e.g. the specificity of the Sec insertion in protein loci and so the efficient and specific prokaryotic expression system for the selenium protein is established, by which native GPX and its mutants with high activity and stability are expressed and screened.

含硒的谷胱甘肽过氧化物酶（GPX）是机体抗氧化防御体系的关键酶，能清除体内活性氧，分解羟自由基和其它有害的过氧化物，防止组织细胞过氧化损伤，治疗氧化应激引起的各种疾病。然而，由于编码其催化基团硒代半胱氨酸（Sec）的密码子UGA通常作为终止密码子，需要多种顺式元件和反式因子共同参与的复杂过程才能翻译为Sec，且通读率在真核不到3%，原核不到5%，这导致天然GPX的来源相当有限且稳定性差，因此科学家一直致力于人工GPX及其模拟酶的研究。本项目通过序列比对、计算机模拟、分子拟合，突变库构建与筛选技术，重新设计Sec特异的tRNA和定向进化大肠杆菌的延伸因子获得适于Sec的特殊翻译系统。实现Sec在UAG工程菌中，通过与常规氨基酸相同的翻译机制参与蛋白质合成，即Sec在蛋白质任意位点的特异性插入，为硒蛋白的表达和研究提供高效特异的原核表达系统，进而表达筛选各型天然GPX及其高活力高稳定的突变体。

含硒的谷胱甘肽过氧化物酶（GPX）是机体抗氧化防御体系的关键酶，能分解羟自由基和其它有害过氧化物，清除体内活性氧，防止细胞和组织过氧化损伤，治疗氧化应激引起的各种疾病。然而，由于编码其催化基团硒代半胱氨酸（Sec）的密码子UGA通常作为终止密码子，需要多种顺式元件和反式因子共同参与的复杂过程才能翻译为Sec，且通读率在真核不到3%，原核不到5%，这导致天然GPX的来源相当有限且稳定性差，因此科学家一直致力于人工GPX及其模拟酶的研究。本项目通过计算机模拟、序列比对、分子拟合，突变库构建与筛选技术，重新设计Sec特异的tRNA和定向进化大肠杆菌的延伸因子，将终止密码子UAG的通读效率由5%提高到70%，获得了适于Sec的特殊翻译系统。实现了Sec在UAG工程菌中，通过与常规氨基酸相同的翻译机制参与蛋白质的合成，即Sec在蛋白质任意位点的特异性插入。最后用UAG指导的新型硒蛋白表达系统成功表达了基因工程重组GPX及其突变体和双功能抗氧化酶。与缺陷型原核表达系统相比，该方法不仅可以增加蛋白产率、降低生产成本，而且不会引起组成蛋白骨架的任何关键氨基酸的突变。酶促反应动力学研究表明，重组人GPX及其突变体和双功能酶的催化机制与天然GPX相同。其中，具有较高活力的重组人GPX1和GPX4在细胞和动物水平都显示出较好的抗氧化效应，修饰分子PEG的引入改善了其体内稳定性和药效学结果。该研究不仅为硒蛋白的表达提供了新方法，而且为该类抗氧化酶的开发与应用奠定了基础。