Abstract: Alternative oxidase (AOX), a terminal oxidase located in the inner mitochondrial membrane, has been identified universally in plant, algae, fungi and protozoan. AOX branches from the cytochrome pathway at the level of the ubiquinone and catalyses the oxidation of ubiquinol and reduction of oxygen to water. The enzyme remains difficult to purify to homogeneity in a stable, active form. The paper expressed the AOX with inducer IPTG in pFLAG-1 Escherichia coli system. pFLAG-1 carry a ompA which secretary the AOX to periplasm. Three strategies have combined to increase the yield of AOX. First strategy involved tuning of the inducer concentration while the second involved alter the growth temperature and the last involved differ the cell concentration. The optimized protocol is that the host cell is DH5α, the temperature is 37℃, the cell concentration is OD₆₀₀ = 0.6 and the IPTG is 0.2 mmol/L. Purification of proteins showed AOX is expressed in unsoluble protein.

Key words: AOX

摘要： 植物交替氧化酶(Alternative Oxidase, AOX) 位于高等植物线粒体内膜, 从细胞色素途径的辅酶Q分岔, 催化4 个电子还原氧分子形成水的另一终端氧化酶。分离纯化有活性的AOX 比较困难。本文研究AOX 原核表达, 选择pFLAG-1 分泌表达载体, 用异丙基硫代-β-D-半乳糖苷( IPTG) 诱导AOX 优化表达,pFLAG-1-AOX 大肠杆菌优化表达条件为: 宿主DH5α、温度37℃、细胞密度OD₆₀₀ = 0.6、IPTG 浓度0 . 2mmol/L, 诱导后60 min 收获细胞; 获得少量可溶的细胞外周质AOX 和大量不溶的AOX, 为深入研究AOX打下基础, 同时为研究膜蛋白原核表达提供依据。

关键词: 交替氧化酶(AOX) , 大肠杆菌pFLAG-1 表达系统, 超量表达FONT