Plant Diversity ›› 2007, Vol. 29 ›› Issue (01): 74-78.

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Optimal Expression of Alternative Oxidase in Escherichia coli

WANG Ya-Ying, MOORE A L, YANG Shen-Chang, TIAN Hui-Qiao   

  1. Agricultural Institute , Zhangzhou

  • Received:2006-09-06 Online:2007-02-25 Published:2007-02-25
  • Contact: TIAN Hui-Qiao

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

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