Over-Expression Of AOX In Escherichia Coli And Purification Of AOX From Arum Maculatum

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. There is non-protonmotive and energy is liberated as heat instead of producing ATP. AOX is a member of di-iron carboxylate proteins. Two structural models exist for AOX and the latest one is a monotopic protein instead of span membrane. Relatively little is known about its 3-dimensional structure. Main reason is that the sufficient quantities of AOX suitable for crystallogram structural studies is unavailable for its unstable.The objective of research is to produce large amount of AOX for cystallogram structure studies. The aim is achieved by over-expression of AOX from Sauromattum guttatum in E.coli and purification of AOX from a plant sauce (Arum maculatum) mitochondrial.The recombined plasmid of pFLAG-1-AOX were transformed into E. coli DH5αfor expression. E. coli DH5αfreshly transformed with the indicated expression plasmid were cultured and harvested as described in Materials and Methods. AOX was over-expressed with IPTG inducing under optimal conditions: temperature 37℃, cell density OD600=0.6 and inducer IPTG concentration 0.2mmol/L.To assay the expression level, the cell were broken by osmotic shock, and the inclusion body fraction separated from a fraction containing both soluble and membrane-bound proteins. The soluble and membrane protein sample was further fractionated to obtain a membrane pellet. Samples were loaded on a denaturing SDS-PAGE on the basis of equal cell density, and expression level was assessed by visual...