title: Expression and crystallization of the plant alternative oxidase.
abstract: The alternative oxidase (AOX) is an integral monotopic membrane protein located on the inner surface of the inner mitochondrial membrane. Branching from the traditional respiratory chain at the quinone pool, AOX is responsible for cyanide-resistant respiration in plants and fungi, heat generation in thermogenic plants, and survival of parasites, such as Trypanosoma brucei, in the human host. A recently solved AOX structure provides insight into its active site, thereby facilitating rational phytopathogenic and antiparasitic drug design. Here, we describe expression of recombinant AOX using two different expression systems. Purification protocols for the production of highly pure and stable AOX protein in sufficient quantities to facilitate further kinetic, biophysical, and structural analyses are also described.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, John Maynard Smith Building, Falmer, Brighton, BN1 9QG, UK.
authors.forename: Benjamin
authors.initials: B
authors.lastname: May
authors.forename: Catherine
authors.initials: C
authors.lastname: Elliott
authors.forename: Momi
authors.initials: M
authors.lastname: Iwata
authors.forename: Luke
authors.initials: L
authors.lastname: Young
authors.forename: Julia
authors.initials: J
authors.lastname: Shearman
authors.forename: Mary S
authors.initials: MS
authors.lastname: Albury
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Moore
mesh_terms: D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization / Q000379:methods; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D019684:Magnoliopsida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D012995:Solubility; D014170:Transformation, Genetic; D000099188:Alternative Oxidase
chemical_list: D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase