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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Chromatography, Gel / methods
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Cloning, Molecular / methods
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Crystallization / methods
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Enzyme Assays / methods
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Escherichia coli / genetics
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Magnoliopsida / chemistry
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Magnoliopsida / enzymology*
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Magnoliopsida / genetics*
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Magnoliopsida / metabolism
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Mitochondrial Proteins / chemistry*
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Mitochondrial Proteins / genetics*
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Mitochondrial Proteins / isolation & purification
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Mitochondrial Proteins / metabolism
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Oxidoreductases / chemistry*
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Oxidoreductases / genetics*
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Oxidoreductases / isolation & purification
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Oxidoreductases / metabolism
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Plant Proteins / chemistry*
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Plant Proteins / genetics*
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Plant Proteins / isolation & purification
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Plant Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Schizosaccharomyces / genetics
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Solubility
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Transformation, Genetic
Substances
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Mitochondrial Proteins
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Plant Proteins
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Recombinant Proteins
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Oxidoreductases
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alternative oxidase