Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli
Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli
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DOI：
10.1124/dmd.109.029520
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发表时间：
2009-12-01
影响因子：
3.9
通讯作者：
Jones, Jeffrey P.
中科院分区：
文献类型：
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作者：
Alfaro, Joshua F.;Joswig-Jones, Carolyn A.;Jones, Jeffrey P.
将人醛氧化酶1(AOX1)亚克隆到适合在大肠杆菌中表达的载体中，并进行了表达。由此产生的蛋白质是有活性的，硫被结合到钼掺杂辅因子中。表达水平适中，但1升细胞可以提供足够的蛋白质来进行生化和动力学表征。镍亲和层析通过在蛋白质的氨基末端添加6个组氨酸来实现部分纯化。动力学分析，包括动力学同位素效应和与黄嘌呤氧化酶的比较，揭示了类似的机制，但有一些细微的差异。该表达系统可用于利用定点突变技术研究人醛氧化酶的结构/功能关系，并为研究AOX1在药物代谢中的作用提供蛋白质。
Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.