Journal article
Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli
Drug metabolism and disposition, Vol.37(12), pp.2393-2398
12/2009
Handle:
https://hdl.handle.net/2376/104884
PMCID: PMC2784701
PMID: 19741035
Abstract
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.
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Details
- Title
- Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli
- Creators
- Joshua F Alfaro - Department of Chemistry, Washington State University, Pullman, WashingtonCarolyn A Joswig-Jones - Department of Chemistry, Washington State University, Pullman, WashingtonWenyun Ouyang - Department of Chemistry, Washington State University, Pullman, WashingtonJoseph Nichols - Department of Chemistry, Washington State University, Pullman, WashingtonGregory J Crouch - Department of Chemistry, Washington State University, Pullman, WashingtonJeffrey P Jones - Department of Chemistry, Washington State University, Pullman, Washington
- Publication Details
- Drug metabolism and disposition, Vol.37(12), pp.2393-2398
- Academic Unit
- Chemistry, Department of
- Publisher
- The American Society for Pharmacology and Experimental Therapeutics
- Grant note
- GM84546 / National Institutes of Health
- Identifiers
- 99900546733401842
- Language
- English
- Resource Type
- Journal article