Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs)

Methods Mol Biol. 2019;1982:61-74. doi: 10.1007/978-1-4939-9424-3_4.

Abstract

The dual oxidase (DUOX) enzymes (DUOX1 and DUOX2) are unique hydrogen peroxide (H2O2)-producing members of the NADPH oxidase (NOX) family, structurally distinguished from their related NOX isoforms by the presence of an additional N-terminal extracellular domain. This region has significant sequence and predicted structural homology to mammalian peroxidases, including myeloperoxidase (MPO) and lactoperoxidase (LPO), therefore justifying the nomenclature of the peroxidase homology domain (PHD). Obtaining detailed structural information and defining a function for this appended region are both critical for elucidation of the uncharacterized mechanism of H2O2 production by DUOX proteins. Purification strategies focused on isolated sections of each DUOX enzyme are a logical means to further characterization, particularly as isolation of the complete membrane-bound enzyme in significant quantities remains unachievable. In this chapter, a reproducible method for production of the homology domain applicable to both human DUOX isoforms is described. The approach utilizes a baculovirus expression vector in insect cell culture to produce secreted recombinant PHD; an appended C-terminal His6 affinity tag was found to be crucial for structural stability. Finally, initial characterization of the activity of the purified PHDs is also described.

Keywords: Baculovirus; Dual oxidase; Hydrogen peroxide; Mammalian peroxidase.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Chromatography, Affinity
  • Cloning, Molecular
  • Dual Oxidases / chemistry*
  • Dual Oxidases / genetics
  • Dual Oxidases / isolation & purification*
  • Dual Oxidases / metabolism
  • Enzyme Activation
  • Gene Expression
  • Genetic Vectors / genetics
  • Humans
  • Hydrogen Peroxide / metabolism
  • Isoenzymes
  • Models, Molecular
  • Protein Conformation
  • Protein Domains*
  • Recombinant Fusion Proteins
  • Sf9 Cells
  • Structure-Activity Relationship

Substances

  • Isoenzymes
  • Recombinant Fusion Proteins
  • Hydrogen Peroxide
  • Dual Oxidases
  • DUOX1 protein, human