Expression in Escherichia coli of the catalytic domain of human proline oxidase

Protein Expr Purif. 2012 Apr;82(2):345-51. doi: 10.1016/j.pep.2012.01.021. Epub 2012 Feb 8.


The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ(1)-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure-function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Catalytic Domain
  • Chromatography, Affinity
  • Cloning, Molecular
  • Escherichia coli
  • Gene Expression
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Peptide Fragments / biosynthesis*
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Proline Oxidase / biosynthesis*
  • Proline Oxidase / chemistry
  • Proline Oxidase / isolation & purification
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Solubility


  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Proline Oxidase