Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

Heng Yen Pan, Mei M. Whittaker, Romaric Bouveret, Anne Berna, François Bernier, James W. Whittaker

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 × 104 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)925-929
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume356
Issue number4
DOIs
StatePublished - May 18 2007

Keywords

  • Cupin
  • Germin
  • Glycan
  • Glycoprotein
  • Oxalate oxidase
  • Pichia pastoris

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Pan, H. Y., Whittaker, M. M., Bouveret, R., Berna, A., Bernier, F., & Whittaker, J. W. (2007). Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris. Biochemical and Biophysical Research Communications, 356(4), 925-929. https://doi.org/10.1016/j.bbrc.2007.03.097