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

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

Research output: Contribution to journalArticle

15 Citations (Scopus)

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

Fingerprint

oxalate oxidase
Pichia
Triticum
Mating Factor
Fermentation
Enzymes
Enzyme activity
Protein Sorting Signals
Recombinant Proteins
Polysaccharides
Proteins
Fusion reactions
Complementary DNA
germin

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris. / Pan, Heng Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, François; Whittaker, James.

In: Biochemical and Biophysical Research Communications, Vol. 356, No. 4, 18.05.2007, p. 925-929.

Research output: Contribution to journalArticle

Pan, HY, Whittaker, MM, Bouveret, R, Berna, A, Bernier, F & Whittaker, J 2007, 'Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris', Biochemical and Biophysical Research Communications, vol. 356, no. 4, pp. 925-929. https://doi.org/10.1016/j.bbrc.2007.03.097
Pan, Heng Yen ; Whittaker, Mei M. ; Bouveret, Romaric ; Berna, Anne ; Bernier, François ; Whittaker, James. / Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris. In: Biochemical and Biophysical Research Communications. 2007 ; Vol. 356, No. 4. pp. 925-929.
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