Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris.
High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-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 (4x10(4) 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.
Pan
Heng-Yen
HY
Department of Environmental and Biomolecular Systems, Oregon Health and Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921, USA.
Whittaker
Mei M
MM
Bouveret
Romaric
R
Berna
Anne
A
Bernier
François
F
Whittaker
James W
JW
D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D006023:Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C058814:germin; D010088:Oxidoreductases; C020598:oxalate oxidase