Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris |
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Authors: | Pan Heng-Yen Whittaker Mei M Bouveret Romaric Berna Anne Bernier François Whittaker James W |
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Institution: | Department of Environmental and Biomolecular Systems, Oregon Health and Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921, USA. |
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Abstract: | 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. |
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Keywords: | Germin Oxalate oxidase Pichia pastoris Glycoprotein Cupin Glycan |
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