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

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 (4 x 10{sup 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.

Authors:
 [1];  [1];  [2];  [2];  [2];  [3]
  1. Department of Environmental and Biomolecular Systems, Oregon Health and Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921 (United States)
  2. IBMP-Institut de Botanique, Strasbourg (France)
  3. Department of Environmental and Biomolecular Systems, Oregon Health and Sciences University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921 (United States). E-mail: jim@ebs.ogi.edu
Publication Date:
OSTI Identifier:
20991343
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 356; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2007.03.097; PII: S0006-291X(07)00547-5; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOTECHNOLOGY; ENZYME ACTIVITY; FERMENTATION; GLYCOPROTEINS; MATING; OXALATES; OXIDASES; PEPTIDES; PERIODATES; SODIUM; WHEAT

Citation Formats

Pan, Heng-Yen, Whittaker, Mei M., Bouveret, Romaric, Berna, Anne, Bernier, Francois, and Whittaker, James W. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.03.097.
Pan, Heng-Yen, Whittaker, Mei M., Bouveret, Romaric, Berna, Anne, Bernier, Francois, & Whittaker, James W. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris. United States. doi:10.1016/j.bbrc.2007.03.097.
Pan, Heng-Yen, Whittaker, Mei M., Bouveret, Romaric, Berna, Anne, Bernier, Francois, and Whittaker, James W. Fri . "Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris". United States. doi:10.1016/j.bbrc.2007.03.097.
@article{osti_20991343,
title = {Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris},
author = {Pan, Heng-Yen and Whittaker, Mei M. and Bouveret, Romaric and Berna, Anne and Bernier, Francois and Whittaker, James W.},
abstractNote = {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 (4 x 10{sup 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.},
doi = {10.1016/j.bbrc.2007.03.097},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 356,
place = {United States},
year = {Fri May 18 00:00:00 EDT 2007},
month = {Fri May 18 00:00:00 EDT 2007}
}
  • Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologousmore » protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0-99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was also used to compare scFv production levels from the periplasm, inclusion bodies, and culture media. The E. coli production system was then used to produce variants of several scFv to determine structure function relationships.« less
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  • Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and themore » production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.« less
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  • No abstract prepared.