Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

Masaki Yamaguchi, Yusuke Tahara, Atsunori Nakano, Tadayoshi Taniyama

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882 bp fragment, close to the size of glucose oxidase (GOD), which is 1818 bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing β-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 °C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.

Original languageEnglish
Pages (from-to)273-278
Number of pages6
JournalProtein Expression and Purification
Volume55
Issue number2
DOIs
Publication statusPublished - Oct 1 2007
Externally publishedYes

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Glucose Oxidase
Pichia
Aspergillus niger
Aspergillus
Genes
Temperature
Secretory Pathway
Enzyme activity
Molecular mass
Electrophoresis
Cell culture
Recombinant Proteins
Yeast
Open Reading Frames
Machinery
Polyacrylamide Gel Electrophoresis
Proteins
Clone Cells
Yeasts
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris. / Yamaguchi, Masaki; Tahara, Yusuke; Nakano, Atsunori; Taniyama, Tadayoshi.

In: Protein Expression and Purification, Vol. 55, No. 2, 01.10.2007, p. 273-278.

Research output: Contribution to journalArticle

Yamaguchi, Masaki ; Tahara, Yusuke ; Nakano, Atsunori ; Taniyama, Tadayoshi. / Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris. In: Protein Expression and Purification. 2007 ; Vol. 55, No. 2. pp. 273-278.
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