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Title 

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

Authors 

Ji Hyun KoMoon Sun HahmKang Hyun AhS W NamBong Hyun Chung

Publisher 

Elsevier

Issue Date 

2002

Citation 

Protein Expression & Purification, vol. 25, no. 3, pp. 488-493

Keywords 

aspergillus nigergene deletiongenes, fungalglucose oxidasesaccharomyces cerevisiaesaccharomyces cerevisiae proteinsaspergillussaccharomyces

Abstract 

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His6 secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His6 was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca++-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His6 per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His6 secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His6 in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Δ mutant-derived GOD-His6 enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Δ mutant-derived GOD-His6 on a protein basis was very similar to that of the hyperglycosylated GOD-His6, although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His6.

ISSN 

1046-5928

Link 

http://dx.doi.org/10.1016/S1046-5928(02)00035-9

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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