PMID: 12182830Aug 17, 2002Paper

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

Protein Expression and Purification
Ji-Hyun KoBong Hyun Chung

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-His(6) 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-His(6) 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-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta muta...Continue Reading

References

Sep 20, 1985·Science·R A SmithD T Moir
Aug 11, 2000·Journal of Biotechnology·E H ParkM S Yang
Apr 15, 1991·Biotechnology and Bioengineering·B G TurnerD T Moir

Related Concepts

SSC1 protein, S cerevisiae
Ca(2+)-Transporting ATPase
Aspergillus niger
SDS-PAGE
Enzyme Stability
Genes, Fungal
Glucose Oxidase
Hot Temperature
Hydrogen-Ion Concentration
Proteins, Recombinant DNA

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