Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain

Protein Expression and Purification
H HeimoI Suominen

Abstract

In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P. pastoris. O-glycosides (studied to our knowledge for the first time in P. pastoris in this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple hands on isoelectric focusing with p1 value...Continue Reading

References

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Citations

Feb 18, 2016·Microbial Biotechnology·Safa Ben AzounHéla Kallel
Dec 6, 2008·General and Comparative Endocrinology·Yamila CarpioMario Pablo Estrada
Dec 3, 2016·World Journal of Microbiology & Biotechnology·Zhen KangJian Chen
Feb 20, 2018·Applied Microbiology and Biotechnology·Fernanda Gobbi AmorimEliane Candiani Arantes
Feb 11, 2005·Yeast·Sue Macauley-PatrickLinda M Harvey
May 2, 2018·Microorganisms·Antonio Milton Vieira GomesNádia Skorupa Parachin
Apr 20, 2001·Journal of Agricultural and Food Chemistry·B K Choi, R Jiménez-Flores

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