Expression inPichia pastorisand Purification ofAspergillus awamoriGlucoamylase Catalytic Domain

Abstract

In this paper we report the expression inPichia pastoris,purification, and characterization of theAspergillus awamoriglucoamylase catalytical domain (GAc).Pichia pastorisproduced 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 fromAspergillusand about 100-fold more than previously achieved bySaccharomyces cerevisiae.The GAc expressed inPichia pastoriswas 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 byP. pastoris.Glucoamylase produced byP. pastorisis N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed inP. pastoris.O-glycosides (studied to our knowledge for the first time inP. pastorisin this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple bands on isoelectric focusing with pIvalues around 3.5, a value that is little higher than that for GAc produced inS. cerevisiae.GAc could be used as a versatile tool in studying protein expression inP. pastoris:as an affinity handle for other secreted proteins produced inP. pastoris,as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing inP. pastoris.