EXPRESSION IN PICHIA-PASTORIS AND PURIFICATION OF ASPERGILLUS-AWAMORIGLUCOAMYLASE CATALYTIC DOMAIN

In this paper we report the expression in Pichia pastoris, purificatio n, and characterization of the Aspergillus awamori glucoamylase cataly tical 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 th at gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expresse d in Pichia pastoris was purified by two independent chromatographic m ethods employing ion exchange or affinity chromatography to apparent h omogeneity. The purified protein has a molecular weight of about 75,00 0 and specific activity of 78 units per milligram protein. The propept ide present in the glucoamylase N terminus was found to be removed cor rectly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosacch arides appear to be larger than in invertase, another glycoprotein het erologously expressed in P. pastoris, O-glycosides (studied to our kno wledge for the first time in P. pastoris in this report) contribute ab out half of the total carbohydrate content in GAc. Purified GAc appear s as multiple bands on isoelectric focusing with pI values around 3.5, a value that is little higher than that for GAc produced in S. cerevi siae. GAc could be used as a versatile tool in studying protein expres sion in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expres sion, and as a model protein in studying protein secretion and process ing in P. pastoris. (C) 1997 Academic Press.