title: Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain.
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 values around 3.5, a value that is little higher than that for GAc produced in S. cerevisiae. GAc could be used as a versatile tool in studying protein expression in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P. pastoris.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, Finland.
authors.forename: H
authors.initials: H
authors.lastname: Heimo
authors.forename: K
authors.initials: K
authors.lastname: Palmu
authors.forename: I
authors.initials: I
authors.lastname: Suominen
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005087:Glucan 1,4-alpha-Glucosidase