Expression of Recombinant Galactose Oxidase by Pichia pastoris

• Published in: Protein expression and purification 2000-10, Vol.20 (1), p.105-111
• Main Authors: Whittaker, Mei M., Whittaker, James W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; DNA, Complementary; Fermentation; galactose oxidase; Galactose Oxidase - genetics; Galactose Oxidase - isolation & purification; Molecular Sequence Data; Pichia - genetics; Pichia pastoris; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2000.1287

Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25°C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications.