Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD₆₅) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD₆₅ at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD₆₅ was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.