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.
Raymond
C K
CK
ZymoGenetics, Inc., Seattle, WA 98102, USA.
Bukowski
T
T
Holderman
S D
SD
Ching
A F
AF
Vanaja
E
E
Stamm
M R
MR
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005800:Genes, Fungal; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014617:Vacuoles
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D010450:Endopeptidases; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; D005968:Glutamate Decarboxylase; D000432:Methanol