Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression

Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol–inducibte AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high–level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kan r gene conferring G4I8–resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug–resistance. Using a G418 growth inhibition screen, we readily isolated a series of transforniants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV–1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single–copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection , using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.