Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

Abstract

The expression of the mouse α -amylase gene in the methylotrophic yeast, P. pas- toris was investigated. The mouse α -amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested with SalI or BglII, and was introduced into P. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SalI or BglII into the HIS4 locus (38 of Mut + clone) or into the AOX1 locus (15 of Mut S clone). Southern blot was carried out in 11 transformants, which showed that the mouse α -amylase gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α -amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α -amylase gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse α -amylase gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.