Expression of Mouse $\alpha-Amylase$ Gene in Methylotrophic Yeast Pichia pastoris

• Published in: Biotechnology and bioprocess engineering Vol. 5; no. 1; pp. 7 - 12
• Main Authors: Uehara Hiroyuki, Choi Du Bok, Park Enoch Y, Okabe Mitsuyasu
• Format: Journal Article
• Language: Korean
• Published: 2000
• Subjects: mouse a-amylase gene; Pichia pastoris; methylotrophic yeast
• ISSN: 1226-8372; 1976-3816

The expression of the mouse $\alpha-amylase$ gene in the methylotrophic yeast, P pastoris was investigated. The mouse $\alpha-amylase$ gene was inserted into the multi-cloning site of a Pichi a 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 GSl15 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SaiII or BglII into the HIS4locus $(38\;of\;Mut^+\;clone)$ or into the AOX1 locus $(15\;of\;Mut^s\;clone)$. Southern blot was carried out in 11 transformants, which showed that the mouse $\alpha-amylase$ gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest $\alpha-amylase$ activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse $\alpha-amylase$ gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse $\alpha-amylase$ gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.