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

Uehara Hiroyuki    (Lab. of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University   ); Choi Du Bok    (Institute of Life Science, Chosun University   ); Park Enoch Y.    (Lab. of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University   ); Okabe Mitsuyasu    (Lab. of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University  );
  • 초록

    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.


  • 주제어

    methylotrophic yeast .   Pichia pastoris .   mouse a-amylase gene.  

  • 참고문헌 (15)

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