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Efficient expression, purification and characterization of mouse salivary α‐amylase secreted from methylotrophic yeast, Pichia pastoris
Author(s) -
Kato Shinya,
Ishibashi Matsujiro,
Tatsuda Daisuke,
Tokunaga Hiroko,
Tokunaga Masao
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.714
Subject(s) - pichia pastoris , biology , yeast , amylase , pichia , biochemistry , microbiology and biotechnology , recombinant dna , enzyme , gene
We constructed a secretion vector of mouse salivary α‐amylase, pPAM, using the AOX1 promoter‐terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris . Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (∼240 µg/ml) of mouse α‐amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90‐fold higher than that of Saccharomyces cerevisiae . Growth temperature of cells was critical for efficient production of α‐amylase. P. pastoris transformants secreted both core‐glycosylated and non‐glycosylated α‐amylase molecules with a glycosylated:non‐glycosylated ratio of about 20:80. Both glycosylated and non‐glycosylated α‐amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non‐glycosylated α‐amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse α‐amylase secreted by P. pastoris . Copyright © 2001 John Wiley & Sons, Ltd.