Efficient expression, purification and characterization of mouse salivary alpha-amylase secreted from methylotrophic yeast, Pichia pastoris.
We constructed a secretion vector of mouse salivary alpha-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 (approximately 240 microg/ml) of mouse alpha-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 alpha-amylase. P. pastoris transformants secreted both core-glycosylated and non-glycosylated alpha-amylase molecules with a glycosylated:non-glycosylated ratio of about 20:80. Both glycosylated and non-glycosylated alpha-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 alpha-amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse alpha-amylase secreted by P. pastoris.
Kato
S
S
Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Ishibashi
M
M
Tatsuda
D
D
Tokunaga
H
H
Tokunaga
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; D000516:alpha-Amylases