title: Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris.
abstract: The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.
authors.affiliation: Graduate School of Agriculture, Kyoto University, Kyoto, Japan. kmizutani@kais.kyoto-u.ac.jp
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Mizutani
authors.forename: Mayuko
authors.initials: M
authors.lastname: Toyoda
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Otake
authors.forename: Soshi
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002712:Chlorides; D018360:Crystallography, X-Ray; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009990:Oryzias; D010843:Pichia / Q000235:genetics; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002712:Chlorides; D029941:Fish Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases