RRC ID 20944
著者 Mizutani K, Toyoda M, Otake Y, Yoshioka S, Takahashi N, Mikami B.
タイトル Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris.
ジャーナル Biochim Biophys Acta
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.
巻・号 1824(8)
ページ 954-62
公開日 2012-8-1
DOI 10.1016/j.bbapap.2012.05.005
PII S1570-9639(12)00094-5
PMID 22613096
MeSH Amino Acid Sequence Animals Chlorides Crystallography, X-Ray Fish Proteins / chemistry* Fish Proteins / genetics Fish Proteins / isolation & purification Genetic Vectors / genetics Homologous Recombination Hydrogen-Ion Concentration Kinetics Molecular Sequence Data Oryzias* Pichia / genetics Protein Denaturation Protein Structure, Tertiary Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Temperature alpha-Amylases / chemistry* alpha-Amylases / genetics alpha-Amylases / isolation & purification
IF 3.411
引用数 7
WOS 分野 BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
酵母 BY2778
メダカ full length cDNA (olvl8b03)