Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

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• 作者：Kimihiko Mizutani ; ^{kmizutani@kais.kyoto-u.ac.jp} ; Mayuko Toyoda ; Yuichiro Otake ; Soshi Yoshioka ; Nobuyuki Takahashi ; Bunzo Mikami
• 关键词：OLAmy ; Oryzias latipes 伪-amylase ; PCR ; polymerase chain reaction ; CBB ; Coomassie Brilliant Blue ; PAGE ; polyacrylamide gel electrophoresis ; GPCR ; G protein-coupled receptor
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2012
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：August, 2012
• 卷：1824
• 期：8
• 页码：954-962
• 文件大小：6127 K

摘要

The medaka fish 伪-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 伪-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 (伪/尾)₈ barrel fold, as do other known 伪-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) 伪-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.