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 ¦Á-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
- 出版时间：August, 2012
- 年：2012
- 卷：1824
- 期：8
- 页码：954-962
- 全文大小：6127 K
文摘
The medaka fish ¦Á-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 ¦Á-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and KM and Vmax 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 (¦Á/¦Â)8 barrel fold, as do other known ¦Á-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) ¦Á-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.