Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris |
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Authors: | Kimihiko Mizutani Mayuko ToyodaYuichiro Otake Soshi YoshiokaNobuyuki Takahashi Bunzo Mikami |
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Institution: | Laboratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan |
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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 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 (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. |
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Keywords: | OLAmy Oryzias latipes α-amylase PCR polymerase chain reaction CBB Coomassie Brilliant Blue PAGE polyacrylamide gel electrophoresis GPCR G protein-coupled receptor |
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