講演・口頭発表等[R]

基本情報

氏名 新垣 知輝
氏名(カナ) シンガキ トモテル
氏名(英語) Shingaki Tomoteru
所属 薬学部 医療薬学科
職名 准教授
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タイトル

The effect of the expression of SmpB in an Escherichia coli cell-free protein synthesis 

講演者

 

会議名

パシフィコ横浜

発表年月日

2014/11/27

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国際共著

 

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概要

Cell-free protein synthesis system was developed as a model to study the mechanism of translation and succeeded to provide a lot of proteins even if its property suppressed cell survival. However, messenger RNA (mRNA) is easily degraded and the ribosomes were stalled by the defective mRNAs. The amount of synthesized protein was decrease by the stalled ribosomes. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) is essential components of the highly conserved tmRNA-SmpB system that has the function of releasing stalled ribosomes from damaged mRNAs. In this study we expressed SmpB in cell-free protein systems. The genes encoding SmpB and tmRNA were amplified by PCR from E. coli BL21DE3 strain genome and cloned into pURE-1 vector using a restriction site-free method. The obtained plasmid was transformed into E. coli JM109 strain. The plasmid was separated and purified. The mRNA of SmpB was expressed with in vitro transcription system.The translation of SmpB were carried out with PURESYSTEM or PURE Frex. The separation of the synthesized proteins were carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the case of PURE Frex, the protein which molecular weight was approximately 40 kDa was shifted when only SmpB was expressed. Meanwhile, the change of mobility was not observed in the case of PURESYSTEM. This protein contains in PURE Frex system and it is supposed that SmpB is interacted with 40 kDa protein regardless of stalled ribosome.

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