32個のプロリン残基を非正規のアミノ酸に置き換えると,高度に活性なDNAポリメラーゼが生じる
Bastian Holzberger1, Andreas Marx
1Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraβe 10, 78457 Konstanz, Germany.
Journal of the American Chemical Society
|October 22, 2010
まとめ
タンパク質工学では,非正規のアミノ酸をDNAポリメラーゼに成功裏に組み込みました. この多化酵素は重要な機能を保持し,将来のタンパク質工学アプリケーションの有望性を示しました.
科学分野:
- バイオケミストリー バイオケミストリー
- 分子生物学は分子生物学である.
- プロテイン工学は,タンパク質の
背景:
- プロテイン工学は,伝統的に20の天然アミノ酸を使用しています.
- 非正規のアミノ酸は,タンパク質の設計に新しい機能性を提供します.
- DNAポリメラーゼは,DNA合成に不可欠な動的酵素である.
研究 の 目的:
- 非正規のアミノ酸を用いて多化DNAポリメラーゼを生成する.
- 非正規のアミノ酸置換が酵素の活性,選択性,安定性に与える影響を評価する.
- 高度にダイナミックな酵素のエンジニアリングにおける非正規のアミノ酸の可能性を調査する.
主な方法:
- KlenTaq DNAポリメラーゼで32個のプロリン残基を (4R) - フロロプロリンで全体的に置き換えること.
- プライマー拡張とPCRアッセイを用いた酵素活性調査.
- 酸化酵素を親の野生型酵素と比較した.
主要な成果:
- プロリン残基を (4R) - フロロロプロリンで効率的に (92%) 置き換えた.
- その結果生じた酸化DNAポリメラーゼは,高い活性を示し,忠誠性と感受性を保持した.
- 酵素は,野生のタイプと比較して,熱安定性がわずかに低下したことを示した.
結論:
- 非正規のアミノ酸の置換は,重要な性質を損なうことなく,大きく,ダイナミックな酵素で実現可能である.
- 酸化DNAポリメラーゼは,さらなるタンパク質工学のための貴重なツールと出発点として機能します.
- この研究は,非正規のアミノ酸の有用性を実証することによって,タンパク質工学のツールキットを拡張します.
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