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SARS-CoV-2 メインプロテアゼを一時的な人工ジモゲンに閉じ込め,高収量発現と簡素化された浄化を可能にします

Pavel Novotný¹, Adéla Moravcová², Veronika Nováková³

¹Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic; Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.

International journal of biological macromolecules

|February 13, 2026

まとめ

3つの発現戦略により,研究用の高品質のSARS-CoV-2メインプロテアゼ (Mpro) が得られます. すべての方法は,生化学的および構造的研究に適した,本物のN-末端を持つ活性Mproを生成します.

科学分野:

バイオケミストリーバイオケミストリー
構造生物学構造生物学とは
ウイルス学ウイルス学ウイルス学

背景:

SARS-CoV-2の複製は,ポリタンパク質処理のためのメインプロテアゼ (Mpro) に依存しています.
アクティブMproは,完全な酵素機能のために本物のN端を必要とします.
高品質のMproは,生化学的,構造的,および抗ウイルス研究に不可欠です.

研究の目的:

活性SARS-CoV-2 Mpro.を発現するための3つの異なるベクトル設計戦略を比較する.
最適な活動のための本物のN端を持つMproを取得するための方法を評価する.
発現したMproの生物物理学的および結晶化研究のための適性を決定する.

主な方法:

自動処理融合: SUMO-Mpro-HisTagコンストラクタは,自己触媒的な放出のためのネイティブ・クリバージ・サイトを備えています.
外部プロテアゼ分裂:HisTag-SUMO-Mpro融合はULP-1プロテアゼを必要とする.
自己処理の変種:HisTag-SUMOは,単純化された浄化のための変異した割れ部位を介してMproとリンクされています.

主要な成果:

この3つの戦略はすべて,本物のN-terminiを持つMproを成功裏に生産しました.
表現されたMproは,比較可能なN端子,円形の二重化スペクトル,運動パラメータ,および熱安定性を示した.
その結果生成されるMproタンパク質は,生体物理学的分析と結晶化に適しています.

結論:

複数の発現戦略により,研究に適した活性SARS-CoV-2 Mproが得られます.
発現戦略の選択は,N末端の改変に敏感な他のプロテアゼにも適応することができます.
これらの方法は,重要な生化学的および構造的調査のための高品質のMproの生産を促進します.

キーワード:

プロテイン浄化タンパク質の浄化 SARS-CoV-2 メインプロテアゼサイモゲン (Zymogen) とは

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