Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni²⁺-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni²⁺–NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability

短鎖ぺプチドタグへの金属配位を利用したタンパク質化学修飾ツールの開発

京都大学新制・課程博士博士(工学)甲第24818号工博第5161号新制||工||1986(附属図書館)京都大学大学院工学研究科合成・生物化学専攻(主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA