Meier Jordan L, Mercer Andrew C, Rivera Heriberto, Burkart Michael D
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, USA.
J Am Chem Soc. 2006 Sep 20;128(37):12174-84. doi: 10.1021/ja063217n.
In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.
体内载体蛋白标记最近已成为融合系统位点特异性修饰和天然产物蛋白质组学新方法的一个有吸引力的目标。为了确定活细胞内共价蛋白质标记的合适伙伴，对泛硫乙胺类似物进行了详细研究。开发了一种快速合成泛酰胺类似物的方法，并用于制备一组用于体外和体内蛋白质标记评估的样品。动力学比较有助于构建结构-活性关系，以确定载体蛋白标记所选择的连接子、染料和生物正交报告基团。最后，生物正交泛硫乙胺类似物在体内被证明能以高特异性靶向载体蛋白，并在粗细胞裂解物中与报告基团进行化学选择性连接。本文展示的方法首次使得无需抗体技术就能对载体蛋白进行可视化和分离，并为载体蛋白融合在化学生物学中的未来应用奠定了基础。
