Watzke Anja, Gutierrez-Rodriguez Marta, Köhn Maja, Wacker Ron, Schroeder Hendrik, Breinbauer Rolf, Kuhlmann Jürgen, Alexandrov Kirill, Niemeyer Christof M, Goody Roger S, Waldmann Herbert
Max-Planck-Institut für molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Bioorg Med Chem. 2006 Sep 15;14(18):6288-306. doi: 10.1016/j.bmc.2006.05.006. Epub 2006 May 24.
Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.
表达蛋白连接（EPL）和基于马来酰亚胺基团的生物共轭（MIC共轭）为蛋白质修饰提供了强大的工具。鉴于位点选择性修饰的蛋白质对于蛋白质功能研究的重要性，开发了一种利用EPL和MIC共轭将标签和报告基团引入蛋白质C末端的灵活方法。我们描述了一种通用构建模块的固相合成，该构建模块配备了荧光标记或不同的官能团。这种通用构建模块允许将不同的标签灵活地掺入蛋白质中，并通过EPL或MIC共轭技术用于将荧光标记引入Rab和Ras GTP酶的C末端。此外，还合成了一种经过适当修饰以将叠氮化物掺入蛋白质中的构建模块。通过施陶丁格连接将叠氮化物功能化的Ras蛋白固定在膦修饰的表面上，从而提供了一种用于蛋白质固定的高度化学选择性连接方法。
