The Texas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX, USA.
Methods Mol Biol. 2022;2530:159-167. doi: 10.1007/978-1-0716-2489-0_11.
Proteins with a functionalized C-terminus are critical to synthesizing large proteins via expressed protein ligation. To overcome the limitations of currently available C-terminus functionalization strategies, we established an approach based on a small molecule cyanylating reagent that chemically activates a cysteine in a recombinant protein at its N-side amide for undergoing nucleophilic acyl substitution with amines. We demonstrated the versatility of this approach by successfully synthesizing RNAse H with its RNA hydrolyzing activity restored and in vitro nucleosome build with a C-terminal posttranslational modified histone H2A. This technique will expand the landscape of protein chemical synthesis and its application in new research fields significantly.
具有功能化 C 末端的蛋白质对于通过表达蛋白连接合成大蛋白至关重要。为了克服当前可用的 C 末端功能化策略的限制，我们建立了一种基于小分子氰基化试剂的方法，该试剂可在重组蛋白的 N-侧酰胺中化学激活半胱氨酸，使其与胺发生亲核酰基取代。我们通过成功合成具有恢复 RNA 水解活性的 RNAse H 和带有 C 末端翻译后修饰组蛋白 H2A 的体外核小体构建，证明了这种方法的多功能性。这项技术将极大地扩展蛋白质化学合成及其在新研究领域应用的范围。
