van Eldijk Mark B, van Hest Jan C M
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.
Methods Mol Biol. 2018;1728:137-145. doi: 10.1007/978-1-4939-7574-7_8.
The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive handles (azides and alkynes) and hydrophobic fluorinated side chains. Here, we describe the efficient residue-specific replacement of methionine by azidonorleucine in an engineered green fluorescent protein using a bacterial expression system to introduce a single reactive site for the strain-promoted azide-alkyne cycloaddition.
非标准氨基酸的引入使蛋白质化学家能够使用范围更广的氨基酸。这种方法显著拓宽了蛋白质工程的范围，允许引入具有非天然功能的氨基酸，如生物正交反应基团（叠氮化物和炔烃）以及疏水性氟化侧链。在此，我们描述了在工程化绿色荧光蛋白中使用细菌表达系统，通过叠氮高亮氨酸高效地对甲硫氨酸进行位点特异性置换，以引入一个用于菌株促进的叠氮化物-炔烃环加成反应的单一反应位点。
