Hohsaka Takahiro, Abe Ryoji, Shiraga Kaori, Sisido Masahiko
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan.
Nucleic Acids Res Suppl. 2003(3):271-2. doi: 10.1093/nass/3.1.271.
Various nonnatural amino acids has been incorporated into proteins by using four-base codons in an E. coli in vitro translation system. Here, design and synthesis of novel fluorescently labeled nonnatural amino acids and their incorporation into proteins were investigated. Transfer RNAs that contained a CCCG anticodon and were aminoacylated with BODIPY FL-labeled amino acids were prepared by a chemical aminoacylation method, and added to an in vitro translation system in the presence of a streptavidin mRNA containing a CGGG codon. SDS-PAGE and Western blot analysis of the synthesized proteins indicate that BODIPY FL-labeled aminophenylalanine derivatives are efficiently incorporated into proteins through the four-base codon decoding.
通过在大肠杆菌体外翻译系统中使用四碱基密码子，各种非天然氨基酸已被整合到蛋白质中。在此，对新型荧光标记非天然氨基酸的设计与合成及其整合到蛋白质中的情况进行了研究。通过化学氨酰化方法制备了含有CCCG反密码子并被BODIPY FL标记氨基酸氨酰化的转运RNA，并在含有CGGG密码子的链霉抗生物素蛋白mRNA存在的情况下添加到体外翻译系统中。对合成蛋白质的SDS-PAGE和蛋白质印迹分析表明，BODIPY FL标记的氨基苯丙氨酸衍生物通过四碱基密码子解码有效地整合到蛋白质中。
