Taira Hikaru, Matsushita Yosuke, Kojima Kenji, Shiraga Kaori, Hohsaka Takahiro
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Biochem Biophys Res Commun. 2008 Sep 19;374(2):304-8. doi: 10.1016/j.bbrc.2008.07.020. Epub 2008 Jul 15.
Incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 can competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia coli K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS-PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricolum Trp(1) tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into proteins in response to the amber codon.
响应琥珀密码子或四碱基密码子将非天然氨基酸掺入蛋白质中是蛋白质研究中的一项有用技术。然而，就琥珀密码子而言，释放因子1可以竞争性地解码相同的密码子，从而抑制非天然氨基酸的掺入。为了改进琥珀密码子介导的掺入，我们对源自大肠杆菌K12和山羊支原体基因组中编码的所有tRNA的琥珀抑制tRNA进行了全面筛选。琥珀抑制tRNA由合成基因合成，用荧光非天然氨基酸进行氨酰化，并添加到大肠杆菌无细胞翻译系统中。荧光SDS-PAGE分析表明，色氨酸tRNA在两种生物体中均表现出高抑制活性。进一步的诱变和筛选表明，具有G1C72A73突变的山羊支原体色氨酸(1)tRNA最适合于响应琥珀密码子将非天然氨基酸高效且特异性地掺入蛋白质中。
