Progress toward the evolution of an organism with an expanded genetic code

Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo. An “orthogonal” suppressor tRNA was derived from Saccharomyces cerevisiae tRNA(2)(Gln). This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E. coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E. coli translational machinery. Importantly, S. cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E. coli, but does not charge E. coli tRNA(Gln). This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E. coli suitable for the delivery of unnatural amino acids into proteins in vivo. A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA. Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids. The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E. coli. Implications of these results are discussed.

针对活体内蛋白质的非天然氨基酸定点掺入通用方法，本研究已完成多项关键性进展。本研究从酿酒酵母（Saccharomyces cerevisiae）的谷氨酰胺tRNA₂（tRNA²(Gln)）中衍生得到一种正交抑制型tRNA（orthogonal suppressor tRNA）。该酵母来源的正交抑制型tRNA，无法被任何大肠杆菌（Escherichia coli）氨酰-tRNA合成酶（aminoacyl-tRNA synthetase）在体外或活体内识别为底物，但可在大肠杆菌的翻译装置中正常发挥功能。尤为重要的是，酿酒酵母来源的GlnRS可在体外及大肠杆菌体内对该酵母正交抑制型tRNA进行氨酰化修饰，但无法氨酰化修饰大肠杆菌的谷氨酰胺tRNA（tRNA(Gln)）。因此，该酵母来源的抑制型tRNA与酿酒酵母GlnRS共同构成了一套在大肠杆菌中完全正交的tRNA/合成酶对，适用于在活体内将非天然氨基酸递送至蛋白质中。本研究开发了一种通用筛选方法，可用于筛选能够将任何核糖体可接纳的分子加载至正交抑制型tRNA上的突变型氨酰-tRNA合成酶。最后，本研究开发了一种快速非放射性筛选方法，用于检测非天然氨基酸的摄取效率，并将其应用于138种待测氨基酸的集合筛选。研究发现，在所检测的谷氨酰胺与谷氨酸类似物中，绝大多数均可被大肠杆菌摄取。本文对上述结果的研究意义展开了讨论。