Institute for Molecular Bioscience and the Australian Institute for Bioengeneering and Nanotechnology, The University of Queensland, St. Lucia, Queensland 4072, Australia.
J Am Chem Soc. 2015 Apr 8;137(13):4404-13. doi: 10.1021/ja5131963. Epub 2015 Mar 30.
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.
遗传密码扩展是合成生物学和蛋白质工程的主要目标。这方面的大多数努力都集中在重新分配终止或解码四联体密码子上。虽然遗传密码的冗余性提供了大量潜在可重新分配的密码子，但由于与同源氨酰-tRNA 的不可避免相互作用，它们的用途受到了限制。为了解决这个问题，我们试图建立一个具有简化的合成 tRNA 补充的体外蛋白质合成系统，从而使一些有意义的密码子正交化。这种定量的体外肽合成测定法使我们能够分析合成 tRNA 解码所有 61 个有意义密码子的能力。我们观察到，除了天冬酰胺、谷氨酸和异亮氨酸的同功受体外，大多数 48 种合成大肠杆菌 tRNA 可以在无细胞系统中支持蛋白质翻译。我们从天然大肠杆菌 tRNA 混合物中纯化为同质功能的天冬酰胺、谷氨酸和异亮氨酸 tRNA，并通过将它们与合成 tRNA 结合，为所有 20 种氨基酸制定了半合成 tRNA 补充。我们进一步证明，这种 tRNA 补充可以将 tRNA 耗尽的大肠杆菌裂解物的蛋白质翻译活性恢复到与总天然 tRNA 相当的水平。为了确认开发的系统可以有效地合成长多肽，我们表达了三种不同的编码超折叠 GFP 的序列。这种新型半合成翻译系统是 tRNA 工程的有力工具，可能使至少 9 个编码丝氨酸、精氨酸、亮氨酸、脯氨酸、苏氨酸和甘氨酸的有意义密码子重新分配成为可能。
