Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis
Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis
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DOI：
10.1021/ja5131963
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发表时间：
2015-04-08
影响因子：
15
通讯作者：
Alexandrov, Kirill
中科院分区：
文献类型：
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作者：
Cui, Zhenling;Stein, Viktor;Alexandrov, Kirill
遗传密码扩展是合成生物学和蛋白质工程的关键目标。这方面的大部分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提供了大量潜在的可重新分配的密码子，但它们的效用因与同源氨酰-tRNA的不可避免的相互作用而减少。为了解决这个问题，我们试图建立一个体外蛋白质合成系统与一个简化的合成tRNA互补，从而正交化的一些有义密码子。这种定量的体外肽合成测定使我们能够分析合成的tRNA解码所有61个有义密码子的能力。我们观察到，除了Asn，Glu和Ile的isoceptor外，大多数48种合成的大肠杆菌tRNA可以支持无细胞系统中的蛋白质翻译。我们从天然大肠杆菌中纯化了同质功能性Asn、Glu和Ile tRNA。大肠杆菌tRNA混合物，并通过将它们与合成的tRNA结合，我们配制了所有20种氨基酸的半合成tRNA互补物。我们进一步证明，这种tRNA互补可以恢复tRNA缺失的E.大肠杆菌裂解物的水平相当的总天然tRNA。为了证实所开发的系统可以有效地合成长多肽，我们表达了编码超折叠GFP的三种不同序列。这种新的半合成翻译系统是tRNA工程的有力工具，并且可能使编码Ser、Arg、Leu、Pro、Thr和Gly的至少9个有义密码子重新分配。
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.