Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.
Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.
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DOI：
10.1016/j.bbagen.2016.12.002
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发表时间：
2017-11
期刊：
影响因子：
--
通讯作者：
Fan C
中科院分区：
文献类型：
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作者：
Gan Q;Fan C
无细胞蛋白质合成为非规范氨基酸（ncAA）与蛋白质的共翻译结合提供了一个强大的平台，以促进生物学研究和生物技术应用。最近，消除释放因子1的活性已被证明可以增加琥珀色密码子对ncAA的掺入。然而，这种方法可能通过近同源抑制促进典型氨基酸的错误结合。我们执行了一个简单的方案，从总tRNA中去除琥珀色密码子的近同源tRNA同受体，并使用磷酸丝氨酸（Sep）结合系统作为验证。通过操纵目标基因的密码子使用和tRNA物种引入无细胞蛋白合成系统，我们提高了Sep在特定位置结合的保真度。通过从总tRNA中去除琥珀色终止密码子的三个近同源tRNA同受体[tRNALys， tRNATyr和tRNAGln(CUG)]，近同源抑制降低了5倍，而不损害无细胞蛋白质合成系统中的正常蛋白质合成。质谱分析表明，ncAA结合的保真度有所提高。在释放因子缺乏系统中，去除琥珀色密码子的近同源tRNA同受体可以增加ncAA对琥珀色停止密码子的结合保真度。我们提供了一种通用的策略来提高ncAA在无细胞蛋白质合成系统中与停止、四重和感觉密码子结合的保真度。
Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems.