Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes
Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes
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DOI：
10.1002/cbic.201402033
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发表时间：
2014-08-18
期刊：
影响因子：
3.2
通讯作者：
Neumann, Heinz
中科院分区：
文献类型：
--
作者：
Lammers, Christoph;Hahn, Liljan E.;Neumann, Heinz
将多种不同的非天然氨基酸结合到同一多肽中仍然是一个重大挑战。正交核糖体是可进化的，因为它们指导单个专用的正交mRNA的翻译，可以提供常规生产这种多肽的途径。正交核糖体工程的最新进展创造了一个原型系统，尽管效率有限，但可以通过基因编码引入两个不同的功能基团。在这里，我们系统地研究了该系统的限制因素，通过测定单个组分的水平和活性；我们确定了barkeri Methanosarcina PylRS是蛋白产量的限制因子。平衡各组分的表达水平可显著提高生长速度和蛋白质产量。该系统的优化可能会增加进化的正交核糖体介导的多种不同的非天然氨基酸结合的范围。
Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.