Engineering Thermal Properties of Elastin-like Polypeptides by Incorporation of Unnatural Amino Acids in a Cell-free Protein Synthesis System
Engineering Thermal Properties of Elastin-like Polypeptides by Incorporation of Unnatural Amino Acids in a Cell-free Protein Synthesis System
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DOI：
10.1007/s12257-015-0190-1
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发表时间：
2015-06-01
影响因子：
3.2
通讯作者：
Kim, Dong-Myung
中科院分区：
文献类型：
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作者：
Catherine, Christy;Oh, Su Jin;Kim, Dong-Myung
基于蛋白质合成的中心法则，传统的蛋白质工程方法要求改变蛋白质结构和功能必须伴随着改变编码蛋白质的基因的核苷酸序列。然而，为每种蛋白质制备模板基因需要大量的时间和精力，从而限制了研究工程蛋白质的通量和范围。在这项研究中，我们描述了使用无细胞蛋白质合成的蛋白质的纯化水平工程。利用氨酰tRNA合成酶在接受结构相似的氨基酸类似物方面的混杂性，将非天然氨基酸引入弹性蛋白样多肽中以代替相应的同源氨基酸。通过引入不同的类似物，并从相同的基因开始，弹性蛋白样多肽的相变温度变得可调。我们的研究结果表明，无细胞蛋白质合成蛋白质工程使用非天然氨基酸，而不需要克隆的有用性。
Based on the central dogma of protein synthesis, traditional methods for protein engineering require that altering protein structure and function must be accompanied by changing the nucleotide sequence of the genes encoding the protein. However, the preparation of a template gene for each individual protein requires a great deal of time and effort, thereby limiting the throughput and scope of studying engineered proteins. In this study, we describe translation-level engineering of proteins using cell-free protein synthesis. Taking advantage of the promiscuity of aminoacyl tRNA synthetases in accepting structurally similar amino acid analogues, unnatural amino acids were introduced into elastin-like polypeptides in place of the corresponding cognate amino acids. Through the incorporation of various analogues and starting from the same gene, the phase transition temperatures of elastin-like polypeptides became tunable. Our results demonstrate the usefulness of cell-free protein synthesis for protein engineering using unnatural amino acids without the need for cloning.