An efficient system for the evolution of aminoacyl-tRNA synthetase specificity
An efficient system for the evolution of aminoacyl-tRNA synthetase specificity
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DOI：
10.1038/nbt742
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发表时间：
2002-10-01
影响因子：
46.9
通讯作者：
Schultz, PG
中科院分区：
文献类型：
--
作者：
Santoro, SW;Wang, L;Schultz, PG
已经探索了将非天然氨基酸掺入蛋白质中的多种策略（1-5），但所有策略在技术可及性、可扩展性、对体内研究的适用性或氨基酸掺入的位点特异性方面都具有局限性。在体内将非天然官能团选择性引入蛋白质内特定位点的能力6，7为蛋白质功能的研究和新型蛋白质的大规模生产提供了潜在的强有力的方法。在这里，我们描述了一个组合的遗传选择和屏幕，允许氨酰-tRNA合成酶底物特异性的快速进化。我们的策略涉及使用“正交”氨酰-tRNA合成酶和tRNA对，其不能与大肠杆菌中的任何内源性合成酶-tRNA对相互作用（8-11）。氯霉素抗性（Cm-r）报告基因用于选择高活性合成酶变体，并且可扩增的荧光报告基因与荧光激活细胞分选（FACS）一起用于筛选具有所需氨基酸特异性变化的变体。这两种报告基因都包含在一个单一的遗传构建体中，消除了质粒穿梭的需要，并允许在几天内完成进化。在进化之后，可重复的荧光报告子允许合成酶活性和选择性的视觉和荧光评价。使用这个系统来探索酪氨酰-tRNA合成酶的氨基酸结合口袋的可进化性，我们确定了三个新的变体，允许选择性地将氨基，异丙基和烯丙基含酪氨酸类似物掺入到所需的蛋白质中。这种新的酶可用于在大肠杆菌中生产毫克/升的非天然氨基酸蛋白质。杆菌
A variety of strategies to incorporate unnatural amino acids into proteins have been pursued(1-5), but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo 6,7, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli(8-11). A chloramphenicol-resistance (Cm-r) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.