A new strategy for the site-specific modification of proteins in vivo
A new strategy for the site-specific modification of proteins in vivo
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DOI：
10.1021/bi0300231
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发表时间：
2003-06-10
期刊：
影响因子：
2.9
通讯作者：
Schultz, PG
中科院分区：
文献类型：
--
作者：
Zhang, ZW;Smith, BAC;Schultz, PG
我们最近开发了一种方法，将非天然氨基酸位点特异性地结合到大肠杆菌中表达的蛋白质中，以响应琥珀色无义密码子。在这里，我们描述了一个正交tRNA-TyrRS对的选择，选择性和有效地将间乙酰- l-苯丙氨酸结合到大肠杆菌的蛋白质中。我们证明含有间乙酰- l-苯丙氨酸或对乙酰- l-苯丙氨酸的蛋白质不仅可以在体外而且可以在活细胞中选择性地用肼衍生物进行标记。标记反应是选择性的，通常产率为75%。在具体的例子中，m-乙酰基- l-苯丙氨酸取代了胞质蛋白Z结构域的Lys7和外膜蛋白LamB的Arg200，并用一系列荧光染料选择性地标记突变蛋白。将非蛋白质成因的“酮柄”遗传结合到蛋白质中，为引入生物物理探针用于体外或体内蛋白质的结构和功能分析提供了有力的工具。
We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-L-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-Lphenylalanine or p-acetyl-L-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-L-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.