Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells
Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells
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DOI：
10.1021/ja071773r
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发表时间：
2007-08-29
影响因子：
15
通讯作者：
Tirrell, David A.
中科院分区：
文献类型：
--
作者：
Kwon, Inchan;Tirrell, David A.
设计的酵母苯丙氨酰-tRNA合成酶（yPheRS（T415 G））激活四种色氨酸（Trp）类似物（6-氯色氨酸（6ClW）、6-溴色氨酸（6 BrW）、5-溴色氨酸（5 BrW）和苯并噻吩丙氨酸（BT）），这些色氨酸类似物不被内源性大肠杆菌利用。大肠杆菌翻译器。将yPheRS（T415 G）和突变型酵母苯丙氨酸琥珀抑制基因tRNA（ytRNA（CUA_UG）（Phe））导入E.大肠杆菌表达宿主允许这些类似物中的三种（6ClW、6 BrW和BT）响应于琥珀终止密码子以至少98%的保真度位点特异性地掺入重组鼠二氢叶酸还原酶中。所有这三种类似物都被引入到青色荧光蛋白变体（CFP 6）的发色团中的Trp 66位置，以研究光谱特性的随之而来的变化。每个类似物引起的荧光发射和吸收最大值的蓝移。在位置66处带有BT的CFP 6变体表现出异常大的斯托克斯位移（56 nm）。一套扩大的遗传编码的色氨酸类似物应该能够设计新的蛋白质与新的光谱特性。
A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNA(CUA_UG)(Phe)) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.