Saks M E, Sampson J R, Nowak M W, Kearney P C, Du F, Abelson J N, Lester H A, Dougherty D A
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 1996 Sep 20;271(38):23169-75. doi: 10.1074/jbc.271.38.23169.
A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.
一种新的tRNA，即THG73，已被设计并评估为一种载体，可利用终止密码子抑制技术将非天然氨基酸位点特异性地掺入体内表达的蛋白质中。该构建体是嗜热四膜虫tRNAGln(CUA)的一种修饰，它天然识别终止密码子UAG。通过对烟碱型乙酰胆碱受体多个位点的突变进行电生理研究，证实THG73在非天然氨基酸掺入的效率和保真度方面均比以前的无义抑制子有了重大改进。与之前用于体内抑制的tRNA相比，THG73被非洲爪蟾卵母细胞内源性合成酶酰化的可能性低至100倍。这有效地消除了体内抑制方法的一个主要问题，即在抑制位点意外掺入天然氨基酸。此外，THG73在卵母细胞系统中掺入非天然氨基酸的效率要高4至10倍。综上所述，这两项进展应能极大地扩展体内无义抑制方法的适用范围。
