期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：24
页码：8882-8887
DOI：10.1073/pnas.0307029101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNATrp ([IMG]f1.gif" BORDER="0">) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNATrp was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the [IMG]f1.gif" BORDER="0"> gene was inserted between the 5' and 3' flanking sequences of the tRNATrp-1 gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate [IMG]f1.gif" BORDER="0"> and no endogenous mammalian tRNAs. Similarly, the [IMG]f1.gif" BORDER="0"> is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-L-tryptophan. The resulting mutant [IMG]f2.gif" BORDER="0"> pair allows the efficient and selective incorporation of 5-hydroxy-L-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.
