NEXT-A (n端扩展与转移酶和ARS)反应。

Masumi Taki, Hiroyuki Kuroiwa, Masahiko Sisido
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引用次数: 5

摘要

L/ f转移酶催化疏水氨基酸从氨基酰基tRNA转移到以赖氨酸或精氨酸为n端的蛋白质的n端。将L/ f转移酶与大肠杆菌苯丙烯酰trna合成酶(ARS)结合,实现了非核糖体n端特异性引入多种非天然氨基酸到蛋白质上。一种非天然氨基酸一旦被突变的ARS原位加载到大肠杆菌tRNA(Phe)上,并依次从tRNA转移到靶蛋白上,即NEXT-A反应。除了在ARS上的alphaA294G突变外,通过NEXT-A反应,alphaat251a、betaG318W、betaA356W双突变均能有效提高引种效率。还演示了NEXT-A反应后Huisgen环加成的蛋白质特异性荧光标记。
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The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

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