Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein
Sense codon-dependent introduction of unnatural amino acids into multiple sites of a protein
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DOI：
10.1006/bbrc.2000.2556
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发表时间：
2000-04-21
影响因子：
3.1
通讯作者：
Takaku, H
中科院分区：
文献类型：
--
作者：
Kanda, T;Takai, K;Takaku, H
由大肠杆菌的粗 S30 提取物驱动的无细胞蛋白质合成已应用于制备在特定位置含有非天然氨基酸的蛋白质。我们开发了通过反义处理灭活粗制大肠杆菌 tRNA 中 tRNA(Asp) 和 tRNA(Phe) 的方法，以及消化 S30 提取物中大部分 tRNA 的方法，而不会对核糖体活性造成本质损害。在本研究中，应用这些方法用非天然氨基酸取代 HIV-1 蛋白酶的 Asp 和 Phe 残基。使用10 mM Mg2+，翻译效率高于其他测试浓度，并且误读效率较低。在反义DNA处理的tRNA混合物和2-萘基丙氨酰-和/或对苯基偶氮苯丙氨酰-tRNA存在下翻译蛋白酶mRNA。结果表明，很大一部分翻译产物在最初由 Asp 或 Phe 占据的所有七个位置上都被取代。 (C) 2000 年学术出版社。
Cell-free protein synthesis, driven by a crude S30 extract from Escherichia coli, has been applied to the preparation of proteins containing unnatural amino acids at specific positions. We have developed methods for inactivating tRNA(Asp) and tRNA(Phe) within a crude E. coli tRNA by an antisense treatment and for digesting most of the tRNA within the S30 extract without essentail damage to the ribosomal activity. In the present study, are applied these methods to the substitution of Asp and Phe residues of the HIV-1 protease with unnatural amino acids. With 10 mM Mg2+, the translation efficiency was higher than that with the other tested concentration, and the misreading efficiency was low, The protease mRNA was translated in the presence of an antisense DNA-treated tRNA mixture and 2-naphthylalanyl- and/or p-phenylazophenylalanyl-tRNA. The results suggest that a good portion of the translation products are substituted at all of the seven positions originally occupied by Asp or Phe. (C) 2000 Academic Press.