Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Nat Chem. 2018 Aug;10(8):831-837. doi: 10.1038/s41557-018-0052-5. Epub 2018 May 28.
Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/tRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmtRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmtRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/tRNA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/tRNA pairs that are mutually orthogonal to the MmPylRS/MmtRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/tRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.
将不同的非天然氨基酸（ncAAs）基因编码到细胞中合成的蛋白质中需要相互正交的氨酰-tRNA 合成酶（aaRS）/tRNA 对。已经对来源于 Methanosarcina mazei（Mm）的吡咯赖氨酸-tRNA 合成酶/tRNA 对进行了工程改造，以掺入各种 ncAAs，通常被认为是遗传密码扩展的理想配对。然而，寻找具有 MmPylRS/MmtRNA 对优点且与内源性 aaRS/tRNA 对和 MmPylRS/MmtRNA 对均正交的新 aaRS/tRNA 对一直具有挑战性。在这里，我们证明了几个缺乏 N 端结构域的 ΔNPylRS/tRNA 对在大肠杆菌中是活跃的、正交的并且能够有效地掺入 ncAAs。我们创建了新的 PylRS/tRNA 对，它们与 MmPylRS/MmtRNA 对是相互正交的，并表明将 MmPylRS 的 ncAA 特异性重新编程的突变移植到新的 PylRS 中可以重新编程其底物特异性。最后，我们表明不同的 PylRS/tRNA 衍生对可以在同一细胞中发挥作用，解码不同的密码子并掺入不同的 ncAAs。
