Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

遗传密码扩展是一种强大的技术，用于将非天然氨基酸位点特异性掺入感兴趣的蛋白质中。该技术依赖于正交氨酰-tRNA 合成酶/tRNA 对，并能够将 100 多种不同的非天然氨基酸掺入细胞中核糖体合成的蛋白质中。吡咯赖氨酰-tRNA 合成酶 (PylRS) 及其来自Methanosarcina的同源 tRNA物种可以说是使用最广泛的正交对。在这里，我们研究了由一个物种的 PylRS 的 N 末端突变引起的非天然氨基酸掺入的有益效果是否可转移到另一个物种的 PylRS。结果表明，MmPylRS N 末端结构域上的保守突变将非天然氨基酸掺入效率提高了五倍。由于 MbPylRS 与 MmPylRS 具有高序列同一性，并且这两个同源物经常互换使用，我们检查了四个 MbPylRS 变体在两个温度下对五个非天然氨基酸的掺入。我们的结果表明，MmPylRS 中有益的 N 末端突变并未提高 MbPylRS 的非天然氨基酸掺入效率。