Biocenter, Johannes Gutenberg University Mainz, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz, Germany; International PhD Programme of the Institute of Molecular Biology (IMB) gGmbH, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz, Germany.
Biocenter, Johannes Gutenberg University Mainz, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz, Germany; Biocenter, IMPRS on Cellular Biophysics, Johannes Gutenberg University Mainz, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz, Germany.
J Mol Biol. 2024 Nov 1;436(21):168728. doi: 10.1016/j.jmb.2024.168728. Epub 2024 Jul 30.
Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically.
通过遗传密码扩展（GCE）技术，可以实现非天然氨基酸（ncAAs）的特异性掺入。已经开发出不同的正交 tRNA 合成酶/tRNA（RS/tRNA）对，以在所需位置引入 ncAA，从而提供可安装到选定蛋白质中的各种功能。RS/tRNA 对的细胞质表达可能导致背景 ncAA 掺入宿主蛋白的问题。正交翻译细胞器（OTOs）的应用，受相分离概念的启发，为哺乳动物细胞中的这个问题提供了解决方案，允许特异性和蛋白质选择性 ncAA 掺入。到目前为止，只有 Methanosarcina mazei（Mm）吡咯赖氨酸-tRNA 合成酶（PylRS）已在 OTOs 中使用，限制了该方法的潜力。在这里，我们探索了使用 OTOs 实现另外四个广泛使用的正交 RS/tRNA 对的可行性，但令我们惊讶的是，它们未能成功实现 mRNA 选择性 GCE。接下来，我们测试了几种实验解决方案，并开发了一种新的苯丙氨酸-RS/tRNA 对，使 ncAA 能够以特异性和蛋白质选择性的方式掺入 OTOs。我们的工作揭示了使用设计细胞器进行酶功能空间工程的未被注意到的设计限制，并提出了一种在体内克服这些限制的策略。然后，我们讨论了一般细胞内工程和特别是使用 GCE 的蛋白质工程的当前限制和未来方向。
