Biocentre, Departments of Biology and Chemistry, Johannes Gutenberg University Mainz, Mainz, Germany.
Institute of Molecular Biology gGmbH, Mainz, Germany.
Methods Mol Biol. 2023;2563:341-369. doi: 10.1007/978-1-0716-2663-4_17.
Engineering new functionalities into living eukaryotic systems is one of the main goals of synthetic biology. To this end, often enzyme evolution or de novo protein design is employed, which each have their own advantages and disadvantages. As complimentary tools, we recently developed orthogonally translating and film-like synthetic organelles that allow to create new enzyme functionalities based on spatial separation. We applied this technology to genetic code expansion (GCE) and showed that it is possible to equip eukaryotic cells with multiple orthogonal genetic codes that enable the specific reprogramming of distinct translational machineries, each with single-residue precision.In this protocol, we describe how synthetic organelles can be used to perform mRNA selective GCE and how they can be further developed to allow the simultaneous incorporation of distinct noncanonical amino acids (ncAAs) into selected proteins and how this can be used to label proteins selectively with fluorescent dyes via bioorthogonal chemistry.
将新功能引入活的真核生物系统是合成生物学的主要目标之一。为此，通常采用酶进化或从头蛋白质设计，这两种方法各有优缺点。作为互补工具，我们最近开发了正交翻译和类膜合成细胞器，可基于空间分离来创建新的酶功能。我们将这项技术应用于遗传密码扩展 (GCE)，并表明可以为真核细胞配备多个正交遗传密码，从而能够特异性地重新编程不同的翻译机器，每个机器的精度均为单个残基。在本方案中，我们将描述如何使用合成细胞器进行 mRNA 选择性 GCE，以及如何进一步开发它们以允许同时将不同的非标准氨基酸 (ncAAs) 掺入选定的蛋白质中，以及如何通过生物正交化学选择性地用荧光染料标记蛋白质。
