The ability to genetically encode non-natural amino acids (nnAAs) into proteins offers an expanded tool set for protein engineering. nnAAs containing unique functional moieties have enabled the study of post-translational modifications, protein interactions, and protein folding. In addition, nnAAs have been developed that enable a variety of biorthogonal conjugation chemistries that allow precise and efficient protein conjugations. These are being studied to create the next generation of antibody-drug conjugates with improved efficacy, potency, and stability for the treatment of cancer. However, the efficiency of nnAA incorporation, and the productive yields of cell-based expression systems, have limited the utility and widespread use of this technology. We developed a process to isolate stable cell lines expressing a pyrrolysyl-tRNA synthetase/tRNApyl pair capable of efficient nnAA incorporation. Two different platform cell lines generated by these methods were used to produce IgG-expressing cell lines with normalized antibody titers of 3 g/L using continuous perfusion. We show that the antibodies produced by these platform cells contain the nnAA functionality that enables facile conjugations. Characterization of these highly active and robust platform hosts identified key parameters that affect nnAA incorporation efficiency. These highly efficient host platforms may help overcome the expression challenges that have impeded the developability of this technology for manufacturing proteins with nnAAs and represents an important step in expanding its utility.

中文翻译：

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开发用于在蛋白质序列中引入非天然氨基酸的高产表达平台。


将非天然氨基酸 (nnAA) 基因编码为蛋白质的能力为蛋白质工程提供了扩展的工具集。含有独特功能部分的 nnAA 使得翻译后修饰、蛋白质相互作用和蛋白质折叠的研究成为可能。此外，已开发出 nnAA，可实现多种双正交缀合化学，从而实现精确有效的蛋白质缀合。这些正在研究中，以创造下一代抗体药物缀合物，以提高癌症治疗的功效、效力和稳定性。然而，nnAA 掺入的效率以及基于细胞的表达系统的产量限制了该技术的实用性和广泛使用。我们开发了一种方法来分离表达能够有效掺入 nnAA 的吡咯赖氨酰-tRNA 合成酶/tRNApyl 对的稳定细胞系。通过这些方法产生的两种不同的平台细胞系用于通过连续灌注产生标准化抗体滴度为3g/L的表达IgG的细胞系。我们证明这些平台细胞产生的抗体含有 nnAA 功能，可以轻松缀合。这些高度活跃和强大的平台宿主的表征确定了影响 nnAA 掺入效率的关键参数。这些高效的宿主平台可能有助于克服阻碍该技术用 nnAA 制造蛋白质的表达挑战，并代表了扩大其实用性的重要一步。