Krebs Simon K, Rakotoarinoro Nathanaël, Stech Marlitt, Zemella Anne, Kubick Stefan
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
Institute for Biotechnology, Technical University of Berlin, Berlin, Germany.
Front Bioeng Biotechnol. 2022 Apr 29;10:873906. doi: 10.3389/fbioe.2022.873906. eCollection 2022.
Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components tyrosyl-tRNA synthetase (TyrRS) and tRNATyr were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).
通过琥珀密码子抑制作用掺入具有生物正交反应基团的非标准氨基酸（ncAA），可以生成具有所需新特性的合成蛋白质。这类修饰分子在基础研究以及癌症治疗和成像等治疗应用中需求很大。为了维持蛋白质功能、实现含ncAA蛋白质的高产率并实现有效偶联，ncAA响应密码子在蛋白质编码序列中的定位至关重要。由于无细胞系统的开放性及其易于操作的特点，无细胞ncAA掺入尤其受到关注。在本研究中，我们报告了一种简单的工作流程，用于在中华仓鼠卵巢（CHO）无细胞系统中探究ncAA掺入效率和蛋白质功能方面的ncAA位置。作为模型，使用成熟的正交翻译组件酪氨酰 - tRNA合成酶（TyrRS）和tRNATyr，以位点特异性方式掺入ncAA对叠氮 - L - 苯丙氨酸（AzF）以响应UAG密码子。对N端融合红色荧光蛋白mRFP1且C端融合绿色荧光蛋白sfGFP的抗表皮生长因子受体（EGFR）单链可变片段（scFv）内的总共七个ncAA位点进行了ncAA掺入效率及对抗原结合影响的研究。所表征的无细胞双荧光报告系统允许筛选具有高掺入效率且能维持蛋白质活性的ncAA掺入位点。它可并行化、可扩展且易于操作。我们提出，所建立的基于CHO的无细胞双荧光报告系统对于抗体 - 药物偶联物（ADC）的开发可能特别有意义。
