Spice Alex J, Aw Rochelle, Bracewell Daniel G, Polizzi Karen M
Department of Chemical Engineering, Imperial College London, London, UK.
Imperial College Centre for Synthetic Biology, Imperial College London, UK.
Synth Syst Biotechnol. 2020 Jun 23;5(3):137-144. doi: 10.1016/j.synbio.2020.06.003. eCollection 2020 Sep.
A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.
在合成生物学方法的加速应用推动下，无细胞蛋白质合成（CFPS）正在复兴。CFPS已成为用于合成基因网络设计、生物传感和按需生物制造的强大平台技术。虽然主要起源于细菌，但人们已经探索了源自多种宿主生物体的无细胞提取物，旨在利用细胞多样性以及与这些生物体相关的有利特性。然而，尽管真核生物产生的无细胞提取物具有改善蛋白质折叠和翻译后修饰的潜力，但由于其产量相对较低，往往被忽视。在此，我们描述了一种无细胞平台的进一步发展，该平台在学术界和生物制药行业都是广泛使用的表达宿主。通过使用简化的实验设计（DOE）方法，我们能够通过改进复杂反应混合物的组成来提高系统的生产力。这在最少的实验运行次数内实现，在设计的限制范围内，无需液体处理机器人。通过这样做，我们能够估计影响系统生产力的主要因素，并分别将萤火虫荧光素酶和生物制药产品人血清白蛋白（HSA）的蛋白质合成提高了4.8倍和3.5倍。这项研究突出了基于无细胞的系统作为一个高产的真核平台，并展示了简化的DOE设计的价值。
