Schloßhauer Jeffrey L, Dondapati Srujan Kumar, Kubick Stefan, Zemella Anne
Fraunhofer Project Group PZ-Syn of the Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Located at the Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany.
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg, 14476 Potsdam, Germany.
Bioengineering (Basel). 2024 Jan 18;11(1):92. doi: 10.3390/bioengineering11010092.
Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast , which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time.
无细胞系统对于筛选应用和生产难以表达的蛋白质特别有吸引力。然而，由于时间要求长、培养成本高以及用能量再生系统补充无细胞反应，细胞裂解物的生产难以大规模实施。因此，本研究利用广泛用于重组蛋白生产的甲基营养酵母，以经济有效的方式实现无细胞合成。评估了敏感的破坏条件，并确定了转运到内质网囊泡中的合适信号序列。开发了一种基于1,6-二磷酸果糖的替代能量再生系统，观察到蛋白质产量增加了约2倍。使用统计实验设计确定了无细胞反应环境的最佳组成。此外，还可以生产功能性离子通道，并使用新型无细胞系统对G蛋白偶联受体进行位点特异性修饰。最后，所建立的无细胞蛋白质生产系统可以在短时间内经济地生产用于生物技术应用的复杂蛋白质。
