Fraunhofer Institute for Biomedical Engineering (IBMT), Branch Potsdam-Golm, Am Mühlenberg 13, 14476, Potsdam, Germany.
Biotechnol Bioeng. 2014 Jan;111(1):25-36. doi: 10.1002/bit.25013. Epub 2013 Sep 6.
Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins.
蛋白质表达系统广泛应用于生物技术和医学领域，用于高效、经济地生产治疗性蛋白。如今，经过培养的中国仓鼠卵巢（CHO）细胞是用于生产复杂治疗性蛋白的主导哺乳动物细胞系。尽管 CHO 细胞具有这种卓越的潜力，但迄今为止，尚未报道基于这些细胞翻译活性裂解物的高产无细胞系统。迄今为止，CHO 细胞提取物仅被用作基础性研究工具，用于了解 mRNA 翻译（Lodish 等人，1974；McDowell 等人，1972）。在本研究中，我们通过建立基于培养的 CHO 细胞提取物的新型无细胞蛋白表达系统来解决这一问题。我们考虑了裂解物制备、体外反应条件的适应以及特定表达载体的构建，以实现高产蛋白。为了获得最佳性能，构建了一种包含 Cricket 麻痹病毒（CrPV）基因间区（IGR）内部核糖体进入位点（IRES）的特定体外表达载体。IGR IRES 被认为直接与真核 40S 核糖体亚基结合，从而绕过无细胞系统中经常出现的翻译起始这一主要瓶颈。表达载体与优化的 CHO 细胞提取物的结合使我们能够在 4 小时内生产约 50µg/mL 的活性萤火虫荧光素。批式无细胞偶联转录-翻译系统具有进行翻译后修饰的潜力，如促红细胞生成素的糖基化。因此，该系统包含易位活性的内源性微粒体，能够将膜蛋白共翻译地整合到生物膜中。因此，所提出的体外翻译系统是一种快速、方便地优化表达构建体、对整合膜蛋白进行特异性标记以及无细胞生产翻译后修饰蛋白的有力工具。
