Liu David V, Zawada James F, Swartz James R
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biotechnol Prog. 2005 Mar-Apr;21(2):460-5. doi: 10.1021/bp049789y.
Escherichia coli extracts activate cell-free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high-density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30,000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large-scale cell-free protein production, especially when combined with high-density fermentation protocols.
大肠杆菌提取物通过提供翻译及其他支持反应所需的催化剂来激活无细胞蛋白质合成系统。近期研究结果表明，高密度发酵可用于提供源细胞，但后续的细胞提取物制备过程需要多个离心和透析步骤，以及昂贵的径流反应。在本文报道的工作中，通过大幅缩短几个步骤，提取物制备方案的时长减少了近50%。此外，通过优化径流孵育，总体试剂成本降低了70%。尽管如此，采用更短、成本更低的方法制备的提取物活性相当。对关键步骤进行了进一步研究，结果表明在标准的30,000g离心过程中核糖体损失最小。此外，蔗糖密度离心分析表明，尽管孵育步骤可显著激活提取物，但核糖体/多聚核糖体解离并非必需。这些见解表明，对于大规模无细胞蛋白质生产，尤其是与高密度发酵方案结合使用时，能够更快速且成本大幅降低地生产出一致性的细胞提取物。
