Kim D M, Choi C Y
Interdisciplinary Program for Biochemical Engineering and Technology, College of Engineering, Seoul National University, Korea.
Biotechnol Prog. 1996 Sep-Oct;12(5):645-9. doi: 10.1021/bp960052l.
This report describes a novel and simple cell-free protein synthesis system. In this paper, we prove that the short duration of protein synthesis in a conventional cell-free protein synthesis system of batch configuration can be attributed both to depletion of energy sources and deactivation of S30 extract by small-molecule byproducts produced during the protein synthesis. The reaction period of cell-free protein synthesis system could be extended through an operation of a continuous-flow cell-free protein synthesis system, which was originally developed by Spirin. However, inspite of the greatly extended reaction period, the final amount of cell-free produced protein was not significantly larger than that can be obtained from a batch system due to the reduced rate of protein synthesis. It was supposed that the reduced rate of protein synthesis in the continuous-flow system was attributed to leakage of translational components through the ultrafiltration membrane during the operation of the continuous-flow system. To solve such a problem of the continuous-flow system, we have developed and operated a novel reactor for cell-free protein synthesis. By use of this system, protein synthesis occurred for at least 14 h, yielding 1.2 mg/mL CAT protein. The present system is superior to the continuous-flow system as well as the conventional batch system in that it enables extremely high productivity without using any complex and hard-to-handle apparatus. As far as we know, the yield of cell-free protein synthesis given above is the best of the results reported to date.
本报告描述了一种新颖且简单的无细胞蛋白质合成系统。在本文中，我们证明了在批式配置的传统无细胞蛋白质合成系统中，蛋白质合成持续时间较短既归因于能量来源的耗尽，也归因于蛋白质合成过程中产生的小分子副产物使S30提取物失活。无细胞蛋白质合成系统的反应期可以通过连续流无细胞蛋白质合成系统的操作来延长，该系统最初由斯皮林开发。然而，尽管反应期大大延长，但由于蛋白质合成速率降低，无细胞产生的蛋白质最终量并不比批式系统显著大。据推测，连续流系统中蛋白质合成速率降低归因于连续流系统运行期间翻译组分通过超滤膜的泄漏。为了解决连续流系统的此类问题，我们开发并运行了一种新型的无细胞蛋白质合成反应器。通过使用该系统，蛋白质合成持续了至少14小时，产生了1.2mg/mL的CAT蛋白。本系统优于连续流系统以及传统批式系统，因为它无需使用任何复杂且难以操作的设备就能实现极高的生产率。据我们所知，上述无细胞蛋白质合成的产量是迄今为止报道的最佳结果。
