Kim D M, Kigawa T, Choi C Y, Yokoyama S
Interdisciplinary Program for Biochemical Engineering and Technology, College of Engineering, Seoul National University, Korea.
Eur J Biochem. 1996 Aug 1;239(3):881-6. doi: 10.1111/j.1432-1033.1996.0881u.x.
We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.
我们用T7噬菌体RNA聚合酶对来自大肠杆菌的无细胞偶联转录/翻译系统进行了改进，实现了高达0.4毫克蛋白质/毫升反应混合物的产量。首先，我们发现磷酸烯醇丙酮酸和聚乙二醇的最佳浓度相互依赖；前者浓度越高，后者浓度也应越高。其次，使用浓缩的30000×g细胞提取物代替传统提取物，显著提高了蛋白质合成的初始速率。这一现象被证明是由于提取物中抑制分子的消除以外的原因。对于使用浓缩提取物的该系统，再次对磷酸烯醇丙酮酸和聚乙二醇的浓度进行了共同优化，从而以0.3毫克/毫升的产量生产氯霉素乙酰转移酶。最后，通过补充氨基酸库，产量进一步提高到0.4毫克/毫升。这种改进的无细胞蛋白质合成系统在产量上优于迄今为止报道的任何其他无细胞系统，包括连续流无细胞系统。
