A highly efficient cell-free protein synthesis system from Escherichia coli
A highly efficient cell-free protein synthesis system from Escherichia coli
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DOI：
10.1111/j.1432-1033.1996.0881u.x
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发表时间：
1996-08-01
期刊：
影响因子：
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通讯作者：
Yokoyama, S
中科院分区：
文献类型：
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作者：
Kim, DM;Kigawa, T;Yokoyama, S
我们用T7噬菌体RNA聚合酶修饰了一个来自大肠杆菌的无细胞偶联转录/翻译系统，并实现了高达0.4 mg蛋白/ml反应混合物的生产率。首先，我们发现磷酸烯醇式丙酮酸和聚（乙二醇）的最佳浓度是相互依赖的;前者的浓度越高，后者的浓度越高。其次，使用浓缩的30 000 Xg细胞提取物代替常规提取物，显著提高了蛋白质合成的初始速率。该现象被证明是由于除从浸提液中消除抑制分子以外的原因。对于具有浓缩提取物的该系统，再次共优化磷酸烯醇丙酮酸和聚（乙二醇）浓度，导致以0.3 mg/ml的生产率产生氯霉素乙酰转移酶。最后，通过补充氨基酸库，生产率进一步提高至0.4 mg/ml。这种改进的无细胞蛋白质合成系统在生产率方面上级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。
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 30 000Xg 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.