Hino Mami, Shinohara Yasuo, Kajimoto Kazuaki, Terada Hiroshi, Baba Yoshinobu
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
Protein Expr Purif. 2002 Mar;24(2):255-9. doi: 10.1006/prep.2001.1570.
To understand the key processes of cell-free protein synthesis, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined under various conditions. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. However, synthesis of A-FABP was not observed when an RNA fragment corresponding to the open reading frame of A-FABP was used in the reaction instead of the expression vector. Northern analysis revealed that the RNA that was added to the reaction mixture promptly underwent degradation. On the contrary, when the expression vector of A-FABP was employed, a strong RNA signal was observed over the entire incubation period. Thus, a continuous supply of RNA is needed in order to account for its loss via degradation to achieve the synthesis of reasonable amounts of A-FABP. Furthermore, the effect of continuous exchange of reaction mixture was also evaluated by measurement of the amount of synthesized A-FABP.
为了解无细胞蛋白质合成的关键过程，在各种条件下检测了快速翻译系统对脂肪型脂肪酸结合蛋白（A-FABP）的合成。通过使用含有T7启动子的A-FABP表达载体实现了A-FABP的合成。然而，当在反应中使用与A-FABP开放阅读框对应的RNA片段代替表达载体时，未观察到A-FABP的合成。Northern分析表明，添加到反应混合物中的RNA迅速发生降解。相反，当使用A-FABP表达载体时，在整个孵育期间观察到强烈的RNA信号。因此，需要持续供应RNA以弥补其因降解造成的损失，从而实现合理量A-FABP的合成。此外，还通过测量合成的A-FABP量评估了反应混合物连续交换的效果。
