Kawarasaki Y, Kawai T, Nakano H, Yamane T
Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Japan.
Anal Biochem. 1995 Apr 10;226(2):320-4. doi: 10.1006/abio.1995.1231.
Several reaction conditions of cell-free protein synthesis such as temperatures, buffers, tRNAs, and creatine phosphate were intensively investigated and optimized to prolong protein synthesis and make it more efficiently in a batch system. As a result of these modifications, the protein synthesis reaction continued for 10 h so that about 30 micrograms of dihydrofolate reductase (DHFR) protein derived from Escherichia coli was synthesized in 1 ml of reaction mixture. In this improved system, translational reactions of other mRNAs such as rabbit beta-globin, Xenopus beta-globin, and tobacco mosaic virus RNA also continued for about 10 h. In addition, protein synthesis directed by uncapped dhfr mRNA containing a viral cap-independent translation initiation-mediating sequence continued for 10 h, resulting in the synthesis of 18 micrograms of DHFR protein per milliliter of reaction mixture.
为了在分批系统中延长蛋白质合成时间并提高其效率，对无细胞蛋白质合成的几个反应条件，如温度、缓冲液、转运RNA和磷酸肌酸进行了深入研究和优化。经过这些改进，蛋白质合成反应持续了10小时，从而在1毫升反应混合物中合成了约30微克源自大肠杆菌的二氢叶酸还原酶（DHFR）蛋白。在这个改进的系统中，其他信使核糖核酸（mRNA）的翻译反应，如兔β-珠蛋白、非洲爪蟾β-珠蛋白和烟草花叶病毒RNA的翻译反应也持续了约10小时。此外，由含有病毒非帽依赖性翻译起始介导序列的无帽dhfr mRNA指导的蛋白质合成持续了10小时，每毫升反应混合物合成了18微克DHFR蛋白。
