Abstract
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 μg 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 β-globin, Xenopus β-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 μg of DHFR protein per milliliter of reaction mixture.
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