A LONG-LIVED BATCH REACTION SYSTEM OF CELL-FREE PROTEIN-SYNTHESIS

Several reaction conditions of cell-free protein synthesis such as tem peratures, buffers, tRNAs, and creatine phosphate were intensively inv estigated 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 mu g o f 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, Xe nopus beta-globin, and tobacco mosaic virus RNA also continued for abo ut 10 h. In addition, protein synthesis directed by uncapped dhfr mRNA containing a viral cap-independent translation initiation-mediating s equence continued for 10 h, resulting in the synthesis of 18 mu g of D HFR protein per milliliter of reaction mixture. (C) 1995 Academic Pres s, Inc.