A highly productive system for cell-free protein synthesis using a lysate of the hyperthermophilic archaeon,...

We report in this study an improved system for cell-free protein synthesis at high temperatures using the lysate of Thermococcus kodakaraensis. Previous work indicated that cell-free protein synthesis of ChiADelta4, a derivative of T. kodakaraensis chitinase, was observed within a temperature range of 40-80 degrees C, and the maximum yield of the ChiADelta4 synthesized was approximately 1.3 microg/ml. To increase productivity of the system, the following approaches were taken. First, the process of lysate preparation was examined, and we found that omitting the preincubation (runoff) step was especially effective to increase the translational activity of lysate. Second, the concentrations of each reaction mixture were optimized. Among them, the requirement of a high concentration of potassium acetate (250 mM) was characteristic to the T. kodakaraensis system. Third, a mutant strain of T. kodakaraensis was constructed in which a heat shock transcriptional regulator gene, phr, was disrupted. By using the lysate made from the mutant, we observed an increase in the optimum reaction temperature by 5 degrees C. Through these modifications to the system, the yield of ChiADelta4 was dramatically increased to 115.4 microg/ml in a batch reaction at 65 degrees C, which was about 90 times higher than that in the previous study. Moreover, in the optimized system, a high speed of protein synthesis was achieved: over 100 microg/ml of ChiADelta4 was produced in the first 15 min of reaction. These results indicate that the system for cell-free protein synthesis based on T. kodakaraensis lysate has a high production potential comparable to the Escherichia coli system.