Cell-free protein synthesis at high temperature using a lysate of a hyperthermophile

We report here the development of a novel system for cell-free protein synthesis that can he operated at high temperatures using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis. With the system, cell-free protein synthesis of ChiA Delta 4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40 degrees C to 80 degrees C, with an optimum at 65 degrees C. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiA Delta 4 folded in a proper tertiary structure. The maximum concentration of active ChiA Delta 4 synthesized was determined to be approximately 1.3 mu g/mL. A time course experiment indicated that the amount of synthesized ChiA Delta 4 saturated within 30 min at 65 degrees C, and energy depletion was suggested to be the main cause of this saturation. We further developed a system for transcription and translation-coupled protein synthesis at high temperatures using a combination of T. kodakaraensis lysate and thermostable T7 RNA polymerase.