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 be operated at high temperatures using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis. With the system, cell-free protein synthesis of ChiADelta4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40 degC to 80 degC, with an optimum at 65 degC. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiADelta4 folded in a proper tertiary structure. The maximum concentration of active ChiADelta4 synthesized was determined to be approximately 1.3 mug/mL. A time course experiment indicated that the amount of synthesized ChiADelta4 saturated within 30 min at 65 degC, 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