Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile.
Abstract Systems for cell-free protein synthesis available today are usually based on the lysates of either Escherichia coli , wheat germ or rabbit reticulocyte, and protein synthesis reactions using these extracts are performed at moderate temperatures (20–40 °C). 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 ChiAΔ4, a derivative of T . kodakaraensis chitinase (ChiA), was observed within a temperature range of 40–80 °C, with an optimum at 65 °C. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiAΔ4 folded in a proper tertiary structure. The maximum concentration of active ChiAΔ4 synthesized was determined to be approximately 1.3 μg/mL. A time course experiment indicated that the amount of synthesized ChiAΔ4 saturated within 30 min at 65 °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.