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

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 degrees 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 ChiA Delta 4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40-80 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/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. (c) 2006 Elsevier B.V. All rights reserved.