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 ChiADelta4, 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 ChiADelta4 folded in a proper tertiary structure. The maximum concentration of active ChiADelta4 synthesized was determined to be approximately 1.3 microg/mL. A time course experiment indicated that the amount of synthesized ChiADelta4 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.
Endoh
Takashi
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
Kanai
Tamotsu
T
Sato
Yuko T
YT
Liu
David V
DV
Yoshikawa
Kenichi
K
Atomi
Haruyuki
H
Imanaka
Tadayuki
T
D001105:Archaea / Q000378:metabolism*; D002474:Cell-Free System; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D010447:Peptide Hydrolases / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D002688:Chitinases; C094590:chitinase ChiA; D010447:Peptide Hydrolases