Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation.
Cell-free protein synthesis has become one of the standard methods for protein expression. One of the major advantages of this method is that PCR-amplified linear DNA fragments can be directly used as templates for protein synthesis. The productivity of cell-free protein synthesis using linear DNA templates is generally lower than that from plasmid DNA templates, especially when using an Escherichia coli cell extract. In the present study, we found that a simple modification of the protocol for cell extract preparation from E. coli, just by altering the cultivation temperature (37 degrees C) of the cells to a moderately lower range (20-34 degrees C), dramatically reduced the linear DNA degradation activity in the cell extract. This modification greatly improved the productivity of cell-free protein synthesis from linear DNA templates. The removal of the RecD protein, one of the components of exonuclease V, from the extract had almost the same effect, indicating that the linear DNA degradation activity in the extract was mainly due to the RecD protein and that its expression level was decreased at the lower cultivation temperature.
Seki
Eiko
E
Systems and Structural Biology Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama 230-0045, Japan.
Matsuda
Natsuko
N
Yokoyama
Shigeyuki
S
Kigawa
Takanori
T
D002474:Cell-Free System; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000302:isolation & purification / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis; D020871:RNA Stability; D015203:Reproducibility of Results; D013696:Temperature; D013698:Templates, Genetic
D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli