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.