Improvement of Escherichia coli cell-free system by utilization of cell extract having additional property. Problems and countermeasures.

In the Escherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E. coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.