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We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong p<i>argC</i> promoter, originally isolated from a moderate thermophilic bacterium <i>Geobacillus stearothermophilus</i>, was used to improve the performance of a cell-free system in extracts of <i>Escherichia coli</i> BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the <i>E. coli</i> RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The <i>E. coli</i> BL21 (DE3) <i>rna</i>::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial p<i>argC</i> and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium <i>Thermotoga</i>, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.