DNA-directed cell-free protein-synthesizing system of Bacillus subtilis

A cell-free coupled system of transcription and translation using cell extracts from Bacillus subtilis and DNA from phage SP82 has been developed. Under optimum conditions, it incorporated approx. 300 pmol methionine during a 1 h incubation. The activity of the system increased linearly as the concentration of S-150 supernatant fraction protein increased from 125 to 325 microgram per assay. The optimum Mg2+ concentration was 12.5-15 mM. Ribosomes required treatment with DNAase in order to reduce endogenous activity, but the S-150 fraction was kept DNAase-free to prevent degradation of exogenously added DNA. The coupled system was sensitive to inhibitors of RNA and protein synthesis. Kinetic studies showed that the number of pmol of nucleotides present in newly synthesized RNA increased linearly for the first 20-min reaction and that the rate of amino acid incorporation increased linearly for the first 30 min. Polyacrylamide gel electrophoresis of the in vitro synthesized products yielded a band pattern that closely resembled the pattern of early phage SP82 proteins produced in vivo.