High-Density, Defined Media Culture for the Production of Escherichia coli Cell Extracts

Cell-free protein synthesis systems use a crude cell extract to produce proteins. While these systems have many advantages over in vivo expression systems, the current procedures for preparation of the cell extract are very labor-intensive. In addition, since they start with low cell density cultures, the methods yield small amounts of final extract. Fed-batch fermentations were performed with a defined medium and a glucose feeding strategy designed to meet metabolic demand but avoid acetate production. We have investigated cultures with cell densities from 3 to 50 OD595 for extract preparation. Furthermore, the effect of growth rate (0.2 to 1.1 /hr) on extract performance was studied in glucose-limited cultures. The glucose feeding strategy was able to control acetate production in some fermentations but not in others. The feeding of amino acids may have caused the variability. Neither cell density nor growth rate had a significant impact on protein synthesis by the cell-free extract. Finally, a relA and spoT null mutant grew more slowly, but still produced extract with the same activity as the parent relA1 spoT1 strain.