Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis

Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyl-transferase (CAT) for up to 6 h in a batch reaction to yield 700 μg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.