The impact of cytoplasmic mimicry on cell-free biology

Cell-free systems have traditionally been used to express recombinant DNA proteins and to study fundamental biological processes. In vitro protein synthesis, which exploits several interrelated molecular subsystems, is perhaps the best characterized example of this field. Bacteria have evolved a complex ensemble of catalysts and other factors for the efficient and precise production of proteins. Harnessing this elegant cellular ensemble to produce a target protein without intact cells offers many advantages over conventional in vivo technologies. However, cell-free protein production has been limited by an inability to supply the intense energy and substrate needs of protein synthesis without causing harmful changes in the chemical environment.;To address this limitation, we report that cytoplasmic mimicry, providing a cell-free reaction environment that more closely resembles the intracellular physicochemical conditions of Escherichia coli (the host organism of our prokaryotic cell-free system), activates innate processes that benefit bioactive protein production. Relative to previous work, this strategy (in the context of non-phosphorylated energy substrates) increases protein production yields (∼0.7mg/mL), provides a durable energy supply, improves system economics, provides for phosphate and pH homeostasis, improves protein folding, and activates natural in vivo processes such as glutamate metabolism and oxidative phosphorylation.;This new cell-free system, which more completely activates in vivo catalytic processes than previously described in vitro systems, also provides a reliable method to study complex biomolecular processes that either cannot be examined or are extremely difficult to address in living cells. For example, we have shown that the protein biosynthesis system behaves as a single, Michaelis-Menten ‘enzyme’ with respect to its critical substrates, ATP and GTP. Furthermore, experiments that probed the relationship between protein production rate and cell extract concentration have demonstrated that in-place condensation of the cell-free reaction mixture leads to increased volumetric productivity.;This work has provided many new insights for the activation and control of S30 extracts to achieve high-level cell-free protein synthesis. It has also provided biophysical insights describing the protein synthesis system. Together these advances both increase the utility of cell-free systems and help to frame many questions that will guide future research towards a better understanding of systems biology.