Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source

Although glucose can be used as an economical and efficient energy source for driving cell-free protein synthesis, use of high concentrations of glucose can cause accumulation of organic acids, which inhibits protein synthesis through the decrease in pH of reaction mixture. In this study, instead of directly using glucose, we used soluble starch as a glucose-releasing energy reservoir molecule. When compared to previously reported methods using glucose as the primary energy source, the use of starch alleviated the problem of a pH drop, thereby allowing a prolonged cell-free protein synthesis. As a result, protein synthesis continued over 12 h in a reaction mixture employing starch as an energy source, and approximately 1.7 ± 0.1 mg/mL of the target protein accumulated in the reaction mixture. Moreover, homeostatic maintenance of the reaction conditions and controlled rate of translation led to substantially larger amounts of proteins being obtained in a soluble form.