Energy systems for ATP regeneration in cell-free protein synthesis reactions.

Supplying energy for cell-free protein synthesis reactions is one of the biggest challenges to the success of these systems. Oftentimes, short reaction duration is attributed to an unstable energy source. Traditional cell-free reactions use a compound with a high-energy phosphate bond, such as phosphoenolpyruvate, to generate the ATP required to drive transcription and translation. However, recent work has led to better understanding and activation of the complex metabolism that can occur during cell-free reactions. We are now able to generate ATP using energy sources that are less expensive and more stable. These energy sources generally involve multistep enzymatic reactions or recreate entire energy-generating pathways, such as glycolysis and oxidative phosphorylation. We describe the various types of energy sources used in cell-free reactions, give examples of the major classes, and demonstrate protocols for successful use of three recently developed energy systems: PANOxSP, cytomim, and glucose.