Impact of Reducing Agents on Protein Synthesis in a Reconstituted Cell-Free Protein Synthesis System

Maintaining proper redox conditions is essential for protein stability and function. In cell-free protein synthesis, reducing agents, such as dithiothreitol and reduced glutathione, are commonly added to mimic the cytosolic environment and prevent unwanted oxidation. The PURE system, which is a fully reconstituted protein synthesis system, also contains reducing agents. Here, we systematically examined how reducing agents affect the protein synthesis in the PURE system. We found that the reducing activity of dithiothreitol decreased during prolonged reactions, leading to the formation of disulfide bonds in synthesized proteins. Dissolved oxygen and contaminating metal ions were identified as major factors causing this loss of activity. Based on these findings, we developed a method to maintain reducing conditions throughout the reaction, ensuring consistent protein quality. Our results provide new insights into redox regulation in cell-free systems and offer a practical strategy for the efficient synthesis of functional proteins, with potential applications in biotechnology and therapeutic protein production.