Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription–Translation System

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.