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.