Towards self-regeneration: exploring the limits of protein synthesis in the 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 exceed the system’s protein synthesis rate. Here we were able to drastically reduce the non-ribosomal 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 non-ribosomal proteins, which is a critical step towards the development of a universal biochemical constructor and living synthetic cell.