A Semicontinuous Prokaryotic Coupled Transcription/Translation System Using a Dialysis Membrane

This report describes a novel and simple cell-free protein synthesis system. In this paper, we prove that the short duration of protein synthesis in a conventional cell-free protein synthesis system of batch configuration can be attributed both to depletion of energy sources and deactivation of S30 extract by small-molecule byproducts produced during the protein synthesis. The reaction period of cell-free protein synthesis system could be extended through an operation of a continuous-flow cell-free protein synthesis system, which was originally developed by Spirin. However, inspite of the greatly extended reaction period, the final amount of cell-free produced protein was not significantly larger than that can be obtained from a batch system due to the reduced rate of protein synthesis. It was supposed that the reduced rate of protein synthesis in the continuous-flow system was attributed to leakage of translational components through the ultrafiltration membrane during the operation of the continuous-flow system. To solve such a problem of the continuous-flow system, we have developed and operated a novel reactor for cell-free protein synthesis. By use of this system, protein synthesis occurred for at least 14 h, yielding 1.2 mg/mL CAT protein. The present system is superior to the continuous-flow system as well as the conventional batch system in that it enables extremely high productivity without using any complex and hard-to-handle apparatus. As far as we know, the yield of cell-free protein synthesis given above is the best of the results reported to date.