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 significantlly 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.