A SEMICONTINUOUS PROKARYOTIC COUPLED TRANSCRIPTION TRANSLATION SYSTEMUSING A DIALYSIS MEMBRANE/

This report describes a novel and simple cell-free protein synthesis s ystem. In this paper, we prove that the short duration of protein synt hesis in a conventional cell-free protein synthesis system of batch co nfiguration can be attributed both to depletion of energy sources and deactivation of S30 extract by small-molecule byproducts produced duri ng the protein synthesis. The reaction period of cell-free protein syn thesis system could be extended through an operation of a continuous-f low 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 reduce d rate of protein synthesis. It was supposed that the reduced rate of protein synthesis in the continuous-flow system was attributed to leak age of translational components through the ultrafiltration membrane d uring the operation of the continuous-flow system. To solve such a pro blem of the continuous-flow system, we have developed and operated a n ovel reactor for cell-free protein synthesis. By use of this system, p rotein synthesis occurred for at least 14 h, yielding 1.2 mg/mL CAT pr otein. The present system is superior to the continuous-flow system as well as the conventional batch system in that it enables extremely hi gh productivity without using any complex and hard-to-handle apparatus . As far as we know, the yield of cell-free protein synthesis given ab ove is the best of the results reported to date.