In-place condensation of reaction mixture using hollow fiber membrane reactors improved productivity of cell-free protein synthesis

Abstract

The authors intended to raise the protein production rate of continuous flow cell-free (CFCF) translation by applying in-place condensation of the reaction mixture containing wheat germ extract (WGE), mRNA, tRNA, amino acids, and high energy biochemicals. A hollow fiber membrane reactor (HFMR) composed of a shell and 80 hollow fibers made of ultrafiltration (UF) membrane was developed for condensing the reaction mixture in in-place manner during CFCF translation. For CFCF translation, first, the extra-capillary space (ECS) of HFMR was filled with the reaction mixture, and then the buffer solution containing the high energy biochemicals and amino acids was continuously fed into the ECS. The protein encoded in mRNA was continuously synthesized in the ECS, and it flowed out of the reactor through the UF membrane and then the hollow fibers, while the components constituting the protein synthesis machinery were held in the ECS. The reaction mixture was condensed in-place when additionally fed into the ECS. The large ratio of surface area to volume of the HFMR made possible the intensive condensation of the reaction mixture. The product protein concentration as well as the production rate increased with the 1.3th power of the condensation ratio. Ten-fold in-place condensation of the reaction mixture raised the protein production rate 20-fold. In contrast, in-place condensation of either WGE or mRNA alone resulted in merely a slight increase in productivity.