Continuously coupled transcription-translation system for the production of rice cytoplasmic aldolase.
A continuously coupled cell-free transcription-translation system was developed for the production of rice cytoplasmic aldolase, an enzyme involved in both glycolytic and gluconeogenic pathways in eukaryotic cells. The system works with a continuous flow of feeding solution containing nucleoside triphosphates and amino acids into a 1-mL reactor containing wheat-germ extract, plasmid DNA, and transcription enzyme, and continuous removal of translation product through an ultrafiltration membrane fitted in the reactor. Addition of free nucleotide primer, m(7)G(5')ppp(5')G, to this reactor was necessary for efficient transcription, thus producing biologically active mRNA for translation. The rate of aldolase synthesis was constant during the continuous translation reaction. It was observed that from 3 h onward only aldolase was synthesized by the system. After 30 h, the total amount of protein synthesized reached 205.6 microg, which is comparable with the amount synthesized (255.6 microg) in the translation system only where separately prepared capped mRNAs were added to the reactor for translation. Autoradiogram and Western blot analyses of the translated product showed a distinct band corresponding to the calculated molecular weight of the protein. These results have shown the establishment of a continuously coupled eukaryotic transcription-translation system for the expression of genes from eukaryotic cells.