Protein production in a test tube using a continuously coupled transcription-translation cell-force system
2002
Tulin, E.E. (Leyte State Univ., ViSCA, Baybay, Leyte 6521 (Philippines). Philippine Root Crops Research and Training Center)
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 fitted in the reactor. The rate of aldolase synthesis was constant during the continuous translation reaction. After 30 h, the total amount of protein synthesized reached 205.6 g, which is comparable with the amount of synthesized (255.6 g) in the translation system only where separately capped mRNAs were added to the reactor for translation. Autoradiogram and Western blot analysis of the translated product showed a distinct band corresponding to the calculated molecular weight of the protein. The technology presented here is useful for the production of proteins of great commercial interest
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