Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm

Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.