Enhanced in vitro translation at reduced temperatures using a cold-shock RNA motif

Cell-free synthesis of recombinant proteins has emerged as an alternative method of protein production although protein yields still cannot compete with in vivo expression techniques. In systems based on S30 extracts of Escherichia coli unfavorable side-reactions are involved in limiting protein yields. Therefore, carrying out cell-free reactions at lower temperatures might be beneficial as side reactions should be decreased. In this study we show that by using the 5'-untranslated region of the cold-shock gene cspA from E. coli as mRNA leader in cell-free reactions, the expression temperature can be decreased and simultaneously leads to an increase in protein yields. A compensation for the lower activity of T7 RNA polymerase at lower temperatures enhances protein synthesis even further. Additionally, this 5'-untranslated region also standardizes the optimal expression temperature of different proteins.