Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

• Fermentation harvest time impacts cell-free protein synthesis extract viability.
• Fermentation media pH can impact cell-free protein synthesis extract viability.
• Cell-free systems needing exogenous components performed best with delayed harvest.

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.