Optimizing protein expression in the One-Pot PURE System: Insights into reaction composition and translation efficiency

Abstract

The One-Pot PURE system forin vitroprotein expression, which results from a co-culture and one-step purification of 36 essential proteins to support gene transcription and translation, can significantly improve the accessibility and affordability of PURE systems. However, replicating this protocol to match the productivity of the traditional PURE system can take considerable time and effort due to variability in the expression level of individual proteins. In this work, we observed unstable PURE protein expression in twoE. coliprotein expression strains, M15/pREP4 and BL21(DE3), and addressed this instability using catabolite repression. We identified key proteins whose concentration in the One-Pot PURE mixture significantly impacted the reaction’s protein expression capacity. Compared to the original method using twoE. coliprotein expression strains, we found that consolidating all expression vectors onto one BL21 (DE3) strain led to more uniform cell growth at the time of protein induction, thereby improving the composition of critical translation initiation factors in the purified mixture for efficient translation. We also found that variations in commercial energy solution formulations could compensate for deficiencies in the One-Pot PURE protein composition. Additionally, our study revealed significant differences in the translation capacity of commercially availableE. colitRNAs, suggesting the potential of optimizing tRNA composition to improve protein translation. Taken together, this work highlights the intricate biochemical interplay influencing protein expression capacity in the One-Pot PURE system and presents strategies to improve its robustness and productivity.