(174ak) Optimizing Protein Expression in One-Pot Pure Systems: Insights into Reaction Composition and Translation Efficiency
AIChE Annual Meeting
2024
2024 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Poster session: Bioengineering
Monday, October 28, 2024 - 3:30pm to 5:00pm
The One-Pot PURE system for in vitro protein expression, in which a mixture of 36 proteins necessary to support transcription and translation reactions is co-cultured and purified in one sitting, can significantly improve the accessibility and affordability of PURE systems. Despite the promise and simplicity of One-Pot PURE, replicating the system to achieve comparable productivity to the PURE system can take considerable time and effort. In this work, we utilized glucose catabolite repression to improve the stability of One-Pot PURE protein expression vectors. We identified a critical set of proteins whose expression levels significantly impact the protein translation rate of One-Pot PURE reactions using liquid chromatography with tandem mass spectrometry (LC-MS/MS). By consolidating all expression vectors onto a single E. coli strain, we achieved more uniform cell growth at the time of protein induction, thereby improving the composition of critical initiation and elongation factors in the purified protein mix for efficient translation. Additionally, we discovered that variations in commercial energy mix formulations could compensate for deficiencies in protein composition in the One-Pot PURE reaction. Furthermore, our findings on different commercially available E. coli tRNAs revealed significant differences in their translation capacity, suggesting potential tRNA optimizations for efficient protein translation. This work highlights the intricate biochemical interplay influencing protein expression capacity in the One-Pot PURE System.