2024 AIChE Annual Meeting

Developing Reactors to Improve Oxygen Availability in Cell-Free Protein Synthesis

In vivo protein production is essential for the pharmaceutical industry and other sectors, but it is limited by the host cell's tolerance to the product and inefficient energy use. Cell-free protein synthesis (CFPS) offers a promising alternative by using cellular components without the constraints of a living cell. CFPS can enhance energy efficiency, as energy can be directed toward specific objectives rather than the multifunctional tasks of living cells. Additionally, CFPS allows the production of biologics too complex or toxic for in vivo methods. However, CFPS remains less efficient than in vivo production.

One of the hypothesized causes of this inefficiency is oxygen availability. Preliminary data suggests oxygen is a limiting factor in E. coli CFPS metabolism, and typical sparging methods hinder protein synthesis. To explore this challenge, we designed a miniature stirred-tank reactor with enhanced gas diffusion and real-time oxygen and green fluorescent protein (GFP) titer sensing. We aim to test how varying oxygen concentrations affect protein titer and metabolism in E. coli CFPS.

While this research is ongoing, we have successfully developed a reactor that supports 4 mL CFPS reactions with real-time measurements of dissolved oxygen and GFP titer. The reactor allows direct sampling to measure adenosine triphosphate (ATP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), and other reaction components.