Lyophilized Escherichia coli-Based Cell-Free Systems for Robust, High-Density, Long-Term Storage

Cell free protein synthesis (CFPS) is a versatile tool for the production and engineering of recombinant proteins. When compared to traditional in vivo protein production, this CFPS system allows for better control over the synthesis environment, higher product selectivity, and faster expression of recombinant genes. Because of these benefits, cell free technology is an excellent platform for high-throughput production of various proteins including virus-like particles, proteins containing unnatural amino acids, cytotoxic proteins, and a variety of biocatalytic enzymes. However, a main disadvantage of cell-free technology is the necessity to store major components, including cell extracts and energy systems, below freezing in bulky aqueous solutions. This requires a substantial capital equipment investment of low or ultralow temperature freezers, continual maintenance of these freezers, emergency backup freezers and procedures, and emergency back-up power. These problems become particularly difficult when transporting materials and when trying to stockpile materials for later use.

In order to address these issues, we have decided to test the effects of a simple and straightforward protocol for the lyophilization of the extracts and energy systems for CFPS. Lyophilization was accomplished by cycling samples through shell freezing and then freeze drying. The samples were then gently ground, aliquoted, and stored at specified temperatures (-80°C, -20°C, 4°C, and room temperature) for particular amounts of time (7, 30, 60, and 90 days).

The lyophilization protocol resulted in the removal of more than 97% of the estimated original liquid content from the extracts. Interestingly, lyophilizing the extracts also decreased bacterial contamination allowing CFPS viability to be retained. In testing the extracts for protein-synthesis viability, it was noted that aqueous extract performed equally well after being stored for 90 days at -80 or -20, suggesting that -80°C storage may not be required for long-term viability. Even at 4°C the aqueous extract maintained all of its activity for 30 days. However, when stored at room temperature the performance of the aqueous extract exponentially decayed with effectively no activity by day 60. The lyophilized extracts we rehydrated and tested initially maintained an average of 85% of the protein synthesis viability of the standard aqueous extract. Extract viability was retained when stored at -80°C, while increasing storage temperatures corresponded to increasing extract degradation rates. However, after storing at room temperature for 90 days, the lyophilized extracts maintained about 20% protein synthesis viability as compared to the aqueous extract which retained less than 2% viability by day 30. In short, the lyophilized extracts retained significantly higher protein synthesis viability than the liquid extracts when stored for more than 30 days above freezing. A similar result was obtained for lyophilized energy systems: after storing at room temperature for 60 days, lyophilized energy systems retained more than 33% of its original viability, over 30% more than its aqueous counterpart.

In conclusion, these techniques allow for high-density storage of cell-free systems that are more robust against temperature and bacterial degradation. Our methods have the potential to decrease storage expenses, allow for longer shelf-life of cell extracts at room temperature, and enable durable portable protein production technologies. These benefits make lyophilized CFPS systems compelling candidates for applications such as pharmacy-on-a-chip microfluidic devices for rapid on-the-site treatment and rapid large-scale vaccine or therapeutic protein production from stockpiled extract.