Preservation of protein expression systems at elevated temperatures for portable therapeutic production

• Published in: Journal of the Royal Society interface Vol. 14; no. 129; p. 20161039
• Main Authors: Karig, David K., Bessling, Seneca, Thielen, Peter, Zhang, Sherry, Wolfe, Joshua
• Format: Journal Article
• Language: English
• Published: England The Royal Society 01.04.2017; The Royal Society Publishing
• Subjects: Air drying; Alcohols; Anti-Bacterial Agents - administration & dosage; Anti-Bacterial Agents - chemical synthesis; Atmospheric conditions; Biotechnology; Cell-Free; Cold-Chain Storage; Desiccation - methods; Drug Compounding - methods; Drug Stability; Drying; Hot Temperature; Indicators and Reagents - chemistry; Life Sciences–Engineering interface; Outbreaks; Point Of Care; Preservation; Protein Engineering - methods; Protein Expression; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa - drug effects; Pyocin; Pyocins - administration & dosage; Pyocins - chemical synthesis; Reagents; Ruggedness; Sugar; Trehalose; Wounds; preservation; pyocin; point of care; cell-free; cold-chain storage; protein expression
• ISSN: 1742-5689; 1742-5662
• DOI: 10.1098/rsif.2016.1039

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability.