Understanding the properties of repair protein p26 for dry-state preservation of biological materials.

The preservation of complex biological materials in the frozen state has been well established and plays a critical role in the advancement of clinical applications and the pharmaceutical industry. Cryopreservation of complex biological materials has several clinical applications, including vaccine preservation, bone marrow transplants, cell therapeutics, and increasing the shelf life of red blood cells (RBCs) for transfusion. However, many challenges are present, including the use of toxic protective agents and the financial burden of maintaining extremely low temperatures between 193.15 and – 77.6 K (Parihar, Kumar et al. 2023). Lyophilization, or freeze-drying, presents an opportunity to mitigate such challenges. However, dry-state storage of more complex water-loss-sensitive systems has yet to be achieved (Weng 2021). We turned to nature for strategies to help improve lyophilization outcomes and allow for storage at ambient temperatures. The animal extremophile Artemia franciscana, brine shrimp, can survive severe dehydration (desiccation) by upregulation of repair proteins that lessen the damage of water loss and during rehydration. One exceptional repair protein, the small heat shock protein (sHSP) p26, was the focus of this study. Protein expression and purification levels were optimized to study the characteristics of p26 that may allow for it to aid in protection during desiccation. We discovered that p26 physiochemical behavior is exceptionally salt-sensitive, likely promoting liquid-liquid phase separation (LLPS) and production of liquid condensates, which is believed to play a role in protection during drying. Characteristics of a secondary sHSP, ArHSP21, were also examined to compare the sequence and characteristics of p26 with ArHSP21 to understand how different molecular chaperones influence protection during stress. Additionally, molecular analysis was utilized to determine whether these proteins function independently or work together to enhance biomolecular protection. These sHSPs were identified as potential candidates to improve lyopreservation outcomes in other systems, including complex water-stress-sensitive biomolecules and systems.