Tardigrade-Derived Strategy for Low-Cost Storage of Cell-Free Expression Lysates

High Resolution Image Download MS PowerPoint Slide Cell-free expression systems (CFESs) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity because of the additives. In this study, we present a CFES that contains a tardigrade-derived cytosolic-abundant heat-soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damage during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity following low-cost room-temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular, for making CFESs more accessible and portable.