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

Cell-free expression systems (CFES) 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 due to 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 damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during 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 CFES more accessible and portable. Graphical abstract