Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems.

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro . However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design-build-test-learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field.