Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA

•PCR products can be used to make proteins quickly and easily in a cell-free system.•Recent advances have widened the application space of such cell-free systems.•The openness of such systems enables rapid, parallel screening of protein libraries.•Applications include proteomic studies, microarray production and rapid screening.

A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems.

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