Protein synthesis using a reconstituted cell-free system.

Most cell-free protein-synthesis systems are based on cell extracts, which often contain undesirable activities. Reconstituted systems, by contrast, are composed of a defined number of purified and recombinant components with minimal nuclease and protease activities. This unit describes the use of a particular commercial reconstituted system, PURExpress. This system allows in vitro synthesis of proteins from mRNA and circular and linear DNA templates, as well as co-translational labeling of proteins. Unique to this system, all recombinant protein components of the system are His-tagged, allowing purification of the synthesized untagged protein by removing the rest of the system's components. Newly synthesized proteins can often be visible on an SDS-PAGE gel and directly assayed for their functions without labeling and purification. Certain components of the system, such as ribosomes or release factors, can be omitted for specific applications. Such "delta" versions of the system are well suited for studies of bacterial translation, assays of ribosome function, incorporation of unnatural amino acids, and ribosome display of protein libraries.