Cell-Free Protein Expression

Cell-free protein expression is performed without the use of living cells. Instead, all components needed to transcribe DNA to RNA and translate the RNA to protein (e.g. ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided in solution for use in vitro. Generally, such solutions are obtained through making a cell lysate from a desired cell type. Cell-free mixtures have been made from both bacterial and eukaryotics cells. Alternatively, the PURExpress® system (NEB #E6800) consists of a mixture of highly-purified components required for transcription and translation in place of using a lysate.

Cell-free systems are generally not practical for large-scale protein expression. However, they are often perfectly suited for a number of applications where the rapid generation of a smaller amount of recombinant protein is desirable. For example, cell-free expression is suitable for high-throughput screening of truncated proteins for structural or functional studies. Other applications for cell-free expression include making proteins that are toxic to expression hosts in vivo, expression of proteins with modified amino acids, incorporation of post-translational modifications, or studies on protein folding.

The PURExpress system is a unique variation of cell-free expression. PURExpress provides all purified components needed to produce a recombinant protein in vitro. With minimal nuclease and protease activity, the PURExpress system preserves the integrity of DNA and RNA templates/complexes and results in proteins that are free of modification and degradation. 

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FAQs for Cell-Free Protein Expression
Protocols for Cell-Free Protein Expression
Application Notes Cell-Free Protein Expression
Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit
25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)
PUREXPRESS® is a registered trademark of New England Biolabs, Inc..
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