Cell-Free Protein Expression

Cell-free protein synthesis (CFPS) is a protein expression approach that enables production of a target protein without the use of living cells. In CFPS, a solution containing all the cellular machinery needed to direct protein synthesis (e.g., ribosomes, tRNAs, enzymes, cofactors, amino acids, etc.) is used to transcribe and translate a supplied nucleic acid template (e.g., plasmid DNA, linear DNA or mRNA).  In a simple biochemical reaction, CFPS generates desired proteins in just hours compared to traditional in vivo protein expression approaches that take several days or longer.

CFPS is well-suited for many applications. The speed and ease of CFPS facilitates high throughput protein expression in applications such as protein engineering, mutagenesis studies, and enzyme screening. In addition, CFPS is often used to generate proteins for biophysical and structure-function analyses. Other CFPS applications include the production of proteins that are toxic to host cells in vivo, expression of proteins with modified or unnatural amino acids, and incorporation of post-translational modifications (in some CFPS systems).  Due to their robust performance, scalability and cost-effectiveness, CFPS systems have also been adopted in metabolic engineering and synthetic biology applications.

There are several different types of CFPS systems.  Many systems are comprised of a solution containing protein synthesis machinery derived from a cell lysate.  Such systems have been made from various types of cells including E. coli, yeast, plant and animal cells.  The NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) is a lysate-based system derived from E. coli cells that have been engineered for high in vitro expression performance.  This system offers high expression levels, the ability to produce high molecular weight proteins, scalability, and is cost-effective for high throughput expression applications.  Alternatively, CFPS can be achieved using purified protein synthesis machinery.  The PURExpress® In Vitro Protein Synthesis Kit (NEB #E6800) is a reconstituted mixture of numerous individually purified components that are required for E. coli transcription and translation.  This system permits the production of proteins in the absence of detrimental E. coli activities (e.g., nucleases and proteases).


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Cell-Free Protein Expression includes these areas of focus:
NEBExpress® Cell-free E. coli Protein Synthesis System
FAQs for Cell-Free Protein Expression
Protocols for Cell-Free Protein Expression
Application Notes for Cell-Free Protein Expression
The PURExpress® System Kit Components
  • His-Tagged Translation Factors
    • Initiation Factors (IF1, IF2, IF3)
    • Elongation Factors (EF-Tu, EF-Ts, EF-G)
    • Release Factors (RF1, RF2, RF3)
    • Ribosome Recycling Factor
    • 20 Aminoacyl tRNA synthetases
    • Methionyl tRNA formyltransferase
  • E. coli Ribosomes
  • E. coli tRNAs
  • Energy Regeneration System
  • NTPs, Amino Acids, Salts, Buffer

In addition, recombinant T7 RNA polymerase is used to couple transcription to translation. The PURE system represents an important step towards a totally defined in vitro transcription/translation system, thus avoiding the “black box” nature of the cell extract-based systems.

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)
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