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.
FAQs for Cell-Free Protein Expression
Protocols for Cell-Free Protein Expression
- Analysis of Synthesized Protein using PURExpress (E3313)
- Analysis of Synthesized Protein using PURExpress (E6800)
- Analysis of Synthesized Protein using PURExpress (E6840)
- Analysis of Synthesized Protein using PURExpress (E6850)
- Determination of Protein Synthesis Yield with PURExpress (E3313)
- Determination of Protein Synthesis Yield with PURExpress (E6800)
- Determination of Protein Synthesis Yield with PURExpress (E6840)
- Determination of Protein Synthesis Yield with PURExpress (E6850)
- Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Protein Synthesis Reaction using PURExpress (E3313)
- Protein Synthesis Reaction using PURExpress (E6800)
- Protein Synthesis Reaction using PURExpress® ∆ (aa, tRNA) Kit (E6840)
- Protein Synthesis Reaction using PURExpress® ∆ RF123 Kit (E6850)
- Protocol for Avoiding Rnase Contamination using Murine Rnase Inhibitor (M0314)
- PURExpress Disulfide Bond Enhancer (E6820)
- Purification of Synthesized Protein using Reverse His-tag Purification
Application Notes Cell-Free Protein Expression
- Use of the PURExpress® in vitro Protein Synthesis Kit, Disulfide Bond Enhancer and SHuffle® Competent E. coli for heterologous in vitro and in vivo cellulase expression.
- Using the PURExpress® In Vitro Protein Synthesis Kit for Heterologous In Vitro Expression and Functional Screening of FMN-dependent Oxidoreductase Variants
Bypassing Common Obstacles in Protein Expression
The Next Generation of Cell-free Protein Synthesis
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
- Protein Expression and Purification Selection Chart
Other Tools & Resources
Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit
NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.