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Cell-Free Protein Synthesis Toolbox

Overview of Cell Free Protein Synthesis Systems

FLEXIQuant Protein Expression Kit

Wheat Germ Cell-Free Expression System to Make Full-Length Isotope-Labeled MS Standards

Further Information!

Protein engineering encompasses an expanding set of methods to produce recombinant proteins for basic and applied research. Currently, cell-based protein engineering approaches are still in wide use despite suffering from multiple problems, including insolubility, low yield, variable expression, low stability, incorrect folding and incorrect disulfide bond formation associated with lack of activity. By contrast, cell-free protein engineering approaches permit direct control over reaction conditions resulting in relatively easy and rapid synthesis of complex proteins, toxic proteins, membrane proteins, and novel proteins with non-natural amino acids.

Two main types of cell-free systems are in common use. The first and older system is based upon crude cell extracts that support transcription and translation. The most common extract sources are Escherichia coli, Saccharomyces cerevisiae, rabbit ret##LINKTEXT##iculocytes, wheat germ, and insect cells. The second type of cell-free system is based upon the Ueda group’s PURE (Protein synthesis Using Recombinant Elements) system. The PURE approach to cell-free protein synthesis is based on modular reconstitution of the translational machinery of the cell from affinity-purified protein components (1-4), including initiation factors (IF1, IF2, IF3), elongation factors (EF-Tu, EF-Ts, EF-G), release factors (RF1, RF2, RF3), ribosome recycling factors, 20 aminoacyl tRNA synthetases, methionyl tRNA formyltransferase and pyrophosphatase.

In some PURE system approaches all protein components (except for ribosomes) harbor 6X His-tags that permit ‘reverse purification’ of target proteins by extracting tagged components by metal affinity chromatography followed by ultrafiltration to remove ribosomes (e.g., PURESYSTEM® from BioComber Co. and PURExpress® from New England Biolabs).

In a second version of the PURE system (PUREfrex® produced by Gene Frontier Corporation in Japan and distributed and supported world wide by Cosmo Bio) all protein components are untagged, permitting the expression and purification of target proteins based upon any desired tag including the His-tag. In both cases, recombinant components are combined with ribosomes and tRNAs isolated from specially engineered E. coli strains, together with all necessary NTPs and amino acids, an ATP-generating catalytic module and recombinant T7 RNA polymerase, creating a self-contained reaction system that can be programmed for protein synthesis using a variety of DNA templates. The advantages of PURE systems include reduced levels of contaminating proteases, nucleases, and phosphatases, greater reproducibility resulting from more defined chemistry, and the flexibility of a modular system. Metabolic side reactions that deplete the amino acid pool in cell extracts can be entirely avoided. Because they are modular, PURE systems support a variety of modifications for specialized applications, including ribosome display and site-selective incorporation of non-natural amino acids. The relative benefits and drawbacks of cell free versus cell-based systems is shown in the table below (ref. 5).

1. Shimizu, Y., Kanamori, T. & Ueda, T. Protein synthesis by pure translation systems. Methods 36, 299–304 (2005).
2. Shimizu, Y. & Ueda, T. in Cell-Free Protein Production (eds. Endo, Y., Takai, K. & Ueda, T.) 607, 11–21 (Humana Press, 2009).
3. Wang, H. H. et al. Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis. ACS Synth. Biol. 1, 43–52 (2011).
4. Whittaker, J. W. Cell-free protein synthesis: the state of the art. Biotechnol Lett 35, 143–152 (2012).
5. Lu, Y. Cell-free synthetic biology: Engineering in an open world. Synthetic and Systems Biotechnology 2, 23–27 (2017).

Cell Free VS Cell-based systems

Feature In vitro cell-free system In vivo cell system
Manipulation of transcription & translation Easy to control in an open environment Hard because of cell membrane as the barrier
Post-translational modification Hard Easy
Self-replication Hard Easy
DNA template Plasmids or PCR products Plasmids or genomes
Synthesis of membrane proteins & complex proteins Easy synthesis by adding surfactants or adjusting the system environment Hard synthesis due to limited intracellular environment
Incorporation of unnatural amino acids into proteins Easy Hard
Ability to only produce the desired products Easy achievement by focusing in the target metabolic pathways Hard achievement due to complicated cellular metabolism
Toxic tolerance High Low
Integration with materials Easy Hard
Design-build-test-learn cyle 2 days 2 weeks
Biomanufacturing High production rate, High product yield & Easy purification process without cell lysis Modest production rate, Modest product yield & Cell lysis prior to product purification
Cost Modest to High Low to Modest

Reconstituted versus lysate-based cell free systems

Application/Benefit Reconstituted Lysate-based
Minimal nuclease and protease activity preserves the integrity of DNA and RNA templates/complexes and results in protein that are free of modification and degradation + -
Ribosome display + -
Translational and/or protein folding studies + -
in vitro directed protein evolution + -
Quickly generate analytical amounts of protein for further characterisation + +
Production of 'difficult' proteins that may be toxic + +
Confirmation of open reading frames + +
Examination of the effects of mutations on ORFs + +
Generation of truncated proteins to identify active domains and functional residues + +
Epitope mapping + +
Ligand-binding region determination/confirmation/competition assays + +
Protein structure analysis + +
Printing protein arrays + +
In vitro expression cloning (IVEC) (functional genomics) + +
Introduction of modified, unnatural or labelled amino acids + +
Isotopic labelling + +
Drug screening (affecting translational rates) + +
Mutation and detection analysis (i.e., enzyme kinetics) + +
Protein:protein interactions (using GST pull-downs) + +
Immunoprecipitation of protein complexes + +
Protein dimerization assays + +
Electrophoretic mobility shift assays (EMSAs) for DNA:protein interactions + +
DNA footprinting and protein cross-linking studies + +
Protein-RNA binding assays + +
Post-translational modification test + +

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