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Cell Free Expression

A cell-free system is an in vitro technology widely used to study biological reactions that happen within cells while reducing the complex interactions found in a whole cell. Subcellular fractions can be isolated by ultracentrifugation to provide molecular machinery that can be used in reactions in the absence of many of the other cellular components.

Figure 1. Cell-free protein synthesis system for producing proteins or (poly)peptide-based materials. (Front. Chem., 2014)Figure 1. Cell-free protein synthesis system for producing proteins or (poly)peptide-based materials. (Front. Chem., 2014)

Cell-free expression systems can be prepared by incubating a number of purified enzymes and coenzymes. Cell-free expression systems are developed as a novel cost-efficient biomanufacturing technology compared to cell-based fermentation used for thousands of years. Cell-free expression systems have several ADVANTAGES suitable in industrial applications:

  • Easily controlled and accessed without membranes.
  • Very high product yields accomplished without the formation of by-products or the synthesis of cell mass.
  • Implementing some biological reactions that living microbes or chemical catalysts unavailable before.
  • Faster reaction rates dependent on enzymatic systems, without the barrier of cellular membrane.
  • Strong toleratance to toxic compounds.
  • Broad reaction conditions, such as high temperature, low pH, the presence of organic solvents or ionic liquids.

Comparison of cell-free protein expression systems

E. Coli cell lysate Rabbit reticulocyte cell lysate Hela cell lysate CHO cell lysate
Yield High Low Medium-high High
Protein modifications None Limited glycosylation Glycosylation and phosphorylation Glycosylation and phosphorylation
Recommended for high MW proteins No No Yes Yes
Produces functional proteins Possible Possible Yes Yes
Comments Robust system Flexible system Higher protein yield per reaction Most economocal cost per miligram produced

Please see Creative Biostructure’s cell-free and cell-based expression services.

Reference:

  1. Y. H. Percival Zhang. Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities. Biotechnology and Bioengineering. March 2010. 105 (4): 663–677. doi:10.1002/bit.22630. PMID 19998281.
  2. New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chemistry. 15: 1708. doi:10.1039/C3GC40625C.
  3. High-Yield Hydrogen Production from Starch and Water by a Synthetic Enzymatic Pathway. PLoS ONE. 2: e456. doi:10.1371/journal.pone.0000456. PMC 1866174Freely accessible. PMID 17520015.
  4. Enzymatic transformation of nonfood biomass to starch. Proceedings of the National Academy of Sciences. 110: 7182–7187. doi:10.1073/pnas.1302420110.
  5. A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nature Communications. 5. doi:10.1038/ncomms4026.
  6. Biohydrogenation from Biomass Sugar Mediated by In Vitro Synthetic Enzymatic Pathways. Chemistry. 18: 372–380. doi:10.1016/j.chembiol.2010.12.019.
  7. Seok Hoon Hong, et al. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis. Front. Chem., 10 June 2014. doi:10.3389/fchem.2014.00034