Cell-Free In Vitro Protein Expression

Cell-Free In Vitro Protein Expression


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Cell-Free In Vitro Protein Expression

As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for the expression of proteins including large proteins and membrane proteins, which are challenging for traditional expression systems. Creative BioMart has developed cell-free in vitro expression systems to meet the increasing demands for the protein synthesis of those particular protein types.

Cell-free in vitro protein expression can be performed in so-called “coupled” systems running RNA transcription and protein translation at the same time in the same reaction mixture. It is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Only two basic conditions are required to establish the system: the template encoding the target protein and a reaction solution containing the necessary transcriptional and translational molecular machinery. Cell extracts supply all or most of the molecules of the reaction solution, including:

  • RNA polymerases for mRNA transcription.
  • Ribosomes for polypeptide translation.
  • tRNA and amino acids.
  • Enzymatic cofactors and an energy source.
  • Cellular components essential for protein folding.

Creative BioMart offers a wide variety of cell-free in vitro systems. Selection of a cell-free system should be according to the biological nature of the protein, application and the template used for protein expression. Our team can guide you through the process of the cell-free expression if you would like to discuss more about your project.


Table 1. Summary of different cell free expression systems

System Advantages Disadvantages
E.coli extract
  • Established system
  • Relatively low cost
  • High translation speed
  • Can be coupled with nanodisc assembly for membrane proteins
  • Genetically modified strains available
  • No post-translational modifications of the eukaryotic systems
  • Codon usage optimization is required
Wheat germ
  • Established system
  • Generally highest yield among eukaryotic cell-free systems
  • High ratio of soluble expression
  • Suitable for the synthesis of large proteins (∼200 kDa)
  • Relatively high cost due to time-consuming extract preparation
  • Some background phosphorylation was observed in protein MS studies
  • No glycosylation
Insect cell lysate
  • Suitable for the synthesis of large proteins
  • Cap independent translation
  • N-glycosylation possible
  • Formation of disulfide bridges
  • Relatively new system
  • Lower protein yield than E. coli or wheat germ systems
  • Relatively high cost
Rabbit reticulocytelysate
  • Established system
  • Mammalian system
  • Cap independent translation
  • Often used in research
  • Sensitive to additives
  • No glycosylation
  • Co-expression of off-target proteins was observed
  • Hemoglobin con. ∼90% of protein
Human cell lysate
  • Some protein modification
  • Low yield
  • Sensitive to additives
Contact us or send an email at for project quotations and more detailed information.

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