Introduction to Cell-Free Protein Expression/Synthesis (CFPE/CFPS)

A form of synthetic biology, cell-free protein expression (CFPE) is a rapid method to produce recombinant proteins in solution without involving cells. Using biomolecular translation machinery extracted from a range of different cell types, CFPE empowers researchers to express a protein of interest within a day. An ideal approach to in vitro synthesis of functional proteins, suitable for a variety of downstream applications, CFPE is a trusted technique which is used across a diversity of research areas.

How does cell-free protein expression work?

CFPE requires a DNA template (either a plasmid or a PCR product) encoding the target protein, and a solution containing all the necessary components to drive its transcription and translation. This solution is a cell-free extract, so-called because it consists only of cytosolic and organelle material; cell membranes have been removed. When the genetic material and the cell-free extract are combined, expression of the target protein can begin.

Which cell types are used to generate the extract?

Early CFPE experiments used cell-free extract derived from E. coli. With rapid growth kinetics, E. coli quickly delivers high protein yields, however some eukaryotic proteins have been found to be insoluble following E. coli CFPE. Advances in the field have since led to the use of cell types including wheat germ, Leishmania tarentolae, rabbit reticulocyte, insect and human cells.

Each cell type offers distinct advantages. For example, using wheat germ, translation of large proteins is possible. Furthermore, wheat germ extract does not contain endogenous mRNAs, which could be translated alongside the target protein. Wheat germ-expressed proteins also maintain their complex 3-dimensional conformation since they are derived from a eukaryotic source.

How does cell-free protein expression differ from other methods of protein production?

The expression of recombinant proteins in live cells is a well-known and widely-used method of protein production. In this approach, DNA encoding a target protein is cloned into an expression vector which is subsequently introduced into a host cell line. A disadvantage of this method is that some proteins may have an activity which is detrimental to the host.

By eliminating the need for cloning or cell culture, CFPE delivers significant time-savings in comparison to recombinant protein expression. Additionally, CFPE is more adaptable to high-throughput applications and can deliver increased overall yields. The lack of a cell membrane also allows for easier manipulation of reaction conditions, for example facilitating the incorporation of labeled amino acids.

What are the applications for cell-free protein expression?

CFPE is applicable to a vast array of studies, including:

  • Characterization of protein-protein interactions
  • Evaluation of protein-nucleic acid interactions
  • Structural analysis
  • Functional comparability of full-length, truncated or mutated proteins
  • Identification of components essential to protein folding, stability or degradation
  • Production of protein microarrays
  • Expression of virus-like particles
  • Identification of drug candidates
  • Ribosome display

The utility of CFPE is likely to expand due to the potential it offers for high-throughput, cost-effective production of large quantities of research-grade proteins.

Are there any limitations to cell-free protein expression?

Generally, CFPE products are more expensive than proteins produced in live cells. This is especially the case if large quantities of material are required, or if the protein does not express particularly well. For this reason, CFPE is typically used to generate proteins for small-scale experiments.

Developed to overcome the cost barrier, while delivering superior stability, higher yield and greater flexibility for a larger variety of target proteins than many commercially-available products, the Next Generation Cell Free Protein Expression Kit (Wheat Germ) (Cat. No. CFPS700) is a cost-effective solution yet with high yield to CFPE. At just $10/reaction with an average yield of 10µg/reaction (50 reactions provided in each kit), this kit offers many advantages, including applicability to high throughput protein production.

Next Generation Cell Free Protein Expression Kit (Wheat Germ)

Suitable for the expression of both prokaryotic and eukaryotic proteins, the Next Generation Cell Free Protein Expression Kit (Wheat Germ) (Cat. No. CFPS700) employs a user-friendly protocol to deliver high yields of target protein within a short timescale.

Particularly suitable for production of proteins with complicated conformation and multiple protein complexes, such as disulfide-bond-containing or integral membrane proteins, the CFPS700 kit has been used successfully by researchers to produce a comprehensive variety of proteins.

Easily scalable protein production using Next Generation Cell Free Protein Expression Kit (Wheat Germ). Cat. No. CFPS700.
Easily scalable protein production using Next Generation Cell Free Protein Expression Kit (Wheat Germ). Cat. No. CFPS700.


The combination of machines, micelles and organelles in our wheat germ extract means that even membrane protein can be soluble without the need to add liposome. In addition, disulfide bonds are induced using our kit. Further advantages include:

  • Rapid procedure – achieve protein expression in just a few hours
  • Straightforward linear scale-up
  • Cheapest CFPE (wheat germ) kit in the market- just $10/reaction, about 45% cheaper than competitors, while enabling highest yield
  • Easy to use with options for C- or N-terminal tag
  • No need for cloning or cell culture
  • Excellent yield – obtain mg amounts of soluble protein from only a few mL reaction volume
  • Open system – add natural or unnatural components to the reaction mixture
  • Ideal for protein labeling
  • Suitable for high-yield expression of toxic proteins
  • Perfect to express multimeric proteins and complexes – subunit ratios can be titrated
  • Proved success in expressing Kinase, Enzyme, Transporter, Receptor and GPCR proteins, from 10kDa to over 220kDa
  • No need to add artificial liposome for membrane proteins
  • Overcomes DNA template instability
  • Animal-free and serum-free
  • High protein solubility due to native conformation and eukaryotic source
  • High stability
  • No requirement for codon optimization, even for expression of transcripts with a high A/T or G/C content

Western blot analysis of a variety of proteins generated using the Next Generation Cell Free Protein Expression Kit (Wheat Germ) Cat. No. CFPS700. Proteins were detected by a tag antibody, such as anti-Flag antibody.
Western blot analysis of a variety of proteins generated using the Next Generation Cell Free Protein Expression Kit (Wheat Germ) Cat. No. CFPS700. Proteins were detected by a tag antibody, such as anti-Flag antibody.


Chosen by researchers

Due to its high protein yield and easy-to-use format, CFPE system (Wheat Germ) was selected as the most suitable choice for a comprehensive protein expression study in the ‘‘Human Protein Factory’’ project at the National Institute of Advanced Industrial Science and Technology (AIST) in Japan.

In this study, published in Nature Methods, the researchers attempted to express 13,364 human proteins and assess their biological activity in two functional categories. Of these, 12996 clones (97.2%) produced protein in the CFPS700 kit and 77% of the 75 tested phosphatases were found to show biological activity.

The CFPS700 kit incorporates several product improvements. For example, the addition of a 3’ URT short translation enhancer (STE) (patented) make transcribed mRNA more stable, resulting in higher protein yield and eliminate the need for cloning. Also, our much-improved wheat germ extract method makes the extract lysate higher quality and better performance consistency.

To achieve high yields of research-grade recombinant proteins while benefiting from an easy-to-use format at a competitive price, look no further than the Next Generation Cell Free Protein Expression Kit (Wheat Germ) (Cat. No. CFPS700) to streamline your research, whatever your downstream application.

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