Cell-Free Protein Synthesis or Expression

What is cell free protein synthesis?

A form of synthetic biology, cell-free protein synthesis 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, CFPS empowers researchers to express a protein of interest within a day. An ideal approach to in vitro translation of functional proteins, suitable for a variety of downstream applications, CFPS is a trusted technique which is used across a variety of research areas.

• How does cell-free protein in vitro translation work?
• Which cell types are used to generate the extract?
• How does CFPS compare to other methods of protein production?
• What are the applications for CFPS?
• Next Generation Cell Free Protein Expression Kit (Wheat Germ)
• Published Study Utilizing our Cell Free Protein Expression System
• ALiCE^® Cell Free Protein Expression Kit (Plant Cell Lysate)

How does in vitro translation work?

CFPS 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 transcription and translation. This solution is a cell-free extract, so-called because it consists only of cytosolic and organelle material while the 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 CFPS 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 CFPS. Advances in the field have since led to the use of cell types including wheat germ, Leishmania tarentolae, rabbit reticulocyte, insect, plant and human cells.

Each cell type offers distinct advantages. For example, using wheat germ extract, 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. When using plant cell extract, yield can be increased up to 3mg/mL and folding of complex proteins and their glycosylation are enabled.

How does cell-free protein synthesis compare to 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 via cell transfection, bacterial transformation, or utilizing viruses by viral transduction. A disadvantage of this method is that some overexpressed proteins may have an activity which is detrimental to the host. Additionally, 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 at high cost.

However, by eliminating the need for cloning or cell culture, CFPS delivers significant time-savings in comparison to recombinant protein expression. Additionally, CFPS 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, including the incorporation of labeled amino acids.

Cell-Free Protein Synthesis applications

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

• Characterization of protein-protein or protein-nucleic interactions
• Structural or functional analyses 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 CFPS is likely to expand, due to the potential it offers for high-throughput, cost-effective production of large quantities of research-grade proteins.

Next Generation Cell Free Protein Expression Kit (Wheat Germ)

Developed to overcome the cost barrier of CFPE, while delivering superior stability, higher yield and greater flexibility for a larger variety of target proteins than many commercially-available products, our Next Generation Cell Free Protein Expression Kit (Wheat Germ) is a cost-effective solution yet with high yield to CFPE. With an average yield of 10µg/reaction (50 reactions provided in each kit), our kit offers many advantages, including applicability to high-throughput protein production and a user-friendly protocol.

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

Figure 1 Easily scalable protein production using MilliporeSigma’s Next Generation Cell Free Protein Expression Kit (Wheat Germ) (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 with options for N- or C-terminal tags. Further advantages include:

• Excellent yield with straightforward linear scale-up: 5.5mL sample scale per kit brings 0.5 - 1 mg of protein at low cost
• Proven expression for diverse proteins with high solubility, including kinases, enzymes, transporters, receptors, GPCR proteins, multimeric proteins and complexes, and toxic proteins ranging from 10 kDa to over 220 kDa
• Rapid protein expression in just a few hours due to eliminating the need for cloning and cell culture, and no requirement for codon optimization, even for expression of transcripts with a high A/T or G/C content
• Open system suitable for addition of natural or unnatural components to the reaction mixture and ideal for protein labeling

Figure 2. 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.

Published Study Utilizing our Cell Free Protein Expression System

Due to its high protein yield and easy-to-use format, the 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 patented 3’ URT short translation enhancer (STE) 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 with 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.

ALiCE^® Cell Free Protein Expression Kit (Plant Cell Lysate)

The ALiCE^® kit is a unique, eukaryotic cell-free system that utilizes cell lysate derived from plant cells. Compared with other eukaryotic cell-free systems on the market, which usually yield 0.1 mg/mL or less, or prokaryotic cell-free systems with yields up to 1mg/mL, the ALiCE^® kit yields an unprecedented 3 mg/mL of protein in batch mode. The power of the ALiCE^® kit lies in its intact organelles, with active mitochondria providing a continuous energy supply. Additionally, microsomes (endoplasmic reticulum reformed into vessels) enable folding and post-translational modifications of complex proteins, including disulfide bonds and glycosylation. Additional advantages for the ALiCE^® kit include:

• Up to 30 times more protein at market competitive prices
• Already validated for over 500 proteins
• Simplified workflow allows for minimal manipulations to start the reaction and included detergents facilitate easy protein recovery (including proteins located in microsomes).

Figure 3. Expression of GLuc. The luciferase of the Gaussia princeps is a globular protein (19 kDa) and its active form has five disulfide bonds. Protein was targeted to the microsomes and produced over 48 hr at 25 °C, 70% RH, 500 rpm and purified via His-Tag affinity chromatography. Activity determination was performed with catalysis of oxidation of coelenterazine to coelenteramide in a light-emitting reaction (lambda em) = 475 nm. Luciferase was concentrated during purification, resulting in higher arbitrary luminescence values.

The ALiCE^® Cell Free Protein Expression Kit (AL0103000) is a radically different approach to cell-free expression and a platform unlike any others. Importantly, your current cell-free expression processes can be easily translated to the ALiCE^® system. The ALiCE^® Cell Free Protein Expression Kit (AL0103000) has been applied for years within Corteva Agriscience, the agrobio division of DowDuPont™, as well as Fraunhofer IME to express over 500 proteins of varying types. This shows that the ALiCE^® kit is applicable for any purpose, be it for biopharmaceuticals, technical enzymes, crop development, metabolic pathways, fundamental research, etc. The ALiCE^® kit has been used to successfully express reporter proteins, antigens, scFv antibodies, allergens, hormones, full-size Abs and membrane proteins.

ALiCE^® is a registered trademark of LenioBio GmbH in Germany.

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