PRODUCT

E. coli High-Yield Cell-Free Protein Expression Kit

Prep-scale CECF protein expression from circular DNA

Built for diverse needs and high‑performance workflows, this cell-free protein expression kit leverages the Continuous Exchange Cell-Free (CECF) principle along with an enhanced E.coli lysate to achieve medium-to-preparative-scale protein yields from plasmid DNA. It supports efficient and scalable production of up to 6-mg protein per run.

High-yield CECF system enables optimized plasmid-based expression scalable from 0.5 mg to 6 mg per 1-mL reaction
Ideal for structural biology workflows including NMR spectroscopy and X-ray crystallography thanks to a preparative-scale yield
Flexible amino acid supply allows substitution of methionine for labeling studies
Optimized for thermomixer-based operation, ensuring consistent CECF exchange and robust yields

Select a Size

E. coli High-Yield Cell-Free Protein Expression Kit (5 Reactions)

#CF31ECXPRO-Med

Specifications Description Quality FAQ Resources

Product Number	CF31ECXPRO
Delivery Format	Tubes
Source	E. coli
Shipping Conditions	Dry Ice
Storage Temperature	-20 C
Stability	24 months from production; refer to expiration dates for details
Function	Continuous-exchange cell-free (CECF) transcription and translation; Plasmid-based protein expression; Expression of toxic or difficult proteins
For Use In	Medium-to-preparative-scale protein production workflows; Structural biology; Isotopic labeling for proteins
Intended Use	This product is intended for research use only

Express Proteins Faster

The E. coli High-Yield Cell-Free Protein Expression Kit is a powerful solution for producing up to 6 mg of protein in a single reaction. Leveraging advanced Continuous Exchange Cell-Free (CECF) technology and a high-yield E. coli lysate, this kit ensures efficient coupled transcription and translation for medium-scale protein expression. Designed for flexibility, it supports the use of custom amino acid mixtures, including the substitution of methionine with selenomethionine for specialized applications.

Ideal for structural biology studies, this kit is optimized for use with a thermomixer, ensuring continuous agitation for maximum protein yield and quality. Whether you're working with standard soluble proteins or challenging membrane proteins, this kit delivers reliable results for your research needs.

CECF Explained:

The Continuous Exchange Cell-Free (CECF) principle uses two semi-permeable membranes to exchange substrates and byproducts between a 1-mL reaction chamber and a 10-mL feeding chamber. This system continuously supplies essential components (e.g., energy, nucleotides, amino acids) while removing inhibitory byproducts, enabling protein expression to continue for up to 24 hours. Without this technology, reactions would stop prematurely due to depletion of substrates or accumulation of byproducts.

Key Features

Toxic Protein Expression: Enables the synthesis of proteins that are difficult or impossible to produce in vivo.
Versatile Protein Expression: Suitable for both soluble and membrane proteins.
Customizable Amino Acid Incorporation: Easily substitute methionine with selenomethionine or create tailored amino acid mixtures.
Advanced CECF Technology: Ensures high-yield protein production with efficient resource utilization.
Optimized for Thermomixers: Continuous agitation enhances the efficiency of the CECF process.

Applications

Medium-Scale Protein Production: Achieve up to 6 mg of protein per reaction for preparative studies.
Structural Biology: Ideal for synthesizing proteins for NMR spectroscopy or X-ray crystallography.
Custom Protein Labeling: Incorporate labeled methionine or other amino acids for specialized research applications.

Custom Component Options

We offer flexible customization to meet your specific needs:

Volume: Optimize packaging sizes to match your application scale.
Concentration: Customize concentrations for optimal performance.
Formulation: Available in custom formats to suit your protocols.

Quality

In vitro protein expression products are manufactured using high quality chemicals and materials.

Component Testing
Each component is tested to ensure the composition and quality meet stated specifications.

Performance Testing
Each product is tested to ensure it meets the stated performance specification.

FAQ

Answers to Commonly Asked Questions

How much protein can I produce with your kits?

The E. coli High-Yield Cell-Free Protein Expression Kit is designed for medium-scale protein production and can produce up to 6 mg of protein in 24 hours. This high yield is achieved using advanced Continuous Exchange Cell-Free (CECF) technology, which sustains protein synthesis over extended periods by continuously supplying substrates and removing inhibitory byproducts. The kit is ideal for applications requiring larger quantities of protein or functional assays, and delivers high-quality proteins suitable for downstream research.

Additionally, the protein expression yield of a given gene is influenced by multiple factors, including and not limited to its mRNA structure, the amount of template DNA utilized per reaction, and the use or not of certain additives. Therefore, for those who seek to maximize the yield, we highly recommend evaluating the expression yield and optimizing the reaction conditions of the gene constructs of interest with the smaller-scale, E.coli Cell-Free Protein Expression Kit first and then upscaling the production with this kit.

Can I use labeled amino acids in cell-free expression?

Yes, the E. coli High-Yield Cell-Free Protein Expression Kit is specifically designed to support the incorporation of labeled amino acids, such as selenomethionine for X-ray crystallography or ¹⁵N/¹³C-labeled amino acids for NMR spectroscopy. Unlike in vivo systems, where labeled amino acids are often metabolized or diluted, this kit provides complete control over the labeling process. The amino acids are supplied separately from other reaction components, allowing researchers to easily substitute labeled amino acids for their unlabeled counterparts. This ensures uniform or selective incorporation, making it easier to produce proteins tailored for structural studies with minimal waste of expensive labeled reagents.

What templates are compatible with your kits?

The E. coli High-Yield Cell-Free Protein Expression Kit is compatible with plasmid DNA templates, which are ideal for medium-scale protein production due to their stability and efficiency in sustained reactions. While linear DNA templates are supported in smaller-scale systems, plasmids are recommended for this kit to maximize yield and reaction longevity.

Templates must include essential regulatory elements such as a T7 promoter, a ribosomal binding site (RBS), and a T7 terminator to ensure efficient transcription and translation.

Do I need special equipment to use these kits?

While the E. coli High-Yield Cell-Free Protein Expression Kit does not require highly specialized equipment, a shaking incubator is recommended to facilitate the CECF process. The shaking motion ensures proper mixing of the reaction and feeding chambers, allowing substrates to flow across the semi-permeable membranes and byproducts to be removed efficiently. This setup ensures sustained protein synthesis for up to 24 hours. A standard laboratory incubator can be used for temperature control, but the shaking incubator is essential for achieving maximum yields.

Can I add supplements to the reaction to improve protein quality?

Yes, the E. coli High-Yield Cell-Free Protein Expression Kit is designed to accommodate the addition of supplements to improve protein quality including solubility, yield, and functionality. For example, detergents like DDM or CHAPS can be added to stabilize membrane proteins, while stabilizers like sorbitol can enhance the solubility of non-membrane proteins. Chaperones such as DnaK or GroE can also be included to assist in proper protein folding. Please refer to Appendix 3 of the User Guide for more details.

The system allows up to 200 µL of supplement solution to be added to the reaction chamber and 2 mL to the feeding chamber, ensuring sustained delivery of supplements throughout the reaction. This flexibility makes it possible to optimize for expressing challenging proteins and producing high-quality, functional results.

If you have additional questions please connect with a member of our team.

RESOURCES

Download the User Guide

CF31ECXPRO User Guide

Related Products

E. coli Cell-Free Protein Expression Kit