Cell-free
technologies.
Accelerating protein expression for Life Sciences, Biotechnology and Pharma.
Cell-free protein expression, also known as acellular protein expression, is an innovative technology based on the use of a reaction mix containing a cell extract that preserves the active transcription-translation machinery, an energy source, and the building blocks required for mRNA and protein assembly.
Combining rapid expression, high experimental flexibility, and linear scale-up capacity, CFPS (Cell-Free Protein Synthesis) has become an efficient solution to accelerate development cycles and reliably produce proteins in a scalable and adaptable way, from early screening phases to larger production volumes.
Increasingly used in both fundamental research and biotechnology, this technology enables the study and production of proteins that are sometimes difficult to obtain using traditional heterologous systems, particularly in contexts such as genetic construct testing, expression of proteins potentially toxic to cells, structural biology, or functional studies.
What is Cell-Free protein expression?
Cell-free protein expression refers to a system in which protein synthesis occurs outside a living cell. The biological machinery required for transcription and translation is extracted from organisms (bacteria, yeast, insect cells, or mammalian cells) and used in vitro in a controlled reaction environment. This principle, often referred to as CFPS (Cell-Free Protein Synthesis) (see glossary), reproduces the fundamental steps of gene expression while eliminating constraints related to cell growth, viability, or cellular regulation.
Used in both academic research and biotechnology, this innovative approach builds upon traditional heterologous expression systems while offering a flexible and adaptable experimental framework for protein production and analysis.
How does a Cell-Free expression expression system work?
General principle of Cell-Free protein synthesis
Cell-free protein synthesis (CFPS) is based on a simple concept: using the cell’s protein synthesis machinery without keeping the cell itself.
After cell lysis, the components required for gene expression remain active within a biological extract called a lysate or cell extract.
This extract is supplemented with an energy source, nucleotides, amino acids and optimized additives.
These elements allow the synthesis of mRNA and recombinant proteins.
In this optimized reaction environment, a DNA or RNA template encoding the protein of interest is directly added. The enzymes and cofactors present then perform the same steps as in living cells: gene reading, messenger RNA production, protein synthesis
The system therefore functions like an “open cell.”
The biochemical environment is experimentally controlled and no longer depends on cell growth, enabling rapid access to protein production.
In vitro transcription and translation
Once the genetic template is added, the reaction follows a sequence similar to natural gene expression:
- Transcription : the DNA template is recognized by RNA polymerase, which synthesizes messenger RNA.
- Translation initiation: the ribosome binds to the mRNA with initiation factors.
- Elongation: tRNAs successively deliver amino acids corresponding to the encoded sequence.
- Termination: the polypeptide chain is released.
All these steps occur in vitro, in an open system. In some cases, mRNA can be added directly, bypassing the transcription step.
This acellular protein expression therefore enables rapid, observable, and tunable protein synthesis, a hallmark of CFPS systems.
STEP 1 – PREPARATION OF THE REACTION MIXTURE
Role of cell lysates
The core of the system is composed of cell lysates (see glossary – lysate). These extracts are obtained after controlled disruption of cells (often E. coli, but also yeast, insect, or mammalian cells), in which the transcription-translation machinery remains functional.
These lysates contain in particular:
- RNA polymerases, responsible for transcribing DNA into mRNA
- initiation, elongation, and termination factors required for translation
- ribosomes, which assemble amino acids into polypeptide chains
- tRNAs and associated enzymes, transporting amino acids to ribosomes for incorporation into proteins
- molecular chaperones, which assist proper protein folding and prevent aggregation
- energy systems and cofactors
These components are essential because they directly determine the system’s capacity to produce correctly synthesized and folded proteins. Protein synthesis properties depend on the cell lysate used, but they can also be enhanced by adding specific additives to the reaction mixture.
Role of reaction additives
Using lysates to produce recombinant proteins outside cells allows direct control over reaction conditions. Medium composition, additives, lipids, cofactors, ligands, or protein partners can be introduced without toxicity constraints or membrane permeability issues. Researchers therefore manipulate the biochemical environment of translation, something difficult to achieve in classical cell culture systems.
CFPS reactions are supplemented with:
- Energy regeneration systems, supplying ATP and GTP required for transcription and translation
- Biological building blocks, such as NTPs and amino acids, enabling mRNA synthesis and protein assembly
- Essential cofactors and salts, stabilizing enzymes and maintaining optimal biochemical conditions
Specific additives can also be included depending on the protein being expressed, such as:
- Detergents or lipids for hydrophobic or membrane proteins
- Ions or cofactors for enzyme stabilization
- Molecular chaperones for correct folding of complex proteins
- Other components adapted to the specific requirements of the target protein
Thanks to this modularity, the CFPS reaction can easily be adapted to recreate a favorable biochemical environment for the expression and functionality of a specific protein.
STEP 2 – PROTEIN EXPRESSION
What Differentiates a cell-free system from a cellular system
DIn classical cellular expression systems, proteins are produced by living cells that must grow, divide, and maintain their metabolism. Gene expression therefore occurs within a complex network of biological regulation.
In a cell-free system, these constraints disappear. There is no intact membrane, no cell viability to maintain, and no biological selection pressure. Protein expression becomes a direct biochemical process rather than a physiological one.
This fundamentally changes the experimental approach. The gene is no longer “expressed by a cell”; it is translated within a controlled environment. Researchers can intervene directly in the reaction, modify conditions during synthesis, or introduce compounds incompatible with living cultures.
Cell-free systems are therefore flexible experimental tools, particularly relevant for screening and characterization of mutant libraries before scaling production in either acellular or cellular systems.
Advantages of cell-free systems compared to cellular systems
Speed
One of the main advantages of CFPS is its speed of implementation. Unlike cellular systems, which require transformation or transfection steps, clone selection, and cell culture, protein synthesis in a cell-free system can start immediately after adding the nucleic acid template to the reaction mixture. Expression timelines are therefore dramatically reduced, from days or weeks to only a few hours.
This speed significantly accelerates testing, optimization, and screening cycles for genetic constructs. Researchers can rapidly evaluate different sequence variants, expression conditions, or reaction additives, facilitating rapid prototyping and faster decision-making during R&D phases.
Speed
The Syn-Xpress™ kits
For example, the Syn-Xpress™ kits we developed enable protein expression:
- in 4 hours in “Flash” mode for rapid testing or screening
- in 16 hours in “HighYield” mode to maximize protein yield
This flexibility allows the reaction duration to be easily adapted to experimental needs while maintaining the speed characteristic of CFPS systems.
Experimental controm enabling expression of complex proteins
In acellular protein expression, synthesis no longer depends on a living cell. The reaction environment is directly accessible and adjustable, transforming protein expression into a controllable experimental parameter. Reaction conditions can be adjusted or enriched depending on the specific needs of the protein being produced.
For example, researchers can add:
- cofactors to stabilize certain enzymes
- lipids or detergents to promote membrane protein synthesis
molecular chaperones to assist folding of complex proteins - labeled or modified amino acids for analytical or structural applications
This open and highly modular system allows fine optimization of expression conditions and provides considerable experimental freedom.
It enables advanced applications such as:
- membrane protein studies
- protein engineering
- incorporation of non-natural amino acids
For example, thanks to our proprietary technology, certain membrane proteins such as CXCR4 or the pro-apoptotic protein Bak can be produced at the milligram scale in about one week (depending on expression levels). This allows researchers to quickly test hypotheses, support structural biology projects, or make earlier decisions about continuing research programs.
Some proteins are difficult or even impossible to produce in cellular systems, especially when they are toxic to host cells. In such cases, their expression may disrupt cell growth or cause cell death, drastically reducing yields and complicating the production of complete functional proteins.
Cell-free systems overcome this limitation. Because no living cells are involved, protein synthesis occurs without cellular viability constraints, allowing the expression of toxic or unstable proteins in a controlled environment.
CFPS is therefore particularly suitable for producing difficult proteins such as certain toxins, highly active enzymes, or proteins that strongly disrupt cellular metabolism.
For more than 15 years, our R&D team has continuously optimized our cell-free technology.
This expertise results in highly reproducible outcomes:
- 80–90% of proteins successfully expressed
- ~80% retain functional activity
This allows researchers to quickly obtain reliable information on expression, solubility, or activity, without going through long biological optimization phases.
Linear scale-up
Finally, CFPS systems offer linear and predictable scale-up. Increasing reaction volumes does not require complex optimization linked to cell growth or metabolism. In most cases, scaling up simply involves proportionally increasing the volume of reaction components, facilitating the transition from small-scale prototyping to larger-scale production.
Our protein designers are currently able to produce quantities covering a wide range, typically from 10 µg to 100 mg, suitable for biochemical analyses as well as structural or functional studies. Production volumes can also be increased to reach gram-scale protein quantities, which is the objective of our Cell-free’Up program.
Applications of cell-free systems
R&D – Rapidly explore and characterize a target
- Rapid production of a protein of interest
- Study of membrane proteins or difficult-to-express proteins
- Analysis of protein-protein interactions
- Functional studies
- Validation of genetic constructs
- Preparation of samples for structural biology
Quickly obtain a characterizable sample to validate experimental hypotheses and secure interpretation of results.
Diagnostics – Develop and validate biological assays
- Production of recombinant antigens
- Reporter enzymes for analytical assays
- Biosensor components
- Easy integration into rapid or miniaturized tests
Obtain functional biomolecules under controlled conditions, facilitating analytical evaluation (sensitivity, specificity, reproducibility).
Screening – Rapidly test numerous candidates
- Screening of protein variants
- Evaluation of mutant libraries
- Optimization of biochemical conditions
- Direct expression from linear DNA
Compare variants in a homogeneous experimental framework and generate comparable data for rational candidate selection.
Why choose Synthelis?
Since 2011, Synthelis Biotech has established itself as a strategic partner for accelerating biological projects, from protein expression to the development of life science tools. Our approach focuses on transforming complex biological challenges into validated industrial solutions.
Cell-Free technology: Speed and Flexibility
Our technology platform redefines standards for speed and customization in biological production:
• Short production cycles: protein production and purification within 24–48 hours after optimization
• High-throughput screening: thousands of mutants expressed and potential hits identified within 7 days
• Ease of use: ready-to-use Syn-Xpress™ kits (E. coli-based) requiring only DNA addition and enabling protein production in 4 h (Flash Mode) or 16 h (HighYield Mode)
• Proven performance: for some protein classes such as GPCR receptors, yields can reach up to 20× higher than cellular systems
Expertise in producing “difficult-to-express” proteins
Lhe added value of Synthelis Biotech lies in its mastery of protein expression where traditional cellular systems reach their limits:
• Expression success: more than 400 complex proteins successfully produced, with expression success rates above 90%
• Biological activity: about 80% of delivered proteins show demonstrated functional activity
• Overcoming in-cell expression bottlenecks: our cell-free system bypasses issues such as inclusion body formation or cytotoxicity encountered in classical E. coli systems
• Complex targets: extensive expertise in expressing membrane proteins (GPCRs, ion channels, transporters), soluble proteins, or proteins requiring post-translational modifications
A comprehensive and complementary offering
Although cell-free expression is our core expertise, we provide a complete ecosystem to support research projects:
• Solutions for everyone: services ranging from mutant screening and feasibility studies to large-scale production, as well as ready-to-use Syn-Xpress™ kits
• Next-generation bioluminescence: development of Hikarazine™ pro-substrates (3–15× brighter than standard luciferin) and LuliFLASH™ technology for ultra-sensitive immunoassays
• Complementary services: classical E. coli expression, protein engineering, and official distribution in France of Berthold bio-analysis instruments
• Tailored support: from protein design to functional validation, covering the entire value chain to accelerate time-to-market
Cell-Free vs Cellular expression: comparison
Do you want to master CFPS technology?
FAQ – Cell-Free protein expression
What is cell-free protein expression (CFPS)?
Cell-free protein synthesis (CFPS), also known as cell-free protein expression, is a protein production method performed outside living cells. It uses cellular extracts containing the natural transcription and translation machinery (enzymes, tRNAs, ribosomes, etc.), supplemented with an energy source, nucleoside triphosphates (NTPs), amino acids, and other additives. Starting from DNA or RNA, the protein of interest is produced directly in vitro. This approach reproduces gene expression without the need for a living cellular unit.
How does a cell-free protein expression system work?
A genetic template (DNA or RNA) is added to an active cell lysate containing the components required for transcription and translation. When starting from DNA (plasmid or linear), messenger RNA is first synthesized, then ribosomes assemble the polypeptide chain. The reaction takes place in an open and controlled environment. Biochemical conditions can be adjusted during synthesis.
What are the advantages of cell-free systems compared to cell-based systems?
Cell-free systems provide rapid results without the need for cell line construction or prolonged culture. The open system allows direct adjustment of the synthesis and folding environment, as well as the addition of cofactors, ligands, or other additives. It also reduces expression failures linked to cytotoxicity, instability, or aggregation. Cell-free expression is particularly well suited for early-stage characterization and experimental validation.
What types of recombinant proteins can be expressed using cell-free systems?
Any type of protein can be expressed using cell-free systems, including membrane proteins through the addition of stabilizers (lipids or detergents), multi-subunit protein complexes through co-translation of several polypeptides, and toxic proteins.
When should cell-free protein synthesis (CFPS) be chosen?
CFPS is particularly relevant when a protein is difficult to express or when rapid results are required. It is used for target validation, interaction studies, labeling, prototyping, and variant screening. It enables the rapid production of a testable protein before considering larger-scale production either in cell-free or cell-based systems.
10 biomyths
about Cell-Free
systems
which you have probably always believed
