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A cell-free system for protein synthesis is a reaction mix that makes protein in a test tube instead of inside living cells. It supplies the translation machinery, an energy source and a template, so a researcher can go from nucleic acid to protein without transforming, transfecting or culturing cells. This page compares the system types on offer, the reaction formats that decide how much protein you get, and the criteria for choosing between them.
Who uses it: bench researchers who want recombinant protein quickly, and groups making proteins that are awkward to express in living cells, such as membrane proteins.
What a cell-free system is
Cell-free protein synthesis (CFPS) produces functional protein “in the test tube, rather than in cells”, as Rosenblum and Cooperman put it in their 2014 FEBS Letters review. The approach has been in use for more than five decades. Thermo Fisher Scientific describes the practical appeal: the technique lets researchers express recombinant proteins more quickly because the steps of transforming, transfecting and culturing cells are skipped.
What is in the reaction
There are two broad ways to supply the machinery. A lysate-based system uses an extract from cells. Extracts have been prepared from bacteria, protozoa, plants, insects and mammals; named examples include E. coli, wheat germ, rabbit reticulocyte lysate, insect cells, HeLa cells and Leishmania tarentolae. A reconstituted system, the best-known example being PUREexpress, instead combines purified components: ribosomes, tRNAs, synthetases, translation factors, amino acids and energy sources.
Types of cell-free system
| System | What it is | Characteristics |
|---|---|---|
| E. coli lysate | Bacterial extract | Described as a robust system with high yield (Thermo Fisher) |
| Rabbit reticulocyte lysate | Mammalian extract | Described as a flexible system with low yield (Thermo Fisher) |
| HeLa lysate | Human-derived extract | Medium-to-high yield, with higher protein yield per reaction (Thermo Fisher) |
| Lipid-bilayer system (MembraneMax) | Lysate plus a lipid bilayer with a scaffold | Designed for membrane-bound proteins (Thermo Fisher) |
| PURE-type system | Purified, defined components | Cost per mg of protein considerably higher than lysate-based kits (Rosenblum and Cooperman) |
Wheat germ, insect-cell and Leishmania tarentolae extracts extracts, named above, are also in use.
Reaction formats and scale
The format of the reaction sets the ceiling on yield and run time. In a plain batch reaction, yield is limited by oxygen depletion and by inhibitory by-products. The main formats are below.
| Format | How it works | Reported yield and run time |
|---|---|---|
| Batch | All reagents in one closed volume | Yield limited by oxygen depletion and inhibitory by-products; lysate reactions reach tens of micromolar target protein |
| Thin-film batch | Batch reaction spread to improve oxygen transfer | As much as 0.5 mg/mL of active protein |
| Two-compartment (continuous exchange) | Extract kept apart from a feeding solution of amino acids, ATP and GTP | Substrate is replenished and by-products removed; translation maintained for tens of hours |
On scale, Rosenblum and Cooperman describe well-plate reactions using volumes as low as 10 µl and a 100-litre reaction that produced 700 mg of protein per litre over 10 hours, with linear scalability.
How to choose
- Native post-translational modifications: Thermo Fisher notes that mammalian systems are more likely to give native modifications.
- Membrane-bound proteins: a lipid-bilayer system such as MembraneMax is the option the Thermo Fisher page lists for them.
- Defined composition: a PURE-type system uses purified components, at a considerably higher cost per mg of protein than lysate-based kits.
- Yield per reaction: Thermo Fisher rates the E. coli system high and rabbit reticulocyte low; HeLa sits at medium-to-high.
- Longer runs: a two-compartment format replenishes substrate and removes by-products, keeping translation going for tens of hours.
Common mismatches
- Choosing the highest-yield bacterial lysate when the protein needs native modifications, which are more likely from mammalian systems.
- Expecting a closed batch reaction to run indefinitely: oxygen depletion and inhibitory by-products cap it.
- Treating cell-free expression as a replacement for downstream work: the product is still a protein that may need purification.
FAQ
What is a cell-free system for protein synthesis?
It is a reaction in which protein is made in a test tube using translation machinery, an energy source and a template, with no living cells involved.
Why use one instead of cells?
It skips transformation, transfection and cell culture, so recombinant protein can be produced more quickly.
What is the difference between a lysate and a PURE system?
A lysate is a cell extract; a PURE-type system is assembled from purified components. The purified system costs considerably more per mg of protein.
Which cell-free system gives the highest yield per reaction?
Thermo Fisher rates E. coli yield as high, HeLa as medium-to-high and rabbit reticulocyte as low.
Which system suits membrane proteins?
Thermo Fisher lists a lipid-bilayer system, MembraneMax, for membrane-bound proteins.
Related pages
For where expressed protein goes next, see protein purification, gene cloning for the template side, what a bioreactor is for the cell-based alternative at scale, bacterial transformation for the step cell-free systems skip, and proteomics.
Sources
- Rosenblum G, Cooperman BS. Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses. FEBS Lett 2014;588(2):261-268. https://pmc.ncbi.nlm.nih.gov/articles/PMC4133780 (checked 2026-10-29).
- Thermo Fisher Scientific. Cell-free protein expression. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/cell-free-protein-expression.html (checked 2026-10-29).
