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Cell-free protein synthesis (CFPS) offers important advantages over traditional in vivo expression because it provides a more open, flexible, and controllable reaction environment. Since there is no living cell to maintain, the researcher can directly adjust variables such as ionic strength, pH, redox conditions, DNA template concentration, cofactors, chaperones, detergents, lipids, or energy substrates without worrying about cell viability. CFPS is also typically faster, allowing protein production in hours rather than requiring cell growth, transformation, and induction steps over longer periods. In addition, it facilitates rapid prototyping of constructs and reaction conditions (Garenne et al., 2021; Jewett et al., 2008).
Another major advantage is that CFPS is particularly useful for proteins that are difficult to express in living cells, such as toxic proteins, membrane proteins, or proteins that require non-standard reaction environments. Because the system is open, reagents can be supplied directly and problematic cellular responses such as toxicity, growth inhibition, or proteolytic stress can be reduced (Garenne et al., 2021; Meyer et al., 2025).
Two cases where cell-free expression is more beneficial than cell-based production are:
| Cases | Description |
|---|---|
| 1) Toxic proteins | They may inhibit growth or kill the host cell during in vivo production (Chipman et al., 2025). |
| 2) Membrane proteins | CFPS allows co-translational insertion into detergents, nanodiscs, or liposomes under defined conditions, improving solubility and functional analysis (Meyer et al., 2025). |
A cell-free expression system generally includes the following components:
| Component: | Description |
|---|---|
| 1) Cell extract or purified transcription–translation machinery | Provides ribosomes, translation factors, tRNAs, aminoacyl-tRNA synthetases, and often metabolic enzymes needed for protein synthesis. In extract-based systems, these components come from lysed cells; in reconstituted systems, they are added as purified factors. (1) |
| 2) DNA or mRNA template | Contains the coding sequence for the target protein and the regulatory elements needed for transcription and/or translation (1). |
| 3) Amino acids | Serve as the building blocks for protein synthesis (1). |
| 4) Nucleotides (ATP, GTP, CTP, UTP) | Required for transcription and for translation-associated energy consumption (1) |
| 5) Energy source and regeneration system | Maintains ATP and GTP availability during the reaction, which is essential because protein synthesis is highly energy demanding (2; 3) |
| 6) Salts and buffer components | Helps to keep suitable ionic strength and pH for enzyme activity and ribosome function, especially magnesium and potassium ions (3) |
| 7) Cofactors and additives | Include chaperones, disulfide-bond helpers, detergents, lipids, nanodiscs, or microsomes depending on the protein being expressed (4; 5) |
Description: 1. Garenne et al., 2021); 2. (Jewett et al., 2008); 3. (Caschera, 2025); 4. (Harris et al., 2020); 5. (Meyer et al., 2025).
Additionally, a view of a CFPS by the article:
Figure 1. CFPS compounds from (Hong et al., 2014)
Energy regeneration is critical in CFPS because transcription and translation consume large amounts of ATP and GTP. Without a continuous energy supply, the reaction quickly slows or stops, lowering protein yield. In addition, some simple high-energy substrates can accumulate inorganic phosphate, which chelates magnesium and impairs ribosomal activity, further reducing productivity (Yavad et al., 2025). One way to ensure continuous ATP supply is to use an ATP-regeneration system based on phosphoenolpyruvate (PEP), which donates phosphate groups for ATP resynthesis. Another effective strategy is to use maltodextrin/polyphosphate-based metabolism in crude extracts, which can support longer-lasting and more cost-effective ATP regeneration through endogenous metabolic enzymes (Caschera & Noireaux, 2015; Chen et al., 2019).
An alternative approach is the use of metabolic energy regeneration systems, such as glucose-based pathways. Anderson et al. (2015) demonstrated that glucose metabolism in eukaryotic cell-free systems enables sustained ATP production through endogenous enzymatic pathways, improving reaction longevity and cost efficiency.
Figure 2. Abstract from: Anderson et al., 2015)
Figure 3. Abstract from: (Mu et al., 2024)
Additionally, recent tools, such as ATP biosensors (Mu et al., 2024), provide insights into the energetic dynamics of biological systems. Although not directly used for ATP regeneration, these biosensors can help optimize cell-free reactions by monitoring ATP availability in real time and guiding adjustments in energy supply strategies.