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Cell-free protein synthesis (CFPS) makes proteins outside intact cells. A DNA template is transcribed into messenger RNA (mRNA), then ribosomes and other translation components read the mRNA and join amino acids into a polypeptide. Whether that chain folds correctly or becomes an active protein depends on the target and the reaction system.
The path from DNA to a protein chain
- Prepare a compatible template. The template carries the gene and the signals the selected reaction needs to recognize it. In a common E. coli system using T7 RNA polymerase, for example, the expression unit typically has a T7 promoter before the coding sequence and a ribosome-binding sequence such as a Shine–Dalgarno element. A start codon marks where translation begins; a stop codon marks where it ends. Depending on the system, the input may be a plasmid, linear DNA, or mRNA. Template-design guidelines explain why these formats and signals are not automatically interchangeable.
- Transcribe DNA into mRNA. RNA polymerase recognizes the promoter and makes an RNA copy of the gene. In a coupled reaction, transcription and translation occur in the same vessel, allowing ribosomes to use the newly produced mRNA.
- Translate the mRNA. A ribosome reads the mRNA three bases at a time, as codons. Transfer RNAs (tRNAs) pair with those codons and deliver their attached amino acids. Aminoacyl-tRNA synthetases charge tRNAs with the appropriate amino acids; initiation, elongation, termination, and recycling factors support the successive stages of translation. A 2024 review of cell-free gene expression describes the machinery and its applications.
- Release the polypeptide. When the ribosome reaches a stop codon, translation terminates and the newly made chain is released. That is the immediate product—not necessarily a correctly folded, soluble, modified, or biologically active protein.
What the reaction needs besides a template
DNA or mRNA supplies instructions, but the reaction also needs the machinery and chemistry to use them: transcription and translation components, amino acids, nucleotide building blocks, salts, cofactors, and a means of regenerating usable energy. Exact ingredients and amounts depend on the formulation; there is no single universal CFPS recipe.
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In extract-based reactions, many cellular components and metabolites are already present in a lysate made from broken-open cells, with additional ingredients supplied according to the formulation. In a purified system, the required machinery and small molecules are assembled more deliberately. These components work together outside an intact cell, so protein production does not depend on growing cells. A User’s Guide to Cell-Free Protein Synthesis provides an overview of the method.
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Both approaches perform the core jobs of transcription and translation, but they differ in how the reaction is assembled and how much of its composition is defined.
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| Comparison | Lysate-based extract | PURE / purified-component system |
|---|---|---|
| What is supplied | Cell extract containing a complex mixture of cellular machinery and metabolites | Purified transcription and translation machinery plus defined small molecules |
| Composition | Complex and less fully specified | More compositionally defined and modular |
| Practical trade-off | Often attractive for cost and broad reaction capacity; lysate batch and background chemistry can affect performance | Greater control and fewer unrelated extract constituents; higher cost is a commonly cited drawback |
| Where it can be useful | General protein prototyping and many expression tasks | Experiments that benefit from defined composition, modular changes, or reduced background |
PURE is described as having fewer contaminating proteases, nucleases, and phosphatases than extract-based systems. Extract composition and performance can vary with the lysate. These are broad distinctions, not a guarantee that one approach will outperform the other for a particular protein: the target and exact formulation matter. See the review of the state of the art and the 2023 review of optimization in cell-free systems.
Choosing a DNA template format
A plasmid is a circular DNA template; linear DNA can be made by PCR and can work robustly in some E. coli lysate reactions. Some commercial lysate and reconstituted products also accept mRNA, while others support particular combinations of template formats. Check the chosen system’s requirements for the input format, promoter, translation-initiation sequence, and any template-protection needs before preparing a reaction. A coding sequence alone may not provide the signals that system needs, and compatibility should not be assumed across products. The 2021 template-design guidelines discuss these considerations.
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When is the synthesized protein actually “finished”?
Translation produces a polypeptide chain. For that chain to become a functional protein, it may also need to fold correctly, remain soluble, form disulfide bonds, insert into a membrane, or receive post-translational modifications. Those outcomes are not guaranteed by translation alone; they depend on the protein and what the reaction system supports. Some targets may need added folding support, an oxidizing environment, membrane mimics, or cellular processing steps not present in a given setup.
A 2015 Nature Protocols method describes synthesizing and measuring the activity of membrane proteins with PURE in a workflow completed within one day, from template DNA preparation to activity measurement. That timing applies to the specific protocol, not to CFPS workflows generally. The protocol details the method and its conditions.
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