1. One of the key advantages of cell‐free protein synthesis (CFPS) over traditional in vivo expression is the high level of experimental control it provides—researchers can directly modulate the reaction environment (e.g., ionic conditions, pH, chaperones, cofactors) without worrying about cellular viability. This flexibility also speeds up optimization and screening because new conditions can be tested rapidly without having to culture cells. Additionally, toxic or otherwise difficult‐to‐express proteins can be synthesized in cell‐free systems without harming host cells, and post‐translational modifications may be more directly manipulated by supplementing relevant factors. Two common scenarios where CFPS offers distinct benefits over in vivo systems are the synthesis of membrane proteins—which can be co‐translated with specific detergents or liposomes—and the rapid synthesis of mutants for high‐throughput protein engineering or functional assays.

2. A typical cell‐free expression system includes an extract (e.g., from E. coli, wheat germ, or insect cells), an energy‐generating system (to supply ATP and GTP), amino acids, buffer salts, cofactors, and the DNA or mRNA template. The extract contains the ribosomes, tRNAs, and translation factors necessary for protein synthesis. The energy system (often a combination of phosphoenolpyruvate or creatine phosphate with the necessary enzymes) sustains ATP regeneration, essential for continuous protein synthesis. Amino acids supply the raw material for polypeptide chain elongation, while buffer salts (like Mg²⁺, K⁺, etc.) maintain optimal ion conditions for enzymatic activity. Finally, the nucleic acid template encodes the protein of interest; in transcription‐coupled systems, a promoter and RNA polymerase may also be added to generate mRNA in situ.

3. Continuous energy provision is critical because translation is an ATP‐intensive process, relying on ATP for both mRNA synthesis (if transcription is included) and the formation of peptide bonds. Without a steady supply of ATP, the translation machinery quickly stalls, reducing protein yield. One popular strategy for ATP regeneration in cell‐free systems is the use of a secondary energy substrate such as phosphoenolpyruvate (PEP), paired with pyruvate kinase, which converts PEP to pyruvate while regenerating ATP. Another method involves the creatine phosphate–creatine kinase system, where creatine phosphate donates a phosphate group to ADP to form ATP. Both methods allow for a longer‐lasting and more stable translation reaction.

4. Prokaryotic cell‐free systems (commonly from E. coli extracts) generally offer high yields and are cost‐effective, but may lack certain eukaryotic post‐translational modifications. This makes them suitable for simpler proteins that do not require extensive folding or glycosylation—such as a bacterial enzyme for industrial applications. In contrast, eukaryotic cell‐free systems (e.g., wheat germ, insect, or mammalian extracts) are better equipped to handle complex folding and modifications. Thus, a mammalian signaling protein requiring glycosylation and proper disulfide bond formation might be successfully produced and properly folded in a eukaryotic CFPS system. The choice often hinges on whether the protein needs eukaryotic chaperones, disulfide isomerases, or glycosylation pathways.

5. To optimize membrane protein expression in a cell‐free setup, you would first choose an appropriate system—often a prokaryotic extract combined with suitable detergents or lipid nanodiscs, or a eukaryotic extract if post‐translational modifications are desired. The experimental design would include screening different detergents or lipid mixtures to maintain the protein in a stable, native‐like conformation. Additional chaperones or cofactors might be supplemented to improve folding. Challenges include protein aggregation due to hydrophobic regions and reduced yields if the detergent disrupts ribosomal function. Careful titration of detergents, optimization of Mg²⁺ and K⁺ concentrations, and possibly employing liposomes or nanodiscs can help solve these issues by providing a more native membrane‐mimicking environment during synthesis.

6. Low yields in a cell‐free system can arise from several factors. First, suboptimal reaction conditions—such as incorrect ion concentrations, pH, or insufficient cofactors—can stall translation. Troubleshooting here involves systematically varying reaction conditions (e.g., Mg²⁺ levels) and ensuring you have adequate energy substrates. Second, the template DNA or mRNA might be degraded or insufficiently transcribed. Checking plasmid or mRNA integrity, and optimizing transcription with a well‐characterized promoter, can address this. Third, protein misfolding or aggregation can lower the overall yield of soluble protein. To resolve this, you could add molecular chaperones, adjust the temperature, or alter reaction additives (e.g., detergents or redox agents). Each issue can generally be diagnosed by running small‐scale time‐course experiments and analyzing both soluble and insoluble fractions for your target protein.