cell-free-expressionlisted

# Cell-Free Protein Synthesis (CFPS) ## System Selection Guide | System | Best For | Yield | PTMs | Disulfides | Cost | |--------|----------|-------|------|------------|------| | **E. coli extract** | Rapid prototyping, prokaryotic proteins | High (100-400 μg/mL) | None | Poor (reducing) | Low | | **E. coli PURE** | Defined conditions, unnatural AAs | Medium (50-150 μg/mL) | None | Controllable | High | | **Wheat germ** | Eukaryotic proteins, membrane proteins | High (100-500 μg/mL) | Limited | Moderate | Medium | | **Rabbit reticulocyte** | Mammalian proteins, post-translational studies | Low (10-50 μg/mL) | Some | Poor | High | | **Insect (Sf21)** | Glycoproteins, complex folds | Medium (50-100 μg/mL) | Glycosylation | Good | High | | **HeLa/CHO** | Native mammalian proteins | Low (10-50 μg/mL) | Full mammalian | Good | Very High | --- ## CFPS Troubleshooting Matrix | Problem | Likely Causes | Design Fix | Reagent Fix | |---------|---------------|------------|-------------| | **No expression** | Rare codons at N-terminus, poor RBS | Codon optimize first 30 codons | Use BL21-CodonPlus extract | | **Low yield** | Strong mRNA secondary structure, template issues | Optimize 5' UTR (ΔG > -5 kcal/mol) | Increase Mg²⁺ (10-18 mM), ATP | | **Aggregation** | Hydrophobic protein, fast translation | Add solubility tags (MBP, SUMO) | Add 0.1% Tween-20, chaperones | | **Inactive protein** | Misfolding, missing cofactors | Slow translation (use rare codons!) | Add GroEL/ES, DnaK/J |