Free SKILL.md scraped from GitHub. Clone the repo or copy the file directly into your Claude Code skills directory.
npx versuz@latest install freedomintelligence-openclaw-medical-skills-skills-cell-free-expressiongit clone https://github.com/FreedomIntelligence/OpenClaw-Medical-Skills.gitcp OpenClaw-Medical-Skills/SKILL.MD ~/.claude/skills/freedomintelligence-openclaw-medical-skills-skills-cell-free-expression/SKILL.md--- name: cell-free-expression description: > Guidance for cell-free protein synthesis (CFPS) optimization. Use when: (1) Planning CFPS experiments, (2) Troubleshooting low yield or aggregation, (3) Optimizing DNA template design for CFPS, (4) Expressing difficult proteins (disulfide-rich, toxic, membrane). license: MIT category: experimental tags: [expression, cfps, validation] --- # 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 | | **Truncation** | Rare codon clusters, mRNA instability | Remove AGG/AGA/CUA clusters | Supplement rare tRNAs | | **Degradation** | Proteolysis | N-terminal Met-Ala | Add protease inhibitors | --- ## Codon Optimization for CFPS ### Codons to Avoid in E. coli CFPS | Codon | Amino Acid | Issue | tRNA Abundance | |-------|------------|-------|----------------| | AGG | Arg | Very rare, stalling | 0.2% | | AGA | Arg | Very rare, stalling | 0.4% | | CUA | Leu | Low abundance | 0.4% | | AUA | Ile | Rare | 0.5% | | CGA | Arg | Inefficient decoding | 0.6% | | CCC | Pro | Can cause pausing | 0.5% | | GGA | Gly | Moderate | 1.1% | ### Design Rules 1. **First 30 codons**: Most critical - use only high-frequency codons 2. **Rare codon clusters**: Avoid 2+ rare codons within 10 nt 3. **Rare codon content**: Keep overall <5% of coding sequence 4. **GC content**: Target 40-60% for balanced expression 5. **Avoid runs**: No >6 consecutive G or C residues (secondary structure) 6. **Strategic slow codons**: Place rare codons between domains (aids folding!) ### When to Use Rare Codons - Domain boundaries (allow cotranslational folding) - Before complex structural elements - When protein is prone to misfolding --- ## mRNA Template Design ### 5' UTR Optimization | Element | Optimal Design | Impact | |---------|----------------|--------| | **RBS (SD sequence)** | AGGAGG, 7-9 nt from start | Ribosome binding | | **Spacing** | 7 nt between SD and AUG | Translation initiation | | **Secondary structure** | ΔG > -5 kcal/mol | Accessibility | | **Upstream AUG** | Avoid (causes false starts) | Reduces truncations | ### Secondary Structure Targets | Region | Ideal ΔG | Impact | |--------|----------|--------| | -30 to +30 around AUG | > -5 kcal/mol | Translation initiation | | Full 5' UTR | > -10 kcal/mol | Ribosome loading | | RBS accessibility | Unpaired | Critical | ### Template Format | Format | Advantages | Disadvantages | |--------|------------|---------------| | **Plasmid** | Stable, high yield | Requires cloning | | **Linear PCR** | Fast, no cloning | May need stabilization | | **mRNA** | Direct translation | Unstable, expensive | --- ## Disulfide Bond Formation ### System Capabilities | System | Native Disulfide Support | Additives Needed | |--------|--------------------------|------------------| | Standard E. coli extract | Poor (DTT present) | IAM, PDI, GSSG/GSH | | Oxidizing E. coli extract | Good | Pre-oxidized glutathione | | Wheat germ | Moderate | Lower DTT, add PDI | | PURE system | Minimal | Full oxidative system | | Insect/Mammalian | Good | Microsome membranes | ### Oxidative Folding Protocol (E. coli extract) ``` 1. Deplete DTT from extract (dialysis or treatment with IAM 5 mM) 2. Add oxidized/reduced glutathione: 4 mM GSSG, 1 mM GSH (4:1 ratio) 3. Add 10 μM PDI (protein disulfide isomerase) 4. Optional: Add 5 μM DsbC (disulfide isomerase) 5. Express at 25°C (not 37°C) for better folding 6. Incubation time: 4-6 hours ``` ### Disulfide-Rich Protein Tips - Start with wheat germ or oxidizing extract - Use PURE system for precise control - Consider co-expression of PDI/DsbC - Verify by non-reducing SDS-PAGE --- ## Expression Prediction from Sequence | Feature | Good | Marginal | Bad | |---------|------|----------|-----| | **Rare codon content** | <3% | 3-8% | >10% | | **First 30 codons rare** | 0 | 1-2 | >2 | | **GC content** | 45-55% | 35-45% or 55-65% | <30% or >70% | | **5' UTR ΔG** | > -3 kcal/mol | -3 to -8 | < -10 kcal/mol | | **Hydrophobic stretches** | <5 consecutive | 5-7 | >8 consecutive | | **N-terminal residue** | Met-Ala, Met-Ser, Met-Gly | Met-Val, Met-Thr | Met-Arg, Met-Lys | | **Cysteine pairs** | Paired (even number) | Mixed | Odd number (free thiols) | --- ## Solubility Enhancement Strategies ### Fusion Tags (ranked by effectiveness) | Tag | Size | Solubility Enhancement | Cleavage | Notes | |-----|------|------------------------|----------|-------| | **MBP** | 40 kDa | Excellent | TEV, Factor Xa | Best overall | | **SUMO** | 11 kDa | Very Good | SUMO protease | Native N-terminus after cleavage | | **NusA** | 55 kDa | Excellent | - | Large size | | **Trx** | 12 kDa | Good | Enterokinase | For disulfide proteins | | **GST** | 26 kDa | Moderate | - | Dimeric | | **His₆** | 1 kDa | Minimal | - | Mainly for purification | ### Buffer Additives for Solubility | Additive | Concentration | Mechanism | |----------|---------------|-----------| | Trehalose | 50-100 mM | Chemical chaperone | | Glycerol | 5-10% | Reduces hydrophobic aggregation | | L-Arginine | 50-100 mM | Suppresses aggregation | | Tween-20 | 0.05-0.1% | Prevents surface adsorption | | Proline | 50 mM | Osmolyte stabilization | ### Chaperone Supplementation | Chaperone System | Target Problem | Concentration | |------------------|----------------|---------------| | GroEL/GroES | General folding | 1-2 μM | | DnaK/DnaJ/GrpE | Aggregation-prone | 1 μM each | | Trigger Factor | Nascent chain | 1-2 μM | | ClpB | Aggregate resolubilization | 0.5 μM | --- ## Temperature Optimization | Temperature | Use Case | Trade-offs | |-------------|----------|------------| | **37°C** | Fast expression, stable proteins | Higher aggregation risk | | **30°C** | Balanced (default) | Good compromise | | **25°C** | Disulfide proteins, complex folds | Slower, better folding | | **18-20°C** | Aggregation-prone proteins | Much slower, best folding | | **16°C** | Cold-shock proteins | Very slow, specialized | --- ## E. coli Extract Preparation (Key Variables) | Variable | Impact | Optimal Range | |----------|--------|---------------| | **Cell density at harvest** | Ribosome content | OD₆₀₀ 2.5-3.5 | | **Lysis method** | Extract activity | Sonication, bead beating | | **Run-off reaction** | Removes endogenous mRNA | 20-80 min at 37°C | | **Mg²⁺ concentration** | Translation fidelity | 10-18 mM | | **K⁺ concentration** | Translation rate | 150-200 mM | | **Energy system** | Sustained synthesis | ATP/GTP, creatine phosphate | --- ## PURE System Specifics ### Advantages - Defined composition (no proteases/nucleases) - Linear DNA templates work well - Unnatural amino acid incorporation - Reproducible between batches ### Limitations - No chaperones (add separately) - No post-translational modifications - Lower yields than crude extracts - Higher cost ### When to Use PURE - Unnatural amino acid incorporation - Studying translation mechanisms - "Clean" proteins needed - Protease-sensitive targets - Linear template expression --- ## Common Artifacts and Solutions ### Low Molecular Weight Bands **Causes**: Premature termination, proteolysis, internal initiation **Solutions**: - Optimize rare codon clusters - Add protease inhibitors - Check for internal AUG codons - Use PURE system ### Higher MW Bands **Causes**: Incomplete termination, read-through, aggregation **Solutions**: - Ensure strong stop codon (UAA preferred) - Check template 3' end - Add release factors (RF1/RF2) - Reduce protein concentration ### No Soluble Protein **Causes**: Aggregation during synthesis **Solutions**: - Lower temperature (25°C → 18°C) - Add chaperones - Use solubility tag - Optimize translation rate --- ## References ### CFPS Overview - [User's Guide to CFPS - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6481089/) - [Optimising Protein Synthesis in Cell-Free Systems - PMC](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9996726/) - [CFPS Systems Comparison - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8258279/) ### Extract Preparation - [Crude Extract Preparation - MDPI Methods](https://www.mdpi.com/2409-9279/2/3/68) - [Simple Rapid Cell-Free Lysate - PLOS One](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165137) - [High-Throughput Extract Preparation - Nature Scientific Reports](https://www.nature.com/articles/srep08663) ### PURE System - [PURE System Evolution - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10521753/) - [PURE System for Membrane Proteins - Nature Protocols](https://www.nature.com/articles/nprot.2015.082) ### Wheat Germ - [Wheat Germ Systems Review - FEBS Letters](https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2014.05.061) - [Wheat Germ for Structural Biology - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8027086/) ### Codon Optimization - [Rare Codons and Solubility - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC2723077/) - [Codon Influence on Expression - Nature](https://www.nature.com/articles/nature16509) - [Synonymous Codon Substitutions Perturb Folding - PNAS](https://www.pnas.org/doi/10.1073/pnas.1907126117) ### Disulfide Formation - [Oxidative Protein Folding in ER - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4312752/) - [PDI-Regulated Disulfide Formation - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7794689/) ### Solubility Tags - [SUMO Fusion for Difficult Proteins - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7129290/) - [Fusion Tags Review - Frontiers Microbiol](https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00063/full) ### Temperature Effects - [Cold Shock Promoters - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9800685/) - [Strategies to Optimize E. coli Expression - PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7162232/)