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技能内容

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

Extract Preparation

PURE System

Wheat Germ

Codon Optimization

Disulfide Formation

Solubility Tags

Temperature Effects

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仓库FreedomIntelligence/OpenClaw-Medical-Skills
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