adaptyvbio/cell-free-expression

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 |

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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 |

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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

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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 |

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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

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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) |

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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 |

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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 |

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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 |

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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

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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

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References

CFPS Overview

• User's Guide to CFPS - PMC
• Optimising Protein Synthesis in Cell-Free Systems - PMC
• CFPS Systems Comparison - PMC

Extract Preparation

• Crude Extract Preparation - MDPI Methods
• Simple Rapid Cell-Free Lysate - PLOS One
• High-Throughput Extract Preparation - Nature Scientific Reports

PURE System

• PURE System Evolution - PMC
• PURE System for Membrane Proteins - Nature Protocols

Wheat Germ

• Wheat Germ Systems Review - FEBS Letters
• Wheat Germ for Structural Biology - PMC

Codon Optimization

• Rare Codons and Solubility - PMC
• Codon Influence on Expression - Nature
• Synonymous Codon Substitutions Perturb Folding - PNAS

Disulfide Formation

• Oxidative Protein Folding in ER - PMC
• PDI-Regulated Disulfide Formation - PMC

Solubility Tags

• SUMO Fusion for Difficult Proteins - PMC
• Fusion Tags Review - Frontiers Microbiol

Temperature Effects

• Cold Shock Promoters - PMC
• Strategies to Optimize E. coli Expression - PMC