/cell-free-expression

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 |

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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 |