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]
source: https://github.com/adaptyvbio/protein-design-skills
Cell-Free Protein Synthesis (CFPS)
System Selection Guide
| System |
Best For |
Yield |
Disulfides |
Cost |
| E. coli extract |
Rapid prototyping |
100-400 μg/mL |
Poor |
Low |
| E. coli PURE |
Defined, unnatural AAs |
50-150 μg/mL |
Controllable |
High |
| Wheat germ |
Eukaryotic proteins |
100-500 μg/mL |
Moderate |
Medium |
| Rabbit reticulocyte |
Mammalian proteins |
10-50 μg/mL |
Poor |
High |
| HeLa/CHO |
Native mammalian |
10-50 μg/mL |
Good |
Very High |
Troubleshooting Matrix
| Problem |
Design Fix |
Reagent Fix |
| No expression |
Codon optimize first 30 codons |
Use BL21-CodonPlus extract |
| Low yield |
Optimize 5' UTR (ΔG > -5 kcal/mol) |
Increase Mg²⁺ (10-18 mM) |
| Aggregation |
Add solubility tags (MBP, SUMO) |
Add 0.1% Tween-20, chaperones |
| Inactive protein |
Slow translation (use rare codons!) |
Add GroEL/ES, DnaK/J |
| Truncation |
Remove AGG/AGA/CUA clusters |
Supplement rare tRNAs |
Codon Optimization Rules
Codons to avoid in E. coli CFPS
| Codon |
AA |
Issue |
| AGG |
Arg |
Very rare, stalling |
| AGA |
Arg |
Very rare, stalling |
| CUA |
Leu |
Low abundance |
Design Rules
- First 30 codons: Use only high-frequency codons
- Rare codon content: Keep <5% of coding sequence
- GC content: Target 40-60%
- No >6 consecutive G or C residues
- Strategic slow codons: Place at domain boundaries for folding!
Expression Prediction
| 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% |
<30% or >70% |
| 5' UTR ΔG |
> -3 kcal/mol |
-3 to -8 |
< -10 kcal/mol |
| Cysteine count |
Even |
Mixed |
Odd |
Solubility Tags
| Tag |
Size |
Enhancement |
Notes |
| MBP |
40 kDa |
Excellent |
Best overall |
| SUMO |
11 kDa |
Very Good |
Native N-terminus after cleavage |
| NusA |
55 kDa |
Excellent |
Large size |
| Trx |
12 kDa |
Good |
For disulfide proteins |
Temperature Optimization
| Temperature |
Use Case |
| 37°C |
Fast expression, stable proteins |
| 30°C |
Balanced (default) |
| 25°C |
Disulfide proteins, complex folds |
| 18-20°C |
Aggregation-prone proteins |
Disulfide Bond Formation (E. coli extract)
1. Deplete DTT (dialysis or IAM 5 mM)
2. Add 4 mM GSSG + 1 mM GSH (4:1 ratio)
3. Add 10 μM PDI
4. Optional: Add 5 μM DsbC
5. Express at 25°C for 4-6 hours
References
By