Agent skill

cell-free-expression

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).

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Install this agent skill to your Project

npx add-skill https://github.com/adaptyvbio/protein-design-skills/tree/main/skills/cell-free-expression

SKILL.md

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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