Experimental Testing

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).

installation instructions

# 1. Ensure Claude Code is installed

View Installation Docs

# 2. Install the marketplace

$/plugin marketplace add adaptyvbio/protein-design-skills

# 3. Install this skill

$/plugin install cell-free-expression@protein-design-skills

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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

First 30 codons: Most critical - use only high-frequency codons
Rare codon clusters: Avoid 2+ rare codons within 10 nt
Rare codon content: Keep overall <5% of coding sequence
GC content: Target 40-60% for balanced expression
Avoid runs: No >6 consecutive G or C residues (secondary structure)
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: