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