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"]
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

1. First 30 codons: Use only high-frequency codons
2. Rare codon content: Keep <5% of coding sequence
3. GC content: Target 40-60%
4. No >6 consecutive G or C residues
5. 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

• User's Guide to CFPS - PMC
• Codon Influence on Expression - Nature