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cell-free-expression

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更新时间2026年3月4日 07:22

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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BioTender-max

BioTender-max/ProteinClaw

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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

同仓库更多 Skills

同仓库

protein-design

BioTender-max/ProteinClaw

Computational protein design toolkit. Use this skill group when: (1) Designing protein binders from scratch, (2) Predicting protein structures (Chai, Boltz, Protenix), (3) Sequence design with ProteinMPNN/LigandMPNN/SolubleMPNN, (4) Quality control and filtering of protein designs, (5) Planning and managing binder design campaigns, (6) Experimental characterization (SPR, BLI, cell-free expression), (7) Searching protein databases (PDB, UniProt, AFDB), (8) Antibody/nanobody design (IgGM, mBER). This is the top-level index. Sub-skills handle specific tools.

2026-03-0411

binder-design

BioTender-max/ProteinClaw

Guidance for choosing the right protein binder design tool. Use this skill when: (1) Deciding between BoltzGen or BindCraft, (2) Planning a binder design campaign, (3) Understanding trade-offs between different approaches, (4) Selecting tools for specific target types.

2026-03-0411

binding-characterization

BioTender-max/ProteinClaw

Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms.

2026-03-0411

boltz

BioTender-max/ProteinClaw

Structure prediction using Boltz-1/Boltz-2, an open biomolecular structure predictor. Use this skill when: (1) Predicting protein complex structures, (2) Validating designed binders, (3) Need open-source alternative to AF2, (4) Predicting protein-ligand complexes, (5) Using local GPU resources. For QC thresholds, use protein-qc. For AlphaFold2 prediction, use alphafold. For Chai prediction, use chai.

2026-03-0411

boltzgen

BioTender-max/ProteinClaw

All-atom protein design using BoltzGen diffusion model. Use this skill when: (1) Need side-chain aware design from the start, (2) Designing around small molecules or ligands, (3) Want all-atom diffusion (not just backbone), (4) Require precise binding geometries, (5) Using YAML-based configuration. For sequence-only design, use proteinmpnn. For structure validation, use boltz.

2026-03-0411

campaign-manager

BioTender-max/ProteinClaw

Goal-oriented binder design campaign planning and health assessment. Use this skill when: (1) Planning a complete binder design campaign, (2) Converting high-level goals into runnable pipelines, (3) Assessing campaign health and pass rates, (4) Diagnosing why designs are failing QC, (5) Estimating time, cost, and expected yields, (6) Selecting between design tools for a specific target. This skill orchestrates the other protein design tools.

2026-03-0411

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