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cell-free-expression

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Cell-Free Protein Synthesis (CFPS)

无细胞蛋白合成（CFPS）

System Selection Guide

系统选择指南

SystemBest ForYieldPTMsDisulfidesCost
E. coli extractRapid prototyping, prokaryotic proteinsHigh (100-400 μg/mL)NonePoor (reducing)Low
E. coli PUREDefined conditions, unnatural AAsMedium (50-150 μg/mL)NoneControllableHigh
Wheat germEukaryotic proteins, membrane proteinsHigh (100-500 μg/mL)LimitedModerateMedium
Rabbit reticulocyteMammalian proteins, post-translational studiesLow (10-50 μg/mL)SomePoorHigh
Insect (Sf21)Glycoproteins, complex foldsMedium (50-100 μg/mL)GlycosylationGoodHigh
HeLa/CHONative mammalian proteinsLow (10-50 μg/mL)Full mammalianGoodVery High
系统适用场景产量PTMs二硫键合成能力成本
E. coli extract快速原型构建、原核蛋白表达高（100-400 μg/mL）无差（还原性环境）低
E. coli PURE明确条件实验、非天然氨基酸掺入中（50-150 μg/mL）无可控高
Wheat germ真核蛋白、膜蛋白表达高（100-500 μg/mL）有限中等中
Rabbit reticulocyte哺乳动物蛋白、翻译后修饰研究低（10-50 μg/mL）部分差高
Insect (Sf21)糖蛋白、复杂折叠蛋白表达中（50-100 μg/mL）糖基化好高
HeLa/CHO天然哺乳动物蛋白表达低（10-50 μg/mL）完整哺乳动物翻译后修饰好极高

CFPS Troubleshooting Matrix

CFPS问题排查矩阵

ProblemLikely CausesDesign FixReagent Fix
No expressionRare codons at N-terminus, poor RBSCodon optimize first 30 codonsUse BL21-CodonPlus extract
Low yieldStrong mRNA secondary structure, template issuesOptimize 5' UTR (ΔG > -5 kcal/mol)Increase Mg²⁺ (10-18 mM), ATP
AggregationHydrophobic protein, fast translationAdd solubility tags (MBP, SUMO)Add 0.1% Tween-20, chaperones
Inactive proteinMisfolding, missing cofactorsSlow translation (use rare codons!)Add GroEL/ES, DnaK/J
TruncationRare codon clusters, mRNA instabilityRemove AGG/AGA/CUA clustersSupplement rare tRNAs
DegradationProteolysisN-terminal Met-AlaAdd protease inhibitors
问题可能原因设计优化方案试剂优化方案
无蛋白表达N端存在稀有密码子、RBS序列不佳优化前30个密码子，仅使用高频密码子使用BL21-CodonPlus提取物
产量低mRNA二级结构过强、模板问题优化5' UTR（ΔG > -5 kcal/mol）提高Mg²⁺浓度（10-18 mM）、补充ATP
蛋白聚集蛋白疏水性强、翻译速度过快添加可溶性标签（MBP、SUMO）添加0.1% Tween-20、伴侣蛋白
蛋白无活性蛋白错误折叠、缺乏辅因子减慢翻译速度（使用稀有密码子！）添加GroEL/ES、DnaK/J
蛋白截短稀有密码子簇、mRNA不稳定移除AGG/AGA/CUA密码子簇补充稀有tRNA
蛋白降解蛋白水解作用N端添加Met-Ala序列添加蛋白酶抑制剂

Codon Optimization for CFPS

CFPS密码子优化

Codons to Avoid in E. coli CFPS

大肠杆菌CFPS中需避免的密码子

CodonAmino AcidIssuetRNA Abundance
AGGArgVery rare, stalling0.2%
AGAArgVery rare, stalling0.4%
CUALeuLow abundance0.4%
AUAIleRare0.5%
CGAArgInefficient decoding0.6%
CCCProCan cause pausing0.5%
GGAGlyModerate1.1%
密码子氨基酸问题tRNA丰度
AGGArg极稀有，导致翻译停滞0.2%
AGAArg极稀有，导致翻译停滞0.4%
CUALeu丰度低0.4%
AUAIle稀有0.5%
CGAArg解码效率低0.6%
CCCPro可能导致翻译暂停0.5%
GGAGly中等丰度1.1%

Design Rules

设计规则

  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!)
  1. 前30个密码子：最关键区域——仅使用高频密码子
  2. 稀有密码子簇：避免10 nt范围内出现2个及以上稀有密码子
  3. 稀有密码子占比：整体占编码序列的比例需低于5%
  4. GC含量：目标40-60%，以实现平衡表达
  5. 避免连续序列：不超过6个连续的G或C残基（防止二级结构形成）
  6. 策略性使用稀有密码子：在结构域之间插入稀有密码子（有助于折叠！）

When to Use Rare Codons

何时使用稀有密码子

  • Domain boundaries (allow cotranslational folding)
  • Before complex structural elements
  • When protein is prone to misfolding
  • 结构域边界处（允许共翻译折叠）
  • 复杂结构元件之前
  • 蛋白易发生错误折叠时

mRNA Template Design

mRNA模板设计

5' UTR Optimization

5' UTR优化

ElementOptimal DesignImpact
RBS (SD sequence)AGGAGG, 7-9 nt from startRibosome binding
Spacing7 nt between SD and AUGTranslation initiation
Secondary structureΔG > -5 kcal/molAccessibility
Upstream AUGAvoid (causes false starts)Reduces truncations
元件最优设计影响
RBS（SD序列）AGGAGG，距离起始密码子7-9 nt核糖体结合
间隔长度SD序列与AUG之间为7 nt翻译起始效率
二级结构ΔG > -5 kcal/mol模板可及性
上游AUG避免存在（会导致错误起始）减少蛋白截短

Secondary Structure Targets

二级结构目标

RegionIdeal ΔGImpact
-30 to +30 around AUG> -5 kcal/molTranslation initiation
Full 5' UTR> -10 kcal/molRibosome loading
RBS accessibilityUnpairedCritical
区域理想ΔG值影响
AUG周围-30至+30 nt区域> -5 kcal/mol翻译起始
完整5' UTR> -10 kcal/mol核糖体加载
RBS可及性未配对至关重要

Template Format

模板格式

FormatAdvantagesDisadvantages
PlasmidStable, high yieldRequires cloning
Linear PCRFast, no cloningMay need stabilization
mRNADirect translationUnstable, expensive
格式优势劣势
质粒稳定、产量高需要克隆操作
线性PCR产物快速、无需克隆可能需要稳定化处理
mRNA直接翻译不稳定、成本高

Disulfide Bond Formation

二硫键形成

System Capabilities

系统能力

SystemNative Disulfide SupportAdditives Needed
Standard E. coli extractPoor (DTT present)IAM, PDI, GSSG/GSH
Oxidizing E. coli extractGoodPre-oxidized glutathione
Wheat germModerateLower DTT, add PDI
PURE systemMinimalFull oxidative system
Insect/MammalianGoodMicrosome membranes
系统天然二硫键合成支持所需添加剂
标准大肠杆菌提取物差（含DTT）IAM、PDI、GSSG/GSH
氧化性大肠杆菌提取物好预氧化型谷胱甘肽
小麦胚芽提取物中等降低DTT浓度、添加PDI
PURE系统极低完整氧化系统
昆虫/哺乳动物提取物好微粒体膜

Oxidative Folding Protocol (E. coli extract)

氧化性折叠方案（大肠杆菌提取物）

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
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

富含二硫键蛋白的表达技巧

  • 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
  • 从小麦胚芽提取物或氧化性提取物开始尝试
  • 使用PURE系统实现精准控制
  • 考虑共表达PDI/DsbC
  • 通过非还原SDS-PAGE验证

Expression Prediction from Sequence

基于序列的表达预测

FeatureGoodMarginalBad
Rare codon content<3%3-8%>10%
First 30 codons rare01-2>2
GC content45-55%35-45% or 55-65%<30% or >70%
5' UTR ΔG> -3 kcal/mol-3 to -8< -10 kcal/mol
Hydrophobic stretches<5 consecutive5-7>8 consecutive
N-terminal residueMet-Ala, Met-Ser, Met-GlyMet-Val, Met-ThrMet-Arg, Met-Lys
Cysteine pairsPaired (even number)MixedOdd number (free thiols)
特征良好一般较差
稀有密码子占比<3%3-8%>10%
前30个密码子中的稀有密码子数量01-2>2
GC含量45-55%35-45% 或 55-65%<30% 或 >70%
5' UTR ΔG值> -3 kcal/mol-3 至 -8< -10 kcal/mol
疏水片段长度<5个连续残基5-7个连续残基>8个连续残基
N端残基Met-Ala、Met-Ser、Met-GlyMet-Val、Met-ThrMet-Arg、Met-Lys
半胱氨酸配对配对（偶数个）混合奇数个（存在游离巯基）

Solubility Enhancement Strategies

溶解度提升策略

Fusion Tags (ranked by effectiveness)

融合标签（按有效性排序）

TagSizeSolubility EnhancementCleavageNotes
MBP40 kDaExcellentTEV, Factor XaBest overall
SUMO11 kDaVery GoodSUMO proteaseNative N-terminus after cleavage
NusA55 kDaExcellent-Large size
Trx12 kDaGoodEnterokinaseFor disulfide proteins
GST26 kDaModerate-Dimeric
His₆1 kDaMinimal-Mainly for purification
标签大小溶解度提升效果切割方式说明
MBP40 kDa极佳TEV、Factor Xa整体效果最佳
SUMO11 kDa很好SUMO蛋白酶切割后保留天然N端
NusA55 kDa极佳-分子量大
Trx12 kDa良好肠激酶适用于二硫键蛋白
GST26 kDa中等-二聚体形式
His₆1 kDa极小-主要用于纯化

Buffer Additives for Solubility

缓冲液添加剂提升溶解度

AdditiveConcentrationMechanism
Trehalose50-100 mMChemical chaperone
Glycerol5-10%Reduces hydrophobic aggregation
L-Arginine50-100 mMSuppresses aggregation
Tween-200.05-0.1%Prevents surface adsorption
Proline50 mMOsmolyte stabilization
添加剂浓度作用机制
海藻糖50-100 mM化学伴侣
甘油5-10%减少疏水聚集
L-精氨酸50-100 mM抑制聚集
Tween-200.05-0.1%防止表面吸附
脯氨酸50 mM渗透稳定剂

Chaperone Supplementation

伴侣蛋白补充

Chaperone SystemTarget ProblemConcentration
GroEL/GroESGeneral folding1-2 μM
DnaK/DnaJ/GrpEAggregation-prone1 μM each
Trigger FactorNascent chain1-2 μM
ClpBAggregate resolubilization0.5 μM
伴侣蛋白系统针对问题浓度
GroEL/GroES通用折叠问题1-2 μM
DnaK/DnaJ/GrpE易聚集蛋白各1 μM
Trigger Factor新生肽链折叠1-2 μM
ClpB聚集体复性0.5 μM

Temperature Optimization

温度优化

TemperatureUse CaseTrade-offs
37°CFast expression, stable proteinsHigher aggregation risk
30°CBalanced (default)Good compromise
25°CDisulfide proteins, complex foldsSlower, better folding
18-20°CAggregation-prone proteinsMuch slower, best folding
16°CCold-shock proteinsVery slow, specialized
温度适用场景权衡
37°C快速表达、稳定蛋白聚集风险更高
30°C平衡方案（默认）良好折中选择
25°C二硫键蛋白、复杂折叠蛋白翻译速度慢，折叠效果更好
18-20°C易聚集蛋白速度慢很多，折叠效果最佳
16°C冷休克蛋白速度极慢，专用场景

E. coli Extract Preparation (Key Variables)

大肠杆菌提取物制备（关键变量）

VariableImpactOptimal Range
Cell density at harvestRibosome contentOD₆₀₀ 2.5-3.5
Lysis methodExtract activitySonication, bead beating
Run-off reactionRemoves endogenous mRNA20-80 min at 37°C
Mg²⁺ concentrationTranslation fidelity10-18 mM
K⁺ concentrationTranslation rate150-200 mM
Energy systemSustained synthesisATP/GTP, creatine phosphate
变量影响最优范围
收获时细胞密度核糖体含量OD₆₀₀ 2.5-3.5
裂解方式提取物活性超声破碎、珠磨破碎
Run-off反应去除内源性mRNA37°C下20-80分钟
Mg²⁺浓度翻译保真性10-18 mM
K⁺浓度翻译速率150-200 mM
能量系统持续合成ATP/GTP、肌酸磷酸

PURE System Specifics

PURE系统细节

Advantages

优势

  • Defined composition (no proteases/nucleases)
  • Linear DNA templates work well
  • Unnatural amino acid incorporation
  • Reproducible between batches
  • 组成明确（无蛋白酶/核酸酶）
  • 线性DNA模板兼容性好
  • 可掺入非天然氨基酸
  • 批次间重复性好

Limitations

局限性

  • No chaperones (add separately)
  • No post-translational modifications
  • Lower yields than crude extracts
  • Higher cost
  • 无内置伴侣蛋白（需单独添加）
  • 无翻译后修饰
  • 产量低于粗提取物
  • 成本更高

When to Use PURE

何时使用PURE系统

  • Unnatural amino acid incorporation
  • Studying translation mechanisms
  • "Clean" proteins needed
  • Protease-sensitive targets
  • Linear template expression
  • 非天然氨基酸掺入
  • 翻译机制研究
  • 需要“无杂质”蛋白
  • 蛋白酶敏感型靶标
  • 线性模板表达

Common Artifacts and Solutions

常见异常及解决方案

Low Molecular Weight Bands

低分子量条带

Causes: Premature termination, proteolysis, internal initiation Solutions:
  • Optimize rare codon clusters
  • Add protease inhibitors
  • Check for internal AUG codons
  • Use PURE system
原因：提前终止、蛋白水解、内部起始 解决方案:
  • 优化稀有密码子簇
  • 添加蛋白酶抑制剂
  • 检查是否存在内部AUG密码子
  • 使用PURE系统

Higher MW Bands

高分子量条带

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
原因：终止不完全、通读、聚集 解决方案:
  • 确保使用强终止密码子（优先选择UAA）
  • 检查模板3'端
  • 添加释放因子（RF1/RF2）
  • 降低蛋白浓度

No Soluble Protein

无可溶性蛋白

Causes: Aggregation during synthesis Solutions:
  • Lower temperature (25°C → 18°C)
  • Add chaperones
  • Use solubility tag
  • Optimize translation rate
原因：合成过程中发生聚集 解决方案:
  • 降低温度（从25°C降至18°C）
  • 添加伴侣蛋白
  • 使用可溶性标签
  • 优化翻译速率

References

参考文献

CFPS Overview

CFPS概述

Extract Preparation

提取物制备

PURE System

PURE系统

Wheat Germ

小麦胚芽系统

Codon Optimization

密码子优化

Disulfide Formation

二硫键形成

Solubility Tags

可溶性标签

Temperature Effects

温度影响