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1
Overcoming obstacles in purification of tagged proteins标签蛋白纯化中的困难与解决方法
Anna HeijbelGlobal Senior Product ManagerGE HealthcareUppsala, Sweden
2
Outline 大纲
Introduction 介绍Affinity tags 亲和标签Expression systems 表达系统
Purification problems 纯化中的难题Protein degradation 蛋白质降解Low yield 低收率Purity 纯度Tag removal 去除标签Protein stability 蛋白质稳定性
Conclusions 结语
3
Introduction
Affinity tags亲和标签
Simplification方便纯化
Increase solubility增加可溶性
Prevent proteolysis防止蛋白水解
GST MBP Strep-tag II His FLAG®
6 aa 8 aa
0.6 mg/ml
Highest
40 mg/ml
Low
Size大小 26 kDa 40 kDa 8 aa
Media 载量Capacity
>10 mg/ml
10 mg/ml 6 mg/ml
Purity纯度
Increased增溶solubility
Price价格 Medium Medium High
4
Introduction
Expression systems表达系统
--+/-+/-Proteolytic蛋白水解cleavage 酶切
+ + + -PTM or Post-translational 翻译后modification修饰
+ + + +/-Secretion分泌表达
--(+)/-+/-Inclusion bodies包涵体
Mammalian cells哺乳细胞
Insect cells昆虫细胞
Yeast酵母Bacteria细菌
- = No+ = Yes
5
Protein expression in E. Coli在大肠杆菌中表达蛋白
Inner membrane细胞内膜
Peptidoglycan肽聚糖
Outer membrane细胞外膜
Lipopolysaccharide脂多糖70 Å
70 Å
70 Å
210 Å
Cytoplasm细胞质
~2000 proteins
~100 proteins
Periplasm胞间质
~10 proteinsMedium培养基
Introduction
6
Outline大纲
Introduction介绍Affinity tags 亲和标签Expression systems表达系统
Purification problems纯化中的难题Protein degradation蛋白降解Low yield低产量Purity纯度Tag removal标签的去除Protein stability蛋白的稳定性
Conclusions结论
7
Protein degradation蛋白的降解
Western blot
Truncated forms with the tag带标签的降解蛋白
Full length protein全蛋白
SDS-PAGE
Protein degradation Low yield Purity Tag removal Protein stability
8
To avoid protein degradation避免蛋白降解
Use a protease-deficient expression host
用蛋白酶缺陷性的表达宿主
Sample preparation样品制备Work fast快速操作
Low temperature 低温Protease inhibitors加入蛋白酶抑制剂DNases/RNasesDNA和RNA酶
Purification 纯化Work fast快速操作
Minimize the handling steps尽量减少操作步骤
3
2
1
9
One way to work fast 快速操作法Conventional purification
Cell lysis
Transfer to tubes
Centrifugation
Collectsupernantant Filtration
Protein purification
Higher yield and biologically active protein 更高产量和高生物学活性蛋白
Cell lysis
Column: HisTrap™ FF crudeResin: Ni Sepharose™ Fast Flow
Purification of unclarified lysateProtein purification
Protein degradation
不澄清的裂解液直接柱纯化
传统纯化方法
10
Fast purification from unclarified sample未澄清的样品的快速纯化
Protein degradation
Column: HisTrap™ FF crude 1ml
Sample: 15 ml unclarified lysate of a histidine-tagged protein expressed in yeast
酵母细胞表达带组氨酸标签的未经澄清的样品15ml
Flow rate: 1 ml/min
System: ÄKTAexplorer™
1 2 3 4
97 00066 000
45 000
30 000
20 10014 400
MrS
Edil. 2x
Edil. 4x
E
11
Low yield低产量
Poor binding结合量低
Inefficient elution不充分的洗脱
Media dependence填料的选择
Protein degradation Low yield Purity Tag removal Protein stability
12
Low Yield低产量 Low yield
Use denaturant or detergent
使用变性剂
Strep II: Addition of Avidin 添加抗生物素蛋白
Optimize conditions
优化条件
Decrease flow rate
减小流速
Tag is not exposed for binding 标签未外露
Factors in the extract interfere with binding e.g. Biotin
提取液中的因素影响结合,如生物素
Binding buffer conditions are not optimal (IMAC)
结合缓冲液条件没有优化(IMAC)
Kinetic between ligand and tag is slow (GST)
配基与标签之间结合动力学很慢
Elution buffer conditions not optimal
洗脱缓冲液条件没有优化
Precipitation (on-column or after elution)
沉淀(柱上或洗脱后)
Optimize conditions优化条件
Smaller sample volume减少上样体积
Linear gradient Step gradient
采用线性梯度洗脱
Poor Binding
Inefficient Elution
13
Low yieldOptimizing buffer conditions优化缓冲液条件 High-throughput buffer screening
高通量缓冲液条件筛选Protein蛋白Histidine tagged Nurr1 ligandbinding domain expressed in E. coli
Parameters studied参数研究Binding buffers结合缓冲液
pH values: pH 6.0 to 8.5 (8 buffers)NaCl: 100 -750 mMGlycerol: 5-10%β-mercaptoethanol: 0.05%
His MultiTrap™ FF
Acknowledgement: R. Steele and B. Grasberger, Johnson & Johnson Pharmaceutical R&D, USA
14
Kinetics: Effect of flow rates 动力学:流速的影响
Low yield
GST: Impact of flow rate during sample application 流速对GST结合影响
Slow binding kinetics GST-glutathione Flow rate during sample application must be low.
GST-谷胱甘肽结合动力学较慢 上样阶段流速一定要低
Binding capacity study using GST-(His)6 on Glutathione Sepharose™ 4 Fast Flow
0
200
400
600
800
1000
0,1 0,3 0,4 1 1,5
RTCold
Yiel
d (r
elat
ive
unit
s)
Flow rate (ml/min)
15
Chromatography media dependence层析填料的选择
Low yield
Media GluthationeSepharose™ 4B
GluthationeSepharose FF
GluthationeSepharose HP
Advantages High Capacity Higher flow ratespossible
High resolution and concentration
Capacity Up to 30 mg Up to 15 mg Up to 10 mg
Bead size 90 µm 90 µm 34 µm
0
1000
2000
3000
4000
mAU
0.0 10.0 20.0 30.0 40.0 ml
GSTrapTM 4B 1 ml
0
500
1000
1500
2000
2500
3000
3500
4000
mAU
0.0 10.0 20.0 30.0 40.0 ml
0
500
1000
1500
2000
2500
3000
3500
4000
mAU
0.0 10.0 20.0 30.0 40.0 ml
GSTrap FF 1ml GSTrap HP 1 ml24.8 mg 8.4 mg 6.3 mg
Sample: 20 ml unclarified E. coli lysate GST-HippocalsinFlow rate: Sample application: 0.3 ml/min,
Equilibration, wash & elution: 1 ml/min
16
Protein degradation Low yield Purity Tag removal Protein stability
Purity纯度
Requirements要求
Raising antibodies > 90-95%
免疫产生抗体
Crystallization > 99%
结晶
Characterization > 99%
性质研究
Purity check纯度检测
kDa
976645
3020.114.4
1 2 3 4 5 6 7
kDa
976645
3020.114.4
1 2 3 4 5 6 7
Purity
SDS-PAGE SuperdexTM 5/150 GL• 50 µl eluate from StrepTrapTM HP• 10 min
17
How to detect?如何检测
Western blot (use anti-”TAG” antibody for proteolytic cleavage)免疫杂交
Co-elution?共洗脱
Heterogeneity due to PTM?转译后修饰产生不均一
Proteolytic cleavage?蛋白水解
Amino acid analysis氨基酸分析
Many bands/peaks of different Mw在不同分子量位置有很多条带或峰
Co-elution共洗脱anti-DnaK anti-GST
Purity
Detergents and/or reducing agents before sonication
在超声前加去垢剂和、或还原剂
1. Chaperone proteins伴侣蛋白DnaK -70 kDa GroEL - 60 kDa
Western blot analysis. Samples: Fractions from second IEX step (first step on Glutathione Sepharose™)
2. Endogenous GST内源性GST
3. Native metal-binding proteins天然金属结合蛋白e.g. Cu/Zn Superoxide dismutase 17 kDa
超氧化物歧化酶
4. Histidine rich proteins 富含组氨酸蛋白e.G Chloramphenicol acetyl transfererase 25 kDa
氯霉素乙酰转移酶
On-column cleavage柱上酶切
Optimize Imidazole conc.优化咪唑浓度
18
19
Purity
To improve purity提高纯度
12345
Multi-step purification多步纯化
Dual tagging of a protein双标签蛋白
Optimize binding and elution conditions优化结合洗脱条件
Optimize metal ion for IMAC优化螯合的金属离子
Refolding and inclusion bodies重折叠和包涵体表达
Two-step purification两步纯化Purity
1
0
1000
2000
3000
4000
mAU
0.0 20.0 40.0 60.0 80.0 mlF3 F4 Wa steA3A5A7A9 Wa ste
1. Affinity step亲和层析Column: HisTrap™ FF 1 ml Sample: 50 ml (His)10-Trx-P 450 in E. coli lysateSystem: ÄKTAexplorer™
20
Column: HiLoad™ 16/60 Superdex™ 200 pg Sample: 5.2 ml eluted pool from HisTrap™System: ÄKTAexplorer ™
2. Gel filtration凝胶过滤
0
100
200
300
400
mAU
20.0 40.0 60.0 80.0 100.0 mlA1A2A3A4A5A6A7A8A9 A11A13A15B14B12B10B8B7B6B5B4B3B2B1C1C2C3C4C5C6C7
1
2
3F
97 000
66 000
45 000
30 000
20 100
14 400
Mr
LMW Start FT wash eluted 1 2 3 F
HisTrap™ FF Superdex™ 200 pg
97 000
66 000
45 000
30 000
20 100
14 400
Mr
97 000
66 000
45 000
30 000
20 100
14 400
Mr
LMW Start FT wash eluted 1 2 3 F
HisTrap™ FF Superdex™ 200 pg
1 3 4 5 6 7 8 92
Dual tagging of a protein双标签蛋白
Run 1. HisTrap HP 1 ml
Run 2. StrepTrap HP 1 ml
Run 3. HisTrap HP 1 ml + StrepTrap HP 1 ml
Combination of a two-step HisTrap™ HP and StrepTrap™ HP purificationSample: 15 ml Strep II-protein-(His)6 in E. coli lysateSystem: ÄKTAxpress™
Run 1 Run 3 Run 2
FT1 FT3 FT2E1 E2E3
Purity2
21Acknowledgement: Martina Nilsson, Biovitrum Stockholm, Sweden
22
0,00
0,50
1,00
1,50
2,00
0 10 20 30 40 50
Imidazole conc. (mM)
Abs
490/
280
0
1
2
3
4
5
6
Abs
490
PurityYield
Purity
Yield
Optimizing imidazole concentration优化咪唑浓度 Yield versus purity产量vs纯度
Column: HisTrap™ HP 1mlSample: GFP-(His)6 expressed in E. coliDetection: 280 and 490 nm
Imidazole concentration
Purity
3
Optimize metal ion for IMAC优化金属离子Purity
4
23
Cu2+ Zn2+ Co2+ Ni2+
Metal ions Ni Co Cu Zn
Histidine tagged proteins +++++ ++++ ++ +++
Untagged proteins ++ + ++++ +
+++++ Strong binding++ Weak binding
Column: HiTrap™ IMAC FF (prepacked with un-charged IMAC Sepharose™ FF)
On-column refolding and purification of a histidine-tagged protein柱上复性和his标签蛋白纯化
Purity
5
Column: HisTrap™ FF 1 ml
Resin: Ni Sepharose™ FF
System: ÄKTAprime™
A280
A280
Elution Gradient: 5 – 500 mM imidazoleFlow rate: 1.0 ml/min
Refolding Gradient: 6 M – 0 M urea30 ml gradient volume Flow rate: 0.5 ml/min
24
Volume
25
Tag cleavage and removal标签的酶切和去除
10C Cleavage siteSepharose™
GST RecombinantProtein
Glutathione
Protein degradation Low yield Purity Tag removal Protein stability
26
GSTRecombinant
ProteinGlutathione
On column tag cleavage柱上酶切
GST GST-tagged PreScissionTM
ProteaseGlutathione SepharoseTM
Still on column after wash
Uncleaved fusion protein
GSTGlutathione
Cleavage site
Endogenous GST
Glutathione
From expression host
Native function of monomeric target protein 单一目标蛋白的天然功能
Removal of endogenous GST去除内源性GSTRemoval of cleavage enzyme去除内切酶
No uncleaved protein没有切开的蛋白
GSTRecombinant
ProteinGlutathioneGlutathione
Endogenous GST
Glutathione
From expression host
GSTRecombinant
ProteinGlutathione
RecombinantProtein
Untagged target protein
Eluted in WASH buffer
27
Tag cleavage
Optimizing tag cleavage优化酶切
On-column cleavage of GST-tagged protein柱上GST标签蛋白的酶切PreScission protease
Incubation time (hours)
% C
leav
ed p
rote
in
Optimize cleavage conditions优化酶切的条件
1. Time for cleavage酶切时间
2. Ratio of protease/target protein酶与目标蛋白的比例
3. Temperature (optimum 4 °C)温度
28
Automatic on-column cleavage and tag removal自动柱上酶切和去除标签
Tag cleavage
Sample: APC1040-(His)6 in E.coli lysateColumn: HisTrap™ HP 1 mlProtease: AcTEV™System: ÄKTAxpress™
Cleaved protein
Regeneration
16 mg
Reference, uncleaved protein
Purified, cleaved protein
Lanes1. Protein markers2. Sample3. Flow through from affinity step4. Eluate from Gel filtration (GF)
29
Protein stability 蛋白的稳定性
Structural studies结构的研究
Characterization性质
Measuring biological activity检测生物学活性
Protein degradation Low yield Purity Tag removal Protein stability
30
Purified protein stability纯化蛋白的稳定性Protein stability
Monitoring condition of protein检测蛋白的状态
Aggregation聚集体
Precipitation 沉淀
Conformation changes结构改变
Check-list检测• Freeze in aliquots分装冷冻• Avoid freeze and thaw避免冻融• Avoid freezing concentrated protein
solution避免冷冻浓缩蛋白溶液• Screen different storage buffers, e.g
pH and salt筛选不同储存缓冲液,如pH和盐种类• Store in 10-50% glycerol储存在10-50%甘油中
Loss of activity活性丢失
凝胶过滤,蒸发光散射,非变性电泳,
Visual目测
Gel filtration, light scattering, native PAGE
31
Conclusions结论
Protein degradation Sample preparation, minimize time
Low yield Optimize binding conditions
Purity Multi-step purification
Tag removal On-column cleavage with tagged
protease
蛋白降解
低产量
标签的去除
纯度
样品准备,缩短时间
优化结合条件
多步纯化
带标签的蛋白酶柱上酶切
THANK YOU!
www.gelifesciences.com/protein-purification
GE, imagination at work, and GE monogram are trademarks of General Electric Company.HiLoad, HisTrap, HiTrap, MultiTrap, PreScission, Sepharose, SpinTrap, StrepTrap ,Superdex, ÄKTAexplorer, ÄKTAprime and ÄKTAxpress are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners.
Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 5,284,933 and US pat 5,310,663, including corresponding foreign patents (assigne: Hoff man La Roche, Inc).
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