overcoming obstacles in purification of tagged proteins 标签

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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

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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

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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

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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

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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

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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

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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

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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

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2

1

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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

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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

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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


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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

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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

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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)

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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

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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

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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

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Purity

To improve purity提高纯度

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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™

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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

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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

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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

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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

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Volume

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Tag cleavage and removal标签的酶切和去除

10C Cleavage siteSepharose™

GST RecombinantProtein

Glutathione


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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

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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

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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)

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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


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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

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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

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overcoming obstacles in purification of tagged proteins 标签

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