Overcoming the most common obstacles in your purificaton of tagged proteins

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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 Heijbel Global Senior Product Manager GE Healthcare Uppsala, Sweden 1

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 2

3 Affinity tags 亲和标签 Introduction 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 Size 大小 26 kda 40 kda 8 aa Media 载量 Capacity Purity 纯度 Increased 增溶 solubility >10 mg/ml 10 mg/ml 6 mg/ml Price 价格 Medium Medium High 6 aa 8 aa 40 mg/ml 0.6 mg/ml Low Highest 3

4 Expression systems 表达系统 Introduction Inclusion bodies 包涵体 Secretion 分泌表达 Bacteria 细菌 Yeast 酵母 +/- +/- (+)/- + Insect cells 昆虫细胞 - + Mammalian cells 哺乳细胞 - + PTM or Posttranslational 翻译后 modification 修饰 Proteolytic 蛋白水解 cleavage 酶切 +/- +/ = Yes -= No 4

5 Protein expression in E. Coli 在大肠杆菌中表达蛋白 70 Å Medium 培养基 ~10 proteins Introduction Lipopolysaccharide 脂多糖 70 Å 210 Å Periplasm 胞间质 ~100 proteins Outer membrane 细胞外膜 Peptidoglycan 肽聚糖 70 Å Cytoplasm 细胞质 ~2000 proteins Inner membrane 细胞内膜 5

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 6

7 Protein degradation 蛋白的降解 SDS-PAGE Western blot Full length protein 全蛋白 Truncated forms with the tag 带标签的降解蛋白 Protein degradation Low yield Purity Tag removal Protein stability 7

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 8

One way to work fast 快速操作法 Conventional purification Cell lysis Transfer to tubes Centrifugation 传统纯化方法 Collect supernantant Filtration Protein degradation Protein purification

9 One way to work fast 快速操作法 Conventional purification Cell lysis Transfer to tubes Centrifugation 传统纯化方法 Collect supernantant Filtration Protein degradation Protein purification Purification of unclarified lysate Cell lysis Protein purification 不澄清的裂解液直接柱纯化 Column: HisTrap FF crude Resin: Ni Sepharose Fast Flow Higher yield and biologically active protein 更高产量和高生物学活性蛋白 9

Protein degradation ast purification from unclarified sample 未澄清的样品的快速纯化 Column: Sample: Flow rate: 1 ml/min System: HisTrap FF crude 1ml 15 ml unclarified lysate of

10 Protein degradation ast purification from unclarified sample 未澄清的样品的快速纯化 Column: Sample: Flow rate: 1 ml/min System: HisTrap FF crude 1ml 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 ÄKTAexplorer M r S E dil. 2x E dil. 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 11

12 Low Yield 低产量 Poor Binding 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 Inefficient Elution 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 ) 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 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 12

Optimizing buffer conditions 优化缓冲液条件 Protein 蛋白 Histidine tagged

coli Parameters studied 参数研究 Binding buffers 结合缓冲液 ph values: ph 6.

5 (8 buffers) NaCl: 100-750 mm Glycerol: 5-10% β-mercaptoethanol: 0.

13 Optimizing buffer conditions 优化缓冲液条件 Protein 蛋白 Histidine tagged Nurr1 ligand binding 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: mm Glycerol: 5-10% β-mercaptoethanol: 0.05% Low yield High-throughput buffer screening 高通量缓冲液条件筛选 His MultiTrap FF Acknowledgement: R. Steele and B. Grasberger, Johnson & Johnson Pharmaceutical R&D, USA 13

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

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 1000 Yield (relative units) ,1 0,3 0,4 1 1,5 Flow rate (ml/min) RT Cold 14

15 Low yield Chromatography media dependence 层析填料的选择 Media Gluthatione Sepharose 4B Gluthatione Sepharose FF Advantages High Capacity Higher flow rates possible Gluthatione Sepharose HP 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 mau GSTrap TM 4B 1 ml mau GSTrap FF 1ml mg 8.4 mg 6.3 mg mau GSTrap HP 1 ml ml ml ml Sample: Flow rate: 20 ml unclarified E. coli lysate GST-Hippocalsin Sample application: 0.3 ml/min, Equilibration, wash & elution: 1 ml/min 15

16 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 Protein degradation Low yield Purity Tag removal Protein stability 16

17 Purity check 纯度检测 Purity kda SDS-PAGE Superdex TM 5/150 GL 50 µl eluate from StrepTrap TM HP 10 min Many bands/peaks of different M w 在不同分子量位置有很多条带或峰 Heterogeneity due to PTM? 转译后修饰产生不均一 Proteolytic cleavage? 蛋白水解 Co-elution? 共洗脱 How to detect? 如何检测 Western blot (use anti- TAG antibody for proteolytic cleavage) 免疫杂交 Amino acid analysis 氨基酸分析 17

Co-elution 共洗脱 anti-dnak anti-gst Purity 1. Chaperone proteins 伴侣蛋白 DnaK -70 kda GroEL - 60 kda Detergents and/or reducing agents before sonication 在超声前加去垢剂和或还原剂 Western blot analysis.

18 Co-elution 共洗脱 anti-dnak anti-gst Purity 1. Chaperone proteins 伴侣蛋白 DnaK -70 kda GroEL - 60 kda Detergents and/or reducing agents before sonication 在超声前加去垢剂和或还原剂 Western blot analysis. Samples: Fractions from second IEX step (first step on Glutathione Sepharose ) 2. Endogenous GST 内源性 GST 柱上酶切 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 Optimize Imidazole conc. 优化咪唑浓度 18

19 To improve purity 提高纯度 Purity 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 19

20 1 Two-step purification 两步纯化 Purity 1. Affinity step 亲和层析 Column: Sample: System: HisTrap FF 1 ml 50 ml (His) 10 -Trx-P 450 in E. coli lysate ÄKTAexplorer 2. Gel filtration 凝胶过滤 Column: Sample: System: HiLoad 16/60 Superdex 200 pg 5.2 ml eluted pool from HisTrap ÄKTAexplorer mau mau F F3 F4 Wa stea3a5a7a9 Wa ste 80.0 ml A1A2A3A4A5A6A7A8A9 A11A13A15B14B12B10B8B7B6B5B4B3B2B1C1C2C3C4C5C6C ml M r LMW Start FT wash eluted F HisTrap FF Superdex 200 pg 20

2 Dual tagging of a protein 双标签蛋白 Purity Combination of a two-step HisTrap HP and StrepTrap HP purification Sample: 15 ml Strep II-protein-(His) 6 in E.

21 2 Dual tagging of a protein 双标签蛋白 Purity Combination of a two-step HisTrap HP and StrepTrap HP purification Sample: 15 ml Strep II-protein-(His) 6 in E. coli lysate System: ÄKTAxpress FT1 E1 FT3 E3 E2 FT2 Run 1. HisTrap HP 1 ml Run 2. StrepTrap HP 1 ml Run 3. HisTrap HP 1 ml + StrepTrap HP 1 ml Run 1 Run 3 Run 2 Acknowledgement: Martina Nilsson, Biovitrum Stockholm, Sweden 21

22 3 Purity Yield Abs 490/280 Optimizing imidazole concentration 优化咪唑浓度 2,00 1,50 1,00 0,50 Purity Yield Yield versus purity 产量 vs 纯度 Abs 490 Column: HisTrap HP 1ml Sample: GFP-(His) 6 expressed in E. coli Detection: 280 and 490 nm Purity 0, Imidazole conc. (mm) 0 Imidazole concentration 22

23 4 Purity Optimize metal ion for IMAC 优化金属离子 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) Cu 2+ Zn 2+ Co 2+ Ni 2+ 23

24 5 On-column refolding and purification of a histidine-tagged protein 柱上复性和 his 标签蛋白纯化 Purity Column: HisTrap FF 1 ml A 280 Refolding Gradient: 6 M 0 M urea 30 ml gradient volume Flow rate: 0.5 ml/min Resin: System: Ni Sepharose FF ÄKTAprime A 280 Elution Gradient: mm imidazole Flow rate: 1.0 ml/min Volume 24

25 Tag cleavage and removal 标签的酶切和去除 Glutathione GST Recombinant Protein Sepharose 10C Cleavage site Protein degradation Low yield Purity Tag removal Protein stability 25

26 On column tag cleavage 柱上酶切 Glutathione Glutathione Cleavage site GST Recombinant Protein Glutathione GST Recombinant Protein Glutathione Sepharose TM GST GST-tagged PreScission TM Protease Glutathione Endogenous GST From expression host Glutathione GST Still on column after wash Glutathione Endogenous GST 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 GST Removal of cleavage enzyme 去除内切酶 No uncleaved protein 没有切开的蛋白 Eluted in WASH buffer Glutathione GST Recombinant Protein Recombinant Protein Uncleaved fusion protein Untagged target protein 26

27 Optimizing tag cleavage 优化酶切 Tag cleavage On-column cleavage of GST-tagged protein 柱上 GST 标签蛋白的酶切 PreScission protease % Cleaved protein 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 Incubation time (hours) 27

28 Tag cleavage Automatic on-column cleavage and tag removal 自动柱上酶切和去除标签 Sample: APC1040-(His) 6 in E.coli lysate Column: HisTrap HP 1 ml Protease: AcTEV System: ÄKTAxpress Cleaved protein Regeneration Reference, uncleaved protein 16 mg Purified, cleaved protein Lanes 1. Protein markers 2. Sample 3. Flow through from affinity step 4. Eluate from Gel filtration (GF) 28

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 29

30 Protein stability Purified protein stability 纯化蛋白的稳定性 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 Gel filtration, light scattering, native PAGE 凝胶过滤, 蒸发光散射, 非变性电泳, Visual 目测 Loss of activity 活性丢失 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 30

31 Conclusions 结论 Protein degradation 蛋白降解 Low yield 低产量 Purity 纯度 Tag removal 标签的去除 Sample preparation, minimize time 样品准备, 缩短时间 Optimize binding conditions 优化结合条件 Multi-step purification 多步纯化 On-column cleavage with tagged protease 带标签的蛋白酶柱上酶切 31

32 THANK YOU!

33 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). A license for the commercial use of GST gene fusion vectors must be obtained from Chemicon International Incorporated, Single Oak Drive, Temecula, CA 92590, USA. StrepTrap HP and StrepTactin Sepharose High Performance are covered by US and equivalent patents and patent applications in other countries. The purchase of StrepTrap HP and StrepTactin Sepharose High Performance includes a license under such patents for non-profit and in-house research only. Please contact IBA (info@ibago. com) for further information on llicenses for commercial use of StrepTactin General Electric Company All rights reserved. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, a General Electric Company. GE Healthcare Bio-Sciences AB, Björkgatan 30, SE Uppsala, Sweden

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1. 請先檢查包裝內容物 AC750 多模式無線分享器安裝指南安裝指南 CD 光碟 BR-6208AC 電源供應器網路線 2. 將設備接上電源, 即可使用智慧型無線裝置進行設定 A. 接上電源 1. 請先檢查包裝內容物 AC750 多模式無線分享器安裝指南安裝指南 CD 光碟 BR-6208AC 電源供應器網路線 2. 將設備接上電源, 即可使用智慧型無線裝置進行設定 A. 接上電源 B. 啟用智慧型裝置的無線 Wi-Fi C. 選擇無線網路名稱 "edimax.setup"