Content GE life science?. of specific types of ligand. : matrices that are chemically modified to facilitate the coupling : covalent attachment of a l

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1 Content GE life science?. of specific types of ligand. : matrices that are chemically modified to facilitate the coupling : covalent attachment of a ligand to a pre-activated matrix to create an affinity medium.? adsorption of wash elute equilibration sample and away bound re-equilibration elution of unbound protein(s) unbound material material begin sample change to application elution >1 1-2 cv x cv 1-2 cv cv 1-2 cv Absorbance Column Volumes (cv) offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest > 90 %

2 DNA NHS activated 4 Fast Flow CNBr activated 4 FF GST, Benzamidine Sepharose, III A - VIII select and Ig Select IgG Content / : pet, phat, pqe,pproex,ptrchis, Co 2+ (Poly His) ph :pgex, pet41, pet42,, (GST) :pmal, MBP (MBP), Strep tag :ppr-iba, pask-iba, OmpA (, Strep- Tactin ),, A/G ph? Group-specific ligands! :

3 Strategy How specific do we really want to be? Usually home-made affinity media Antigen Antibody Elution protocols based Hormone Receptor on general principles Protein G IgG antibodies Keep it simple! Often ready-made affinity media Standard, tested elution protocols First choice Low ph, salt, GuHCl, Urea etc Usually decrease ph, increase ionic strength or decrease polarity adding up to 10 % dioxane or up to 50 % ethylene glycol (Competing binding substances) Usually extremes of ph or chaotropic agents (Competing free ligand) + + Reconstituting NADHBlue Sepharose. Elution of glycoproteins from Con A Sepharose by α-d-methylmannoside + A, α(1,6) N- - +

4 : Hitrap 1ml and 5ml. : :, HiTrap ph ph : : : Troubleshooting Some of the target molecule elutes as a broad, low peak while still under binding conditions A280 A280 Binding Binding Flow through (unbound material) Elution Eluted target Flow through (unbound material) Elution Eluted target ml ml Allow time for sample to bind and/or apply sample in aliquots. Stop flow for a few minutes between each sample application. Troubleshooting ph : Target elutes in a broad, low peak Try different elution conditions. Stop flow intermittently during elution. ml A 280 Flow (unbound through material) Elution Binding Wait Eluted target A280 Binding Flow through (unbound material) Elution Eluted target ml In competitive elution, increase competitor concentration in elution.

5 Content 10 col. vol. 0.1 M Tris-HCl, 0.5 M NaCl, ph col. vol. 0.1 M NaAc, 0.5 M NaCl, ph 4.5 Use chaotropic agents or detergents if needed 10 His Ni Sepharose His His His His His His rprotein cleavage site Ni Sepharose 6 Fast Flow : 90um TEV protease Ni Sepharose High performance : 34um IMAC Sepharose HP/ 6FF Chelating Sepharose HP/FF: His MultiTrap HP/FF His SpinTrap His HisCys His GraviTrap HisTrap CysTry Try FF crude 1 ml/5 ml HisTrap FF/HP 1 ml/5 ml HisPrep FF 16/10 Ni Sepharose 6 Fast Flow Ni Sepharose High Performance Ni Sepharose / Up to 0.5mg Up to 1 mg 40 mg/column 40 mg/ml 40 mg/ml 800 mg/column 40 mg/ml 40 mg/ml ; ; / HisTrap, ÄKTA

6 Ni Sepharose Ni Sepharose MBP-(His)6 : 0.25 ml : 0.25 ml/min : 1 mg/ml (pure protein) (+4 C) -- mg MBP-His/ml gel Ni-NTA ( ) Ni-NTA Ni Sepharose Ni HP Sepharose HP ( ) ( ) ( ) Ni Sepharose 8 M Q Ni Sepharose 60 Q Ni Sepharose Ni Sepharose mau 20-40mM 1. HisTrap FF 2. HiLoad 16/60 Superdex 200 pg mau Mr mM EDTA PH F3 F4 WasteA3A5A7A9 Waste ml F 2 A1A2A3A4A5A6A7A8A9A11A13A15B14B12B10B8B7B6B5B4B3B2B1C1C2C3C4C5C6C ml F HisTrap FF Superdex 200 pg Ni Sepharose mau Mr :IMAC/Chelating Sepharose HP/FF His His- SDS-PAGE ml/min 2 ml/min 4 ml/min Cu 2+ Zn min Co 2+ (ml/min) (min) (mg) : 2: 3: 1 ml/min 4: 2 ml/min 5: 4 ml/min Cu 2+ Zn 2+ Co 2+

7 Ni Sepharose Ni mM IMAC/Chelating Sepharose HP/FF GST GST - Glutathione Sepharose GST- M r Glutathione Sepharose GST GST rec-protein 10C Factor Xa: M r / Thrombin: M r PreScission Protease: M r Thrombin, Factor Xa at ºC GSTrap FF HiTrap Benzamidine FF (high sub) PreScission Protease at + 4ºC GSTrap FF - - PreScission protease GST 4 AC- -DS-AC PreScission 5. HiTrap 6. Protease Desalting GSTrap FF HP 7. PreScission protease 1. GSTrap FF HP 2. GST- 3. PreScission Protease 4. PreScission Protease

8 x g x g Thrombin 10a GST thrombin 5. HiTrap 6. 10a Desalting GSTrap FF HP 7. M r GSTrap FF HP 2. AC- -DS-AC GSTrap FF HP thrombin 10a AC- -AC 4. HiTrap Benzamidine FF (high 5. sub) thrombin 10a Glutathione Sepharose 12C-Linker / GST GST MicroSpin 400 µg,, Purification Module Sepharose Bead Glutathione Sepharose MicroPlex 24 Vacuum ( 48 ) GSTrap FF/HP/4B 1 ml mg, GSTrap FF/HP/4B 5 ml mg, Glutathione Sepharose High performance Glutathione Sepharose 4 Fast flow Glutathione Sepharose 4 B Glutathione Glutathione Sepharose 4B 5 mg / ml Glutathione Sepharose 4 ~10 mg / ml FF/HP GST MultiTrap 4B & FF 0.5 mg/(gst),, / 2 1 Tag 2 E.coli BL21 pgex Vector Leaved by LonOmpT E.coli BL21

9 纯化优化 zoom in on GST-Glutathione Interaction 总结 vectors E. coli BL21 Anti-GST Antibody Protease GST 96-Well Detection Module Glutathione Sepharose 纯化过程优化因素流速样品浓度 GST-标记蛋白质的浓度从表达到纯化一步到位洗脱推荐还原型谷胱苷肽的浓度 20mM Glutathione Sepharose添加剂兼容洗脱时的 ph 8 不同形式的产品适合快速和重复性高的要求使用针筒蠕动泵或者 ÄKTA 系统产品特性 Structure of MBP bound to maltose MBP-标签主要用来提高溶解性蛋白的表达量 MBP标签蛋白的纯化 MBP-标签大小为42.5 kd 因为MBP-标签太大,经常会在纯化后用下列酶切除标签: The figure was taken from the work of J.A. Hall, T.E. Thorgeirsson, J. Lu, Y.-K. Shin & H. Nikaido, Journal of Biological Chemistry, 272, (1997) Factor Xa, Genease, Enterokinase 经常会放在所要表达蛋白的N-端主要用在原核生物 E. coli表达产品特性 MBPTrap HP 1 ml and 5 ml 应用 ÄKTAprime 上简单纯化MBP-MCAD 蛋白第一步亲和填装了Dextrin Sepharose High Performance填料高载量大约10 mg/ml 填料高纯度 Sample: MBP-MCAD in E. coli lysate, 15 比较温和的洗脱条件 10 mm 麦芽糖 ml ph 稳定性 > ph 7.0 可重复的实验结果 Flow rate: 5 ml/min (0.5 ml/min during sample application) 非常容易放大操作简单: 注射器蠕动泵 ÄKTAdesign 清洗非常方便 0.5M NaOH Column: MBPTrap HP 5 ml Binding : 20 mm Tris-HCl, 200 mm NaCl, 1 mm EDTA, 1 mm DTT, ph 7.4 Elution : 10 mm maltose in binding Acknowledgements: Dr. Esther M. Meier, Dr. von Haunersches Kinderspital Munich, Germany 9

10 4.2 MBPTrap HP 0.5 M NaOH ÄKTAprime MBP-MCAD S F MBPTrap HP Superdex 200 pg Start material E1 E2 E3 E4 E5 E6 Column: Superdex TM 200 pg in XK 16/20 Sample: Eluted pool (2 ml) from MBPTrap HP 5 ml Flow rate: 0.4 ml/min Buffer: 20 mm HEPES, 200 mm NaCl, ph 7.0 SDS-PAGE analysis Strep(II) 5.1 Strep(II)-, 8 aa, 1 kd (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) Strep-Tactin N-C- 5.1 StrepTrap HP 1 ml and 5 ml StrepTactin High Performance 6mg/ml 2.5 mm 0.5M NaOH 10 min ph > ph mM His : ÄKTAdesign StrepTrap HP Reagent Concentration Reagent Concentration Reduction agents Others DTT, -mercaptoethanol 50 mm Ammonium sulphate, (NH 4 ) 2 SO 4 2 M Non-ionic detergents CaCl 2 max. 1 M Triton X-100, Tween 20 2% EDTA 50 mm Ionic detergents Guanidine max. 1 M SDS 0.1% Glycerol max. 25% Zwitterionic detergents Imidazole 500 mm CHAPS 0.1% MgCl 2 1 M Urea max. 1 M Acknowledgements: IBA GmbH Germany

11 Sample: Strep(II)-protein-(His)6, Mr: Flow rate: 0.8 ml/min System: ÄKTAxpress Run 1. HisTrap HP 1 ml Run 2. StrepTrap HP 1 ml Run 3. HisTrap HP 1 ml + StrepTrap HP 1 ml Lane: 1. Marker 2. Flow through, individual HisTrap HP 3. Eluted pool, 4. Flow through, HisTrap HP + StrepTrap HP 5. Eluted pool, 6. Eluted pool, individual StrepTrap HP 7. Flow through, ProteinA/G HisTrap HP HisTrap HP + StrepTrap HP StrepTrap HP Acknowledgements: Martina Nilsson, Biovitrum Stockholm, Sweden VL CL VH CH IgG CH VH } CL VL } Fab Fc VL CL VH CH CH VH CH CL VH VL CL VL F(ab)2 Fab Species Protein A Protein G binding binding Human IgA variable - IgD - - IgE IgG IgG IgG IgG IgM* variable - Avian egg yolk IgY - - Cow Dog ++ + Goat - ++ Guinea Pig IgG IgG Hamster + ++ Horse Koala - + Llama - + Species Protein A Protein G binding binding Monkey (rhesus) Mouse IgG IgG 2a IgG 2b IgG IgM* variable - Pig Rabbit Rat IgG IgG 2a IgG 2b - ++ IgG Sheep +/- ++ IgGProtein A Protein G ELISA ++ = + = -= Protein G Argarose: from sea weed to separation meida protein G from GE Healthcare protein GE. coli IgG Mr , The pi of of protein G is 4.1 and the ph stability New PrA media launches: 1. Protein G Sepharose High Performance 34um 2. Protein G Sepharose 4 Fast Flow 90um rprotein A 1996: rprotein A Sepharose 4FF (single-point attached thioether bond) 2001: Mabselect (single-point attached thioether bond) 2005: Mabselect SuRe, Xtra (single-point attached thioether bond),,

MabSelect family MabSelect TM family Mabselect Xtra MabSelect SuRe MabSelect Xtra MabSelect TM DBC 10 % (mg/ml) 50 MabSelect Xtra MabSelect MabSelect SuRe 40 ProSep-vA Ultra 30 ProSep-rA 20 10 0 0 2

= MabSelect SuRe > ProSep va-ultra >> ProSep ra High Capacity Alois Jungbauer and Rainer Hahn MabSelect TM family Mabselect SuRe CIP 40 GuHCl 3 GuHCl MabSelect TM family 100-100- 100-300 0.

12 MabSelect family MabSelect TM family Mabselect Xtra MabSelect SuRe MabSelect Xtra MabSelect TM DBC 10 % (mg/ml) 50 MabSelect Xtra MabSelect MabSelect SuRe 40 ProSep-vA Ultra 30 ProSep-rA Capture the lead in the purification of antibodies Residence time (min) MabSelect TM Xtra has higher capacity then other existing Protein A media on the market MabSelect Xtra > MabSelect = MabSelect SuRe > ProSep va-ultra >> ProSep ra High Capacity Alois Jungbauer and Rainer Hahn MabSelect TM family Mabselect SuRe CIP 40 GuHCl 3 GuHCl MabSelect TM family M NaOH 40 3 MAb Cell culture Wave Reactor CIEX Sp/ Capto S AIEX Q/ Capto Q Cell removal & Clarification Hollow fiber 0.22u MabSelect SuRe Virus Inactivation & Filtration Capto adhere AIEX Capto Q* Capto adhere Thanks! Yuntao.wu@ge.com Pool for Final Filtration UF/DF Kvick system *WO 2004/ (Lonza)

Overcoming the most common obstacles in your purificaton of tagged proteins

Overcoming the most common obstacles in your purificaton of tagged proteins 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 Outline 大纲 Introduction 介绍 Affinity tags 亲和标签 Expression