Tagged protein purification - tips and hints

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In this presentation, GE Life Sciences scientists take you through the basics of tagged protein purification with tips and tricks to guide you to successful purification; covering affinity tags, expression systems and planning both expression and purification of a protein. How to deal with some of the most cited problems in tagged protein purification; including protein degradation, low yield, purity, tag removal and protein stability.

Content
Introduction
Affinity Chromatography - principles
Tags & Expression systems -strategies
Tips & Hints
Protein degradation
Low yield
Purity
Tag cleavage & removal
Protein stability
Summary

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  • GEHC have different IMAC purification products. Depending on the requirements and purposes of your purification of a His-tagged target protein and by what system, and how it has been expressed, different media is better suited.Here the different IMAC products and their “best use” are shown.For high purities the Talon products should be considered whereas the Ni Sepharose ones have higher capacities. As I already mentioned the Ni Sepharose excel is well suited for purification from complex eukaryotic cell media.xxxxxxxxxx
  • Tagged protein purification - tips and hints

    1. 1. Imagination at work. GE Healthcare Life Sciences 2013 Tagged Protein Purification - Tips & Hints
    2. 2. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems -strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    3. 3. Protein purification strategy Tagged protein Un-tagged protein Expression Simple Purification ONE STEP Affinity 80-90 % purity Capture Intermediate Purification Polishing (CiPP) Multi-Step Purification Introduction
    4. 4. Basic principle LigandMatrix Affinity tag Recombinant Protein Cleavage Site + Binding Elution Affinity tag Recombinant Protein Cleavage Site LigandMatrix Affinity tag Recombinant Protein Cleavage Site Introduction
    5. 5. Simple purification of tagged proteins 1 2 3 4 Introduction
    6. 6. Affinity tags Advantages • Simplification • Increase solubility • Prevent proteolysis • Detection Disadvantages • Tag removal • Interfere with structure/function GST MBP Strep-tag™ II His Strep-tag II- His Size 26 kDa 40 kDa 8 aa 6 aa 14 aa Media capacity 30 mg/ml 10 mg/ml 6 mg/ml 40 mg/ml NA Purity Increased solubility Introduction = Low = Highest
    7. 7. Expression systems Bacteria Yeast Insect cells Mammalian cells Inclusion bodies +/− (+)/− − − Secretion +/− + + + PTM or post-translational modification − + + + Proteolytic cleavage +/− +/− − − + = Yes − = No Introduction
    8. 8. Protein expression in E. coli Inner membrane Peptidoglycan Outer membrane Lipopolysaccharide 70 Å 70 Å 70 Å 210 Å Cytoplasm ~2000 proteins ~100 proteins Periplasm ~10 proteins Medium Introduction
    9. 9. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    10. 10. Protein degradation Western blotSDS-PAGE Truncated forms with the tag Full length protein Protein degradation Protein degradation
    11. 11. 1. Use a protease-deficient expression host 2. Work Fast To avoid protein degradation Protein degradation Conventional purification Cell lysis Transfer to tubes Centrifugation Collect supernantant Filtration Protein purification Higher yield and biologically active protein Cell lysis Column: HisTrap™ FF crude Resin: Ni Sepharose™ Fast Flow Purification of unclarified lysate Protein purification
    12. 12. Fast purification from unclarified sample Protein expressed in Pichia pastoris Protein degradation Column: HiTrap™ TALON® crude 1 ml Sample: 20 ml unclarified Pichia pastoris containing hydrolase, GEHC2-(His)6 (Mr 33 700), prepared by sonication 1 LMW-SDS Marker Kit 2. Start material 3. Flowthrough 4. Wash 5. Eluate
    13. 13. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    14. 14. Low yield Poor binding Inefficient elution Low yield
    15. 15. Low yield Low yield Tag is not exposed for binding Factors in the extract/cultivation medium interfere with binding Binding buffer conditions are not optimal Kinetic between ligand and tag is slow Poor binding Elution buffer conditions not optimal Precipitation Inefficient elution
    16. 16. Low yield Cultivation media issues Ni Sepharose™ excel Ni Sepharose™ 6FF Purification of His8-murine plasminogen activator inhibitor (mPAI-1-(his)8) secreted into CHO cell culture medium 1. LMW-SDS Marker Kit 2. Start material 3. Ni Sepharose excel, flowthrough 4. Ni Sepharose excel, wash 5. Ni Sepharose excel, eluate 6. Ni Sepharose 6 FF, flowthrough 7. Ni Sepharose 6 FF, wash 8. Ni Sepharose 6 FF, eluate
    17. 17. Optimizing buffer conditions Protein Histidine tagged Nurr1 ligand binding domain expressed in E. coli Parameters studied Binding buffers pH values: pH 6.0 to 8.5 (8 buffers) NaCl: 100 -750 mM Glycerol: 5-10% b-mercaptoethanol: 0.05% Low yield High-throughput buffer screening His MultiTrap™ FF Acknowledgement: R. Steele and B. Grasberger, Johnson & Johnson Pharmaceutical R&D, USA β-mercaptoethanol 5% glycerol 10% glycerol Increasing NaCl
    18. 18. Kinetics: effect of flow rates GST: Impact of flow rate during sample application Slow binding kinetics Flow rate during sample application must be low 0 100 200 300 400 500 600 700 800 900 1000 0.1 0.3 0.4 1 1.5 RT ColdYield(relativeunits) Flow rate (ml/min) Binding capacity study using GST-(His)6 on Glutathione Sepharose™ 4 Fast Flow Low yield
    19. 19. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    20. 20. Requirements Raising antibodies > 90-95% Crystallization > 99% Characterization > 99% Purity
    21. 21. • 50 µl eluate from StrepTrap™ HP • 10 min Purity check Purity SDS- PAGE kDa 97 66 45 30 20.1 14.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 kDa 97 66 45 30 20.1 14.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Superdex™ short columns Co-elution?Heterogeneity due to PTM? Proteolytic cleavage? Many bands/peaks of different Mw
    22. 22. To improve purity Multi-step purification Dual tagging of a protein Optimize metal ion for IMAC Optimize binding and elution conditions 2 1 4 3 Purity
    23. 23. Two-step purification 1. Affinity step 2. Gel filtration 1 Column: HisTrap™ FF 1 ml Sample: 50 ml (His)10-Trx-P 450 in E. coli lysate System: ÄKTAexplorer™ Column: HiLoad™ 16/60 Superdex™ 200 pg Sample: 5.2 ml eluted pool from HisTrap System: ÄKTAexplorer 0 1000 2000 3000 4000 mAU 0.0 20.0 40.0 60.0 80.0 ml F3 F4 WasteA3A5A7A9 Waste 0 100 200 300 400 mAU 20.0 40.0 60.0 80.0 100.0 ml A1A2A3A4A5A6A7A8A9A11A13A15B14B12B10B8B7B6B5B4B3B2B1C1C2C3C4C5C6C7 1 2 3 F Purity 97 000 66 000 45 000 30 000 20 100 14 400 Mr LMW Start FT wash eluted 1 2 3 F HisTrap Superdex 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 200pg 1 3 4 5 6 7 8 92
    24. 24. Dual tagging of a protein2 Acknowledgement: Martina Nilsson, Biovitrum Stockholm, Sweden 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 FT1 FT 3 FT2E1 E2E3 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™ Purity
    25. 25. Optimize metal ion for IMAC3 Cu2+ Zn2+ Co2+ Ni2+ Cu2+ Zn2+ Co2+ Ni2+ Column: HiTrap™ IMAC FF (prepacked with un-charged IMAC Sepharose™) Purity
    26. 26. Optimize binding & elution conditions Purity Column: HisTrap™ excel 1ml Sample: PRCP-(His)9 secreted into SAFC EX-CELL 405 medium Detection: 280 nm 4
    27. 27. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    28. 28. Tag cleavage and removal Tag cleavage & removal Cleavage site Affinity tag Recombinant Protein Cleavage Site LigandMatrix Sepharose ™ Cleavage site
    29. 29. On column tag cleavage - overview • Native function of monomeric target protein • Removal of endogenous GST • Removal of cleavage enzyme • No uncleaved protein Glutathione Sepharose™ Glutathione Cleavage site GST Recombinant Protein Endogenous GST Glutathione Glutathione From expression host GST Recombinant Protein Glutathione GST-tagged PreScission™ Protease GST Recombinant Protein Endogenous GST Glutathione Glutathione From expression host GSTGlutathione Still on column after wash Uncleaved fusion protein Untagged target protein Eluted in WASH buffer Recombinant Protein Tag cleavage & removal
    30. 30. Optimizing tag cleavage On-column cleavage of GST-tagged protein PreScission™ protease Optimize cleavage conditions 1. Time for cleavage 2. Ratio of protease/target protein 3. Temperature (optimum 4 C) Incubation time (hours) %Cleavedprotein 10 units PreScission Protease/mg protein 20 units PreScission Protease/mg protein 40 units PreScission Protease/mg protein Tag cleavage & removal
    31. 31. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hints – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    32. 32. Protein stability Structural studies Characterization Measuring biological activity Protein stability
    33. 33. Purified protein stability Monitoring condition of protein Aggregation Gel filtration, light scattering, native PAGE Precipitation Visual Conformation changes Loss of activity Check-list • Freeze in aliquots • Avoid freeze and thaw • Avoid freezing concentrated protein solution • Screen different storage buffers, e.g pH and salt • Store in 10-50% glycerol Protein stability
    34. 34. Content Introduction – Affinity Chromatography - principle – Tags & Expression systems - strategies Tips & Hintss – Protein degradation – Low yield – Purity – Tag cleavage & removal – Protein stability Summary
    35. 35. Summary Initial considerations Expression host, sample preparation, affinity tag Protein degradation Sample preparation, minimize time Low yield Optimize binding conditions Purity Multi-step purification Tag removal On-column cleavage with tagged protease
    36. 36. goo.gl/GlvSDl To learn more – download the free handbook A range of guides full of practical tips and in-depth information about common methodologies used in the lab
    37. 37. GE, imagination at work, and GE monogram are trademarks of General Electric Company. GSTrap, HiLoad, HisTrap, HiTrap, PreScission, Sepharose, StrepTrap , Superdex, ÄKTAexplorer, ÄKTAprime and ÄKTAxpress are trademarks of GE Healthcare companies. AcTEV is a trademark of Invotrogen. FLAG is a registered trademark of Sigma-Aldrich™ Biotechnology LP and Sigma-Aldrich Co. Strep-tag is a registered trademark of Institut für Bioanalytik GmbH 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, 28820 Single Oak Drive, Temecula, CA 92590, USA. StrepTrap High Performance is covered by US 6 103 493 and equivalent patents and patent applications in other countries. The purchase of StrepTrap HP 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. © 2009-2011 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. www.gelifesciences.com/protein-purification GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden

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