Tagged protein purification - tips and hints

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Information about Tagged protein purification - tips and hints
Education

Published on February 22, 2014

Author: GEproResearch

Source: slideshare.net

Description

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

Tagged protein purification - Tips & Hints GE Healthcare Life Sciences 2013 Imagination at work.

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

Protein purification strategy Simple Purification ONE STEP Affinity 80-90 % purity Tagged protein Multi-Step Purification Capture Intermediate Purification Polishing Expression (CiPP) Un-tagged protein Introduction

Basic principle Ligand Cleavage Site Matrix + Affinity tag Recombinant Protein Binding Cleavage Site Matrix Ligand Affinity tag Recombinant Protein Elution Cleavage Site Affinity tag Recombinant Protein Introduction

Simple purification of tagged proteins 1 2 3 4 Introduction

Affinity tags Advantages Disadvantages • • • • • Tag removal • Interfere with structure/function Simplification Increase solubility Prevent proteolysis Detection GST Size Media capacity MBP Strep-tag™ II His Strep-tag IIHis 26 kDa 40 kDa 8 aa 6 aa 14 aa 30 mg/ml 10 mg/ml 6 mg/ml 40 mg/ml NA Purity Increased solubility = Low = Highest Introduction

Expression systems Bacteria Yeast Inclusion bodies +/− Secretion PTM or post-translational modification Proteolytic cleavage (+)/− Insect cells − Mammalian cells − +/− + + + − + + + +/− +/− − − + = Yes − = No Introduction

Protein expression in E. coli Medium ~10 proteins Lipopolysaccharide 70 Å 70 Å Outer membrane Peptidoglycan 210 Å Periplasm ~100 proteins Inner membrane 70 Å Cytoplasm ~2000 proteins Introduction

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

Protein degradation Full length protein Truncated forms with the tag SDS-PAGE Western blot Protein degradation Protein degradation

To avoid protein degradation 1. Use a protease-deficient expression host 2. Work Fast Conventional purification Cell lysis Transfer to tubes Centrifugation Collect supernantant Filtration Protein purification Purification of unclarified lysate Cell lysis Protein purification Column: HisTrap™ FF crude Resin: Ni Sepharose™ Fast Flow Higher yield and biologically active protein Protein degradation

Fast purification from unclarified sample Protein expressed in Pichia pastoris 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 Protein degradation

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

Low yield Poor binding Inefficient elution Low yield

Low yield Poor binding 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 Inefficient elution Elution buffer conditions not optimal Precipitation Low yield

Cultivation media issues Ni Sepharose™ excel Purification of His8-murine plasminogen activator inhibitor (mPAI-1-(his)8) secreted into CHO cell culture medium Ni Sepharose™ 6FF 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 Low yield

Optimizing buffer conditions High-throughput buffer screening Protein Histidine tagged Nurr1 ligand binding domain expressed in E. coli Parameters studied Binding buffers pH values: buffers) NaCl: Glycerol: b-mercaptoethanol: pH 6.0 to 8.5 (8 100 -750 mM 5-10% 0.05% His MultiTrap™ FF β-mercaptoethanol 5% glycerol 10% glycerol Increasing NaCl Acknowledgement: R. Steele and B. Grasberger, Johnson & Johnson Pharmaceutical R&D, USA Low yield

Kinetics: effect of flow rates GST: Impact of flow rate during sample application Flow rate during sample application must be low Binding capacity study using GST-(His)6 on Glutathione Sepharose™ 4 Fast Flow Yield (relative units) Slow binding kinetics 1000 900 800 700 600 500 400 300 200 100 0 RT Cold 0.1 0.3 0.4 1 1.5 Flow rate (ml/min) Low yield

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

Requirements Raising antibodies > 90-95% Crystallization > 99% Characterization > 99% Purity

Purity check Superdex™ short columns 50 µl eluate from StrepTrap™ HP SDSPAGE • • 10 min kDa 97 66 45 30 20.1 14.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Many bands/peaks of different Mw Heterogeneity due to PTM? Proteolytic cleavage? Co-elution? Purity

To improve purity 1 Multi-step purification 2 Dual tagging of a protein 3 Optimize metal ion for IMAC 4 Optimize binding and elution conditions Purity

1 Two-step purification 1. Affinity step 2. Gel filtration 50 ml (His)10-Trx-P 450 in E. coli System: ÄKTAexplorer™ HiLoad™ 16/60 Superdex™ 5.2 ml eluted pool from System: HisTrap™ FF 1 ml Sample: lysate Column: 200 pg Sample: HisTrap Column: ÄKTAexplorer mAU 4 00 m AU 3 00 4000 3000 2 00 1 2000 2 3 4 5 7 6 8 9 F Mr 1000 0 F3 0 .0 2 0 .0 F4 40 .0 6 0 .0 W a ste 3A 5A 7A 9 A 8 0 .0 3 1 1 00 2 0 2 0 .0 W a ste ml A 1 2 3 4 5 6 7 8 9 A 1 1A 1 3A 1 5B 1 4 1 2 1 0B 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 AAAAAAAA B B B B B B B B B CCCCCCC 4 0 .0 6 0 .0 8 0 .0 1 0 0 .0 ml 97 000 66 000 45 000 30 000 20 100 14 400 LMW Start FT wash eluted HisTrap FF 1 2 3 F Superdex 200pg Purity

2 Dual tagging of a protein 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 FT 3 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 Purity

3 Optimize metal ion for IMAC Column: HiTrap™ IMAC FF (prepacked with un-charged IMAC Sepharose™) Cu2+ Zn2+ Cu2+ Co2+ Ni2+ Zn2+ Co2+ Ni2+ Purity

4 Optimize binding & elution conditions Column: Sample: Detection: HisTrap™ excel 1ml PRCP-(His)9 secreted into SAFC EX-CELL 405 medium 280 nm Purity

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

Tag cleavage and removal Sepharose ™ Ligand Cleavage Site Matrix Affinity tag Recombinant Protein Cleavage site Cleavage site Tag cleavage & removal

On column tag cleavage - overview Cleavage site Glutathione Glutathione Glutathione Sepharose™ Glutathione GST Recombinant Protein Glutathione GST Recombinant Protein GST-tagged PreScission™ Protease Endogenous GST From expression host Glutathione • • Native function of monomeric target protein Still on column after wash Removal of endogenous GST Glutathione Removal of cleavage enzyme • No uncleaved protein Tag cleavage & removal Endogenous GST From expression host Glutathione • GST GST Recombinant Protein Uncleaved fusion protein Eluted in WASH buffer Recombinant Protein Untagged target protein

Optimizing tag cleavage On-column cleavage of GST-tagged protein PreScission™ protease % Cleaved protein Optimize cleavage conditions 10 units PreScission Protease/mg protein 1. Time for cleavage 2. Ratio of protease/target protein 3. Temperature (optimum 4 C) 20 units PreScission Protease/mg protein 40 units PreScission Protease/mg protein Incubation time (hours) Tag cleavage & removal

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

Protein stability Structural studies Characterization Measuring biological activity Protein stability

Purified protein stability Monitoring condition of protein Aggregation Precipitation Conformation changes Gel filtration, light scattering, native PAGE Visual 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

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

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

Get your free Protein Science Handbooks www.gelifesciences.com/protein-purification

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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