GE Healthcare Purifying Challenging Proteins Principles and Methods Handbooks from GE Healthcare GST Gene Fusion System Gel Filtration Handbook Principles and Methods 18-1157-58 18-1022-18 Affinity Chromatography Recombinant Protein Principles and Methods Purification Handbook 18-1022-29 Principles and Methods 18-1142-75 Antibody Purification Handbook Protein Purification 18-1037-46 Handbook 18-1132-29 Percoll Methodology and Applications Hydrophobic Interaction and 18-1115-69 Reversed Phase Chromatography Principles and Methods Ion Exchange Chromatography 11-0012-69 & Chromatofocusing Principles and Methods 2-D Electrophoresis 11-0004-21 using immobilized pH gradients Principles and Methods Purifying 80-6429-60 Challenging Proteins Principles and Methods Microcarrier Cell Culture 28-9095-31 Principles and Methods 18-1140-62 Purifying Challenging Proteins Principles and Methods Contents Introduction ............................................................................................................................................................... 5 Challenging proteins ........................................................................................................................................................5 Outline ....................................................................................................................................................................................5 Common acronyms and abbreviations ..................................................................................................................6 Symbols .................................................................................................................................................................................7 Chapter 1 Membrane proteins ..........................................................................................................................................................9 Introduction ..........................................................................................................................................................................9 Classification of membrane proteins ....................................................................................................................10 Purification of integral membrane proteins for structural and functional studies .........................11 Starting material .............................................................................................................................................................12 Small-scale expression screening of histidine-tagged membrane proteins from E. coli lysates .........................................................................................................................................................15 Cell harvest ......................................................................................................................................................................17 Cell disruption and membrane preparation ......................................................................................................18 Solubilization ....................................................................................................................................................................20 Purification ........................................................................................................................................................................29 Purification of non-tagged membrane proteins .............................................................................................38 Purity and homogeneity check ...............................................................................................................................40 Conditioning ......................................................................................................................................................................43 Proteomic analysis of membrane proteins ........................................................................................................45 References .........................................................................................................................................................................49 Chapter 2 Multiprotein complexes ...............................................................................................................................................51 Introduction .......................................................................................................................................................................51 Pull-down assays ...........................................................................................................................................................53 Isolation of native complexes ...................................................................................................................................61 Isolation of recombinant protein complexes ....................................................................................................63 References .........................................................................................................................................................................67 Chapter 3 Inclusion bodies ..............................................................................................................................................................69 Optimizing for soluble expression ..........................................................................................................................69 Strategies for handling inclusion bodies .............................................................................................................70 Isolation of inclusion bodies ......................................................................................................................................71 Solubilization ....................................................................................................................................................................72 2 Handbook 28-9095-31 AA Refolding ...........................................................................................................................................................................72 Refolding using gel filtration .....................................................................................................................................74 Analysis of refolding .....................................................................................................................................................82 References .........................................................................................................................................................................82 Appendix 1 Principles and standard conditions for different purification techniques ...........................................83 Affinity chromatography (AC) ...................................................................................................................................83 Ion exchange chromatography (IEX) ....................................................................................................................84 Hydrophobic interaction chromatography (HIC) ............................................................................................86 Gel filtration (GF) chromatography ........................................................................................................................87 Reversed phase chromatography (RPC) .............................................................................................................88 Appendix 2 Manual and automated purification .....................................................................................................................89 Tagged recombinant proteins for simple purification ..................................................................................89 Manual purification techniques ...............................................................................................................................89 Automated purification using ÄKTAdesign chromatography systems ................................................90 Appendix 3 Column packing and preparation .........................................................................................................................93 Column selection ............................................................................................................................................................95 Appendix 4 Conversion data: proteins, column pressures .......................................................................................................... 96 Column pressures ..........................................................................................................................................................96 Appendix 5 ...................................................................................................................................................................97 Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa .....................97 Appendix 6 Amino acids table ...........................................................................................................................................................98 Related literature .............................................................................................................................................