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  • 18-1142-75

    Edition AB

    Protein Amplification and Simple Purification

    The Recombinant Protein Handbook

  • Antibody Purification Handbook 18-1037-46

    The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75

    Protein Purification Handbook 18-1132-29

    Ion Exchange Chromatography Principles and Methods 18-1114-21

    Affinity Chromatography Principles and Methods 18-1022-29

    Hydrophobic Interaction Chromatography Principles and Methods 18-1020-90

    Gel Filtration Principles and Methods 18-1022-18

    Handbooks from Amersham Biosciences

    Reversed Phase Chromatography Principles and Methods 18-1134-16

    Expanded Bed Adsorption Principles and Methods 18-1124-26

    Chromatofocusing with Polybuffer and PBE 50-01-022PB

    Microcarrier cell culture Principles and Methods 18-1140-62

  • 1

    The Recombinant Protein Handbook Protein Amplification

    and Simple Purification

  • 2

    Contents

    Introduction ............................................................................................................. 5 Symbols and abbreviations .............................................................................................................. 5

    CHAPTER 1 .............................................................................................................. 6 Choice of host for protein amplification ......................................................................... 6

    Choice of vectors ......................................................................................................... 7 Vectors for non-fusion proteins ......................................................................................................... 7 Vectors for fusion proteins ............................................................................................................... 8 Choice of fusion tag ........................................................................................................................ 8

    CHAPTER 2 .............................................................................................................. 9 Protein amplification ................................................................................................... 9 Sample extraction .......................................................................................................................... 9 Troubleshooting protein amplification ............................................................................................... 9

    CHAPTER 3 ............................................................................................................ 13 GST fusion proteins ................................................................................................... 13 Amplification ............................................................................................................................... 13 Purification .................................................................................................................................. 14 Detection of GST fusion proteins .................................................................................................... 21 Purification and detection troubleshooting ...................................................................................... 28 Tag removal by enzymatic cleavage ................................................................................................. 30 PreScission Protease cleavage and purification ................................................................................ 31 Thrombin cleavage and purification ................................................................................................ 35 Factor Xa cleavage and purification ................................................................................................ 37 Removal of thrombin, Factor Xa or other serine proteases ................................................................. 39

    CHAPTER 4 ............................................................................................................ 41 (His)6 fusion proteins ................................................................................................. 41 Amplification ............................................................................................................................... 41 Purification .................................................................................................................................. 41 Detection of (His)6 fusion proteins .................................................................................................. 53 Purification and detection troubleshooting ...................................................................................... 56 Tag removal by enzymatic cleavage ................................................................................................. 58

    CHAPTER 5 ............................................................................................................ 59 Handling inclusion bodies .......................................................................................... 59 Solubilization of inclusion bodies ................................................................................................... 59 Refolding of solubilized recombinant proteins ................................................................................. 60

    CHAPTER 6 ............................................................................................................ 63 Harvesting and extraction of recombinant proteins ........................................................ 63

    CHAPTER 7 ............................................................................................................ 67 Buffer exchange and desalting of recombinant proteins ................................................. 67

    CHAPTER 8 ............................................................................................................ 71 Simple purification of other recombinant proteins ......................................................... 71 Ready to use affinity purification columns ....................................................................................... 71 Making a specific purification column ............................................................................................ 73 Purification .................................................................................................................................. 75

  • 3

    CHAPTER 9 ............................................................................................................ 77 Multi-step purification of recombinant proteins (fusion and non-fusion) .......................... 77 Selection and combination of purification techniques ....................................................................... 78

    Appendix 1 ............................................................................................................ 86 Map of the GST fusion vectors showing reading frames and main features ....................... 86

    Glutathione S-transferase (GST) .................................................................................. 87

    Appendix 2 ............................................................................................................ 88 Amino acids table ...................................................................................................... 88

    Appendix 3 ............................................................................................................ 90 Protein conversion data .............................................................................................. 90

    Appendix 4. ........................................................................................................... 90 Centrifuges, rotors and carriers for use with MicroPlex 24 .............................................. 90

    Appendix 5 ............................................................................................................ 91 Characteristics, cleaning and storage of Glutathione Sepharose ...................................... 91

    Characteristics, cleaning and storage of Chelating Sepharose ......................................... 92

    Appendix 6 ............................................................................................................ 93 Column packing and preparation ................................................................................. 93

    Appendix 7 ............................................................................................................ 95 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ...... 95

    Appendix 8 ............................................................................................................ 96 Selection of purification equipment ............................................................................. 96

    Appendix 9 ............................................................................................................ 97 Principles and standard conditions for purification techniques ....................................... 97 Affinity Chromatography (AC) ......................................................................................................... 97 Ion Exchange (IEX) ....................................................................................................................... 97 Hydrophobic Interaction Chromatography (HIC) .....................................................................

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