Protein Chromatography Methods and Protocols

Preface......Page 5
Contents......Page 7
Contributors......Page 9
Part I: Overview Chapters......Page 11
 Chapter 1: A Synopsis of Proteins and Their Purification......Page 12
  References......Page 23
  1 Introduction......Page 24
   2.1 Matrix Choice......Page 25
    2.2.1 Agarose-Based Support Media......Page 27
    2.2.3 Hydroxylated Meth-Acrylic Polymer Resins......Page 28
  3 Applications of Gel-Filtration Chromatography......Page 29
   3.1 Separation of Proteins and Peptides......Page 30
   3.4 Virus Particles......Page 31
   3.6 Absolute Size-Exclusion Chromato-graphy (ASEC)......Page 32
  References......Page 33
   1.1 Emergence and Evolution of Immunoaffinity Chromatography......Page 35
   1.2 Antibody: Structure and Production......Page 36
  2 Affinity Ligands......Page 38
  3 Matrices......Page 43
   3.1 Immobilization Chemistries......Page 45
  4 Elution Conditions......Page 47
  5 Characterization......Page 49
   5.1 Applications......Page 50
  6 Conclusion......Page 52
  References......Page 53
  1 Introduction......Page 60
  2 Protease Classification......Page 61
    3.2.2 Targeting of Expressed Protein......Page 63
    3.2.4 Use of Fusion Proteins......Page 64
    3.3.6 Alternative Approaches......Page 65
   4.1 Protease Inhibitor Selection and Preparation......Page 67
    4.3.1 Additional Inhibitors......Page 68
   4.4 Supplementary Chemical Compounds Including Enzymes......Page 69
   4.5 Protease Inhibition During Chromato-graphy......Page 70
   5.2 Post-­chromatographic Analysis......Page 72
  6 Conclusion......Page 73
  References......Page 74
  1 Introduction......Page 77
   1.1 Protein Purification Using Chromatography......Page 79
   1.2 Scale-Up of Protein Chromatography......Page 81
   2.1 Chromato graphy Media......Page 83
   2.2 Column Packing at Large Scale......Page 84
   2.3 Cleaning of Chromatography Media......Page 86
   2.4 Production Equipment......Page 87
   2.5 Non-­chromatographic Factors Applicable at Large Scale......Page 88
  References......Page 89
  1 Introduction......Page 91
   1.1 Historical Difficulties in Antibody Generation Technology......Page 92
  2 Display Technologies as an Alternative Source of Specific Antibodies......Page 93
   2.2 Why Is Phage Display Not More Heavily Used?......Page 96
  3 Why Chickens (Gallus gallus)?......Page 97
   3.1 Harnessing the Chicken Immune Response via Phage Display......Page 99
  4 Toward Affinity Chromatography with Antibody Fragments......Page 101
  References......Page 103
   1.1 The Vital Importance of Protein Stability......Page 106
   1.2 Definition and Measurement of Protein Stability......Page 107
   1.4 A Stability-­Activity Trade-Off?......Page 109
    1.5.1 Rational Mutations......Page 111
    1.5.2 Directed Evolution......Page 112
    1.5.3 Inference and Prediction......Page 113
   1.7 Conclusion......Page 117
  2 Materials......Page 122
   3.2 Thermal Inactivation......Page 123
   3.3 Measurement of Oxidative Stability......Page 124
   3.4 Chemical Modification of a Protein......Page 125
  4 Notes......Page 127
  References......Page 129
  1 Introduction......Page 135
   2.2 Glutathione S-Transferase......Page 137
   2.3 Small Ubiquitin-­Related Modifier......Page 138
   2.4 Other Solubility-­Enhancing Fusion Partners......Page 140
   3.1 Immobilized Metal Affinity Chromatography......Page 141
   3.2 Other Affinity Tags......Page 144
   3.3 TAP-Tagging......Page 146
  4 Tagging Proteins and Structural Studies......Page 147
  5 Concluding Remarks......Page 153
  References......Page 154
Part II: Protocols Chapters......Page 161
  1 Introduction......Page 162
  2 Materials and Equipment......Page 165
   3.2 Avoidance of Proteolysis......Page 167
   3.3 Use of Stabilizing Additives......Page 168
    3.3.1 Addition of Salts......Page 169
    3.3.3 Substrates and Specific Ligands......Page 170
    3.3.5 Extremely Dilute Solutions......Page 171
   3.4 Low-­Temperature Storage......Page 172
    3.6.1 Start Up......Page 173
    3.6.2 Filling and Loading......Page 174
    3.6.3 The Freeze-Drying Operation......Page 175
    3.6.4 Termination of Run and Removal of Product......Page 176
    3.6.6 Using Simpler Freeze-Dryers......Page 177
   3.7 Lyophilization: Freezing and Annealing......Page 178
   3.8 Lyophilization: Primary Drying......Page 180
   3.9 Lyophilization: Secondary Drying......Page 181
   3.11 Modulated Differential Scanning Calorimetry (mDSC) as an Aid to Lyo philization......Page 182
   3.12 Stability Analysis and Accelerated Degradation Testing......Page 184
  4 Notes......Page 185
  References......Page 191
  1 Introduction......Page 194
   2.1 Recombinant/Native Protein Extraction......Page 195
   2.5 Protein Solubilization......Page 196
   3.1 Computational Prediction of Protein Solubility......Page 197
   3.3 Recombinant/Native Protein Extraction......Page 198
   3.5 Protein Precipitation Using Trichloroacetic Acid......Page 199
   3.7 Protein Solubilization......Page 200
    3.7.1 Protein Solubilization Detergent Screen......Page 201
  4 Notes......Page 202
  References......Page 210
  1 Introduction......Page 212
   1.1 Basic Principles of Ion-Exchange Chromatography......Page 213
    1.2.1 Selection of Ion-­Exchange Matrix......Page 215
    1.2.2 Selection of Buffer Conditions......Page 216
    1.2.4 Selection of Elution Format......Page 219
   2.4 Assay of Post-­DEAE Fractions......Page 220
   3.2 Ammonium Sulfate Precipitation......Page 221
   3.4 Assay of Post-­DEAE Fractions......Page 222
  4 Notes......Page 223
  References......Page 225
  1 Introduction......Page 227
   2.5 The Lowry Protein Assay......Page 230
   2.7 Immunoassay: Indirect ELISA......Page 231
   2.8 Sodium Dodecyl Sulfate-­Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins......Page 232
   2.10 Western Blotting......Page 233
    2.11.1 Preparation of Biotinylated DNA Label......Page 234
    2.11.5 Assay Format I (Additional Components)......Page 235
   3.1 Protein Determination by UV Light Absorption......Page 236
   3.2 Preparation of a Standard Curve......Page 237
   3.4 The Bradford Protein Assay......Page 238
   3.5 The Lowry Protein Assay......Page 239
   3.6 The Bicinchoninic Acid (BCA) Assay......Page 240
    3.7.1 Antigen Coating to Microtiter Plates......Page 241
   3.8 Sodium Dodecyl Sulfate-­Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins......Page 242
   3.9 Staining of SDS-­PAGE Gels......Page 243
    3.9.2 Silver Staining of SDS-PAGE Gels......Page 244
   3.10 Western Blotting......Page 245
    3.10.2 Staining of Proteins Immobilized on Nitrocellulose Filters......Page 246
   3.11 qIPCR Assay......Page 247
    3.11.2 Preparation of Antibody/DNA Label Conjugate......Page 248
    3.11.4 qIPCR Assay Set Up......Page 249
    3.11.6 Assay Format II......Page 250
    3.11.9 Real-Time PCR (qPCR) Assay......Page 251
  4 Notes......Page 252
  References......Page 256
  1 Introduction......Page 258
    2.2.1 Lysis and Clarification......Page 260
    2.2.6 Column Regeneration and Storage......Page 261
    3.2.2 Capture......Page 262
    3.2.4 Protein Characterization......Page 264
    3.2.6 Column Regeneration and Storage......Page 266
  4 Notes......Page 267
  References......Page 273
  1 Introduction......Page 275
  2 Materials......Page 281
    2.1.2 SDS-­Polyacrylamide Gel Electrophoresis of Proteins......Page 282
    2.1.3 Coomassie Blue Staining......Page 283
    2.3.1 Denaturing Solutions......Page 284
    2.3.2 Batch Purification......Page 285
  3 Methods......Page 286
   3.1 High-Level Expression of Recombinant His-Tagged Proteins......Page 287
    3.1.1 Protein Analysis......Page 288
    3.1.4 Western Blotting......Page 289
     Immunological Probing......Page 290
   3.2 Determination of Protein Solubility......Page 291
     Under Native Conditions......Page 293
   3.4 Downstream Processes; His-Tag Removal by Proteolytic Cleavage and Protein Re-folding......Page 295
   3.5 Large-Scale Growth for Preparative Purification of Recombinant Protein......Page 296
  4 Notes......Page 297
  References......Page 301
  1 Introduction......Page 304
   1.2 Protein G......Page 305
   1.4 Protein A/G......Page 307
    2.1.1 Ammonium Sulfate Precipitation......Page 308
    2.2.1 Isotyping of Monoclonal Antibodies......Page 309
    3.1.2 Protein A Chromatography......Page 310
    3.2.3 Iso-strip Lateral Flow Assay Kit......Page 312
    3.2.4 Ammonium Sulfate Precipitation of Monoclonal Antibodies for Protein G Purification......Page 313
    3.2.6 Protein L Affinity Chromatography......Page 314
  4 Notes......Page 315
  References......Page 316
  1 Introduction......Page 318
   2.3 Phage Library Generation......Page 319
   2.5 Output Screening......Page 320
  3 Methods......Page 321
   3.2 cDNA Preparation from Spleen and Bone Marrow......Page 322
   3.4 PCR Assembly of Chicken scFv Libraries......Page 323
   3.5 Cloning the Chicken scFv-Phagemid Library......Page 325
   3.6 Protocol for the Rescue of scFv-­Phagemid Libraries......Page 327
   3.7 Phage Selection Protocol......Page 328
   3.8 Screening of Output Clones......Page 333
  4 Notes......Page 334
  References......Page 336
  1 Introduction......Page 338
   2.5 Kinetic Studies and Data Analysis......Page 340
   3.1 Instrument Cleaning and Surface Preparation......Page 341
   3.3 Immobilization......Page 342
    3.4.1 Regeneration Scouting......Page 344
    3.4.2 Surface Performance Test......Page 345
   3.5 Kinetic Studies and Data Analysis......Page 346
  4 Notes......Page 347
  References......Page 351
  1 Introduction......Page 354
   1.1 Automated HIC Column Selection......Page 356
   3.1 Typical Methodology......Page 358
  4 Notes......Page 359
  References......Page 362
  1 Introduction......Page 363
   3.1 Sample Preparation......Page 365
    3.2.1 Ion-Exchange FPLC......Page 366
    3.2.2 Scouting FPLC Methods......Page 367
   3.3 Automated AKTA FPLC......Page 368
  4 Notes......Page 369
  References......Page 371
  1 Introduction......Page 372
   2.3 Sample Preparation from 1D PAGE Gels (In-Gel Digestion)......Page 374
   2.6 Two-Dimensional Strong Cation Exchange—Reverse-Phase Chromatography......Page 375
   3.1 Immuno­depletion of Serum Sample......Page 376
   3.2 Protein In-Gel Digestion Protocol......Page 378
   3.3 One-Dimensional Reverse-Phase Chromatography Using a 60 min Separation Time......Page 379
   3.5 Two-Dimensional Strong Cation Exchange (SCX) Reverse-Phase Chromatography (RPC) for Complex Protein Mixtures......Page 381
  4 Notes......Page 383
  References......Page 384
  1 Introduction......Page 386
   1.1 Considerations for Membrane Protein Purification......Page 387
   1.2 Peripheral Membrane Protein Extraction......Page 388
   1.3 Integral Membrane Protein Extraction......Page 389
   1.4 Removal of Detergents from Membrane Protein Fractions......Page 391
   2.1 Fractionation of Peripheral and Integral Membrane Proteins Using High pH......Page 392
   3.2 Extraction of Membrane Proteins Using Butanol......Page 393
   3.5 Removal of Detergent with Low Micelle Size and High CMC by Dialysis......Page 394
  4 Notes......Page 395
  References......Page 397
  1 Introduction......Page 398
   2.1 Media......Page 400
    3.2.1 Lysate Preparation......Page 401
    3.2.4 Purification of Nisin by HPLC......Page 402
   3.4 Mass Spectrometry of Purified Peptide......Page 403
    3.5.1 Well Diffusion Assay......Page 404
    3.5.2 Minimum Inhibitory Concentration Assay......Page 405
  References......Page 406
  1 Introduction......Page 408
  2 Materials......Page 412
   3.1 Typical Methodology for Lectin Affinity Chromatography......Page 413
   3.2 Immobilizing a Lectin onto Cyanogen Bromide-­Activated Sepharose......Page 414
  4 Notes......Page 415
  References......Page 417
Index......Page 418