Elsevier S & T

Phage Display of Peptides and Proteins: A Laboratory Manual

Kay, Brian K.; Winter, Jill; McCafferty, John

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Table of contents

Cover
Contentsv
Contributorsxiii
Forewordxvii
Prefacexxi
Chapter 1. Biology of the Filamentous Bacteriophage1
Introduction to the Life Cycle of the Bacteriophage1
The Genome and Its Products4
The Ff Bacteriophage Particle9
The Infection Process11
The Assembly Process13
References16
Chapter 2. Principles and Applications of Phage Display21
Introduction21
Phage Display of Natural Peptides22
Phage Display of Random Peptides23
Phage Display of Protein and Protein Domains25
Conclusion28
World Wide Web Page28
References29
Chapter 3. Vectors for Phage Display35
Phagemid38
Markers45
Other Viral Display Vectors49
Proteolysis49
Valency50
Issue of Biological Selection51
References52
Chapter 4. Microbiological Methods55
Protocol 1: Spreading Techniques55
Protocol 2: Titering Bacteriophage57
Protocol 3: Preparing Phage Particles62
Protocol 4: Preparation of Single-Stranded DNA from Viral Particles63
Protocol 5: Preparation of Vector RF DNA64
References65
Chapter 5. Construction of Random Peptide Libraries in Bacteriophage MI367
Introduction67
Protocol 1: Assembly of Double-Stranded DNA Insert from Degenerate Oligonucleotides69
Protocol 2: Preparation of Linearized Vector DNA72
Protocol 3: Ligation of the Vector and Insert DNA74
Protocol 4: Electroporation of Ligated DNA into Bacteria75
Protocol 5: Amplification and Harvest of Library Phage77
References77
Chapter 6. Construction and Screening of Antibody Display Libraries79
General Introduction and Background79
Production of Antibody Genes and Preparation of Libraries81
Protocol 1: Preparation of Lymphocytes as Single-Cell Suspensions from Mouse Spleen Cells83
Protocol 2: Preparation of Human Peripheral Blood Lymphocytes83
Protocol 3: Preparation of Mouse or Human Antibody cDNA84
Construction of Antibody V Gene Repertoire85
Protocol 4: Preparation and Purification of Primary VH and VL PCR Products90
Protocol 5: Preparation and Purification of Linker Fragments92
Protocol 6: Assembly of Human Single-Chain Fv Antibody Fragments93
Protocol 7: Restriction Digestion of Assembled PCR Fragments95
Protocol 8: Preparation of Vector DNA96
Protocol 9: Ligation of Insert into Vector97
Manipulation of Libraries98
Protocol 10: Rescuing Phagemid Libraries99
Protocol 11: Selection of Phage-Antibody Libraries by Panning in "Immunotubes"99
Protocol 12: Selection of Phage-Antibody Libraries by Biotin Selection101
Protocol 13: Introducing Diversity by Chain Shuffling103
Screening and Expression of Selected Clones105
Protocol 14: Induction of Soluble Antibody Fragments from Phagemid Vectors106
Protocol 15: Preparation of Periplasmic Fraction and Purification of Antibodies by Immobilized Metal107
Protocol 16: Small-Scale Rescue of Phagemid Particles Using Helper Virus108
Protocol 17: ELISA Using either Soluble Fragments or Phage Particles109
References110
Chapter 7. Phagemid-Displayed Peptide Libraries113
Introduction113
Materials114
Construction of Phagemid Vector pIll.PEP115
Protocol 1: Generation of the Two Fragments Needed to Construct the Gene III Minigene115
Protocol 2: Construction of the Gene III Fusion Gene by Splice Overlap Extension (SOE) and PCR Ampli117
Protocol 3: Introduction of NotI Linker in Place of the Gene III Spacer117
Preparation of Phagemid Peptide Libraries118
Protocol 1: Synthesis and Purification of Random Oligonucleotides for Library Synthesis119
Protocol 2: Preparation and Purification of Template ssDNA119
Protocol 3: Preparation and Amplification of Phagemid Libraries121
Protocol 4: Preparation of Electrocompetent Cells122
Protocol 5a: Panning Libraries on mAb Using Acid Elution123
Protocol 5b: Panning Phagemid Libraries by Competitive Elution123
Troubleshooting Phagemid Peptide Library Synthesis124
References125
Chapter 8. Multiple Display of Foreign Peptide Epitopes on Filamentous Bacteriophage Virions127
Introduction127
Vectors128
Protocol 1: Cloning Protocols132
Protocol 2: PCR Protocols133
Protocol 3: Preparation and Purification of Bacteriophages134
Analysis of Bacteriophage Particles136
Immunization and Antibody Preparation138
Concluding Remarks138
References139
Chapter 9. Phage Libraries Displaying Random Peptides Derived from aTarget Sequence141
Introduction141
Protocol 1: Fragmentation of the Target Gene143
Protocol 2: Modifying Termini of DNase-Generated Fragments for Cloning144
Protocol 3: Final Size Selection and Removal of Linkers145
Protocol 4: Ligation, Electroporation, and Harvesting the Library146
Protocol 5: Characterization of the Library147
Protocol 6: Affinity Selection of Phage-Displayed Peptides148
References149
Chapter 10. Display and Selection of Proteins on Genetic Packages151
Introduction151
Summary of the DSPGP Method152
Nomenclature155
General Considerations on Proteins157
Reasons to Use (Nonantibody) Proteins as Binding Molecules158
Choice of Protein Domain to Be Displayed161
Designing a Display Genetic Package167
Variegation of Displayed Proteins177
Selection of Binding Domainsm„Procedures181
Selection of Binding Domainsm„Examples: BPTI Derivatives Binding hNE187
Troubleshooting188
Conclusions189
References189
Chapter 11. A cDNA Cloning System Based on Filamentous Phage: Selection and Enrichment of Functional195
Introduction and Rationale195
The pJuFo Vector196
Considerations for cDNA Cloning into pJuFo and Preparation of Phage Particles199
Protocols199
Screening of Phage205
Practical Aspects of Screening Phage Expressed by pJuFo System209
Applications of the pJuFo Vector209
Example of a Positive Control System211
Potential and Limitations of the pJuFo System212
References212
Chapter 12. Multicombinatorial Libraries and Combinatorial Infection: Practical Considerations for V215
Introduction and Rationale215
The ATT System217
Full-Scale Library Constructionm„Optimization of the System221
References225
Chapter 13. Screening Phage-Displayed Random Peptide Libraries227
Introduction227
General Considerations228
Protocol 1: Affinity Purification with Target Immobilized on ELISA Plates232
Protocol 2: Affinity Purification with Target Immobilized on Paramagnetic Beads235
Protocol 3: Affinity Purification by Capture of Biotinylated Target:Phage Complexes238
Protocol 4: Isolation of Affinity-Purified Phage Clones243
Protocol 5: Propagation of Individual Phage Clones245
Protocol 6: Rapid Confirmation of Binding Activity of Affinity-Purified Phage Clones by ELISA246
Protocol 7: Quantitative Characterization of Binding Activity of Phage Clones by Titering248
Protocol 8: Identification of Binding Clones by Filter Lifts250
References252
Chapter 14. Substrate Phage255
A Suitable Library of Random Peptides Displayed on the Surface of Phage Particles255
A Method for in Vitro Modification of Peptide Substrates257
A Method for Separating Modified Peptides from Unmodified Peptides257
Caveats, Troubleshooting, and Hints258
References259
Chapter 15. Phage Display: Factors Affecting Panning Efficiency261
Introduction261
Variations in Display Level262
Effect of Affinity on Panning Efficiency264
Effect of Affinity and Display Level on Discrimination between Binders270
Selection with Soluble Antigen: Theoretical Aspects271
Conclusion274
References275
Chapter 16. Preparation of Second-Generation Phage Libraries277
Oligonucleotide-Mediated Site-Directed Mutagenesis280
Cassette Mutagenesis285
Error-Prone PCR286
DNA Shuffling287
Conclusion289
References290
Chaper 17. Nonradioactive Sequencing of Random Peptide Recombinant Phage293
Introduction293
Amplification of Phage DNA296
Sequencing of Amplified DNA297
Chemiluminescent Detection298
Troubleshooting Guide303
References303
Chaper 18. Measurement of Peptide Binding Affinities Using Fluorescence Polarization305
Introduction to Fluorescence Polarization305
Selection of a Fluorescence Polarization Instrument307
Time Required to Determine Peptide Binding Constants307
Labeling Peptides with Fluorescein309
Analysis and Purification of Labeled Peptides Using Thin-Layer Chromatography314
Peptide Elution316
Quenching317
Binding of cdc2Hs Peptide to Monoclonal Anti-cdc2Hs317
Results319
Discussion322
A Common Pitfall in Fluorescence Polarization Equilibrium Binding Experiments323
Competition Experiments324
References326
Appendix327
Index339
Color Plate SectionColor Plat

Book details

Vendor Elsevier S & T
SKU 9780124023802
ISBN-13 9780080538709
Author Kay, Brian K.; Winter, Jill; McCafferty, John
Category Science
Subject Biotechnology

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Both novices and experts will benefit from this insightful step-by-step discussion of phage display protocols.
Phage Display of Peptides and Proteins: A Laboratory Manual reviews the literature and outlines the strategies for maximizing the successful application of phage display technology to one's research. It contains the most up-to-date protocols for preparing peptide affinity reagents, monclonal antibodies, and evolved proteins.

Key Features
* Prepared by experts in the field
* Provides proven laboratory protocols, troubleshooting, and tips
* Includes maps, sequences, and sample data
* Contains extensive and up-to-date references

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