Phage display : a practical approach

Phage display : a practical approach / edited by Tim Clackson, Henry B. Lowman.

This new book aims to enable researchers to design and undertake all aspects of a phage display project, from designing an experimental strategy and constructing a library to performing selections and analyzing the results. All of the protocols and chapters are extensively cross-referenced, allowing...

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Bibliographic Details
Other Authors: Clackson, Tim, Lowman, Henry B.
Format: eBook
Language:English
Published: Oxford ; New York : Oxford University Press, ©2004.
Series:Practical approach series ; 266.
Subjects:
Bacteriophages.
Viral proteins.
Microbial biotechnology.
SCIENCE > Life Sciences > Biology.
SCIENCE > Life Sciences > Microbiology.
Bacteriophages
Microbial biotechnology
Viral proteins
Online Access:Click for online access
Table of Contents:
  • Cover
  • Contents
  • Protocol list
  • List of abbreviations
  • List of contributors
  • 1 Introduction to phage biology and phage display
  • 1 Introduction
  • 2 Biology of filamentous phage
  • 2.1 Introduction
  • 2.2 Structure of the phage particle
  • 2.3 Infection
  • 2.4 Replication
  • 2.5 Genes and gene expression
  • 2.6 Physiology of phage assembly
  • 2.7 The mechanics of phage assembly
  • 3 Coat proteins used for display
  • 3.1 pVIII
  • 3.2 pIll
  • 4 Starting a phage display project
  • 4.1 Feasibility of display
  • 4.2 Phage or phagemid vector?
  • 4.3 Polyvalent or monovalent display?
  • 4.4 Helper phage
  • 4.5 General protocols for phage preparation and quantitation
  • 5 General principles of a phage display project
  • 5.1 Making a library
  • 5.2 Selection
  • 5.3 Analysis of clones
  • 6 Common problems
  • 6.1 Library quality
  • 6.2 Expression editing
  • 6.3 Over-selection
  • 7 Alternative display systems
  • 8 Commercial sources of phage display libraries and kits
  • References
  • 2 Constructing phage display libraries by oligonucleotide-directed mutagenesis
  • 1 Introduction
  • 2 Considerations for library design
  • 2.1 Site-directed mutagenesis
  • 2.2 Degenerate codon design
  • 2.3 Theoretical versus actual diversity
  • 3 Oligonucleotide-directed mutagenesis
  • 3.1 Oligonucleotide-directed mutagenesis versus cassette mutagenesis
  • 3.2 The chemistry and biology of oligonucleotide-directed mutagenesis
  • 3.3 Construction of an inactive template
  • 4 Library construction and storage
  • 4.1 Preparation of single-stranded DNA template
  • 4.2 In vitro synthesis of heteroduplex CCC-dsDNA
  • 4.3 E.coli electroporation and production of library phage
  • 4.4 Library storage and reinfection
  • 5 Biological reagents
  • References
  • 3 In vitro DNA recombination
  • 1 Introduction
  • 2 Background to in vitro DNA recombination
  • 2.1 Use of in vitro DNA recombination in directed evolution
  • 2.2 Applications of in vitro DNA recombination
  • 2.3 Recombination statistics
  • 3 Methods for in vitro DNA recombination
  • 3.1 Stemmer method
  • 3.2 Random DNA fragmentation with endonuclease V from E. coli
  • 3.3 Random priming recombination
  • 3.4 Staggered Extension Process (StEP)
  • 3.5 In vitro heteroduplex formation and in vivo repair (heteroduplex recombination)
  • 3.6 Choice of recombination method
  • 3.7 Removal of background
  • 3.8 Technical tips
  • References
  • 4 Phage selection strategies for improved affinity and specificity of proteins and peptides
  • 1 Introduction
  • 2 Vector considerations
  • 2.1 Monovalent and polyvalent phage display
  • 2.2 Confirming display
  • 2.3 Protein expression from phagemid vectors
  • 2.4 Vector construction and phagemid preparation
  • 3 Library design
  • 3.1 Hard randomization
  • 3.2 Soft randomization
  • 4 Target presentation
  • 4.1 Direct immobilization
  • 4.2 Solution binding
  • 4.3 Blocking
  • 4.4 Pilot selection
  • 5 Selection
  • 5.1 Binding buffer considerations
  • 5.2 Stringency of selection
  • 5.3 Competitive selection
  • 5.4 Elution of bound phage
  • 5.5 Amplification
  • 5.6 Monitori.

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