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

Includes bibliographical references and index.

Print version record.

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.

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 readers to move beyond the specific examples provided in order to customize the procedures for their own protein or selection system of interest. Phage Display is an up-to-date, . comprehensive and integrated experimental guide to the technique, which is essential reading for anyone currently using.

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