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

Published Oxford ; New York : Oxford University Press, ©2004

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Description 1 online resource (xxiv, 332 pages) : illustrations Series Practical approach series ; no. 266 Practical approach series ; 266. 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 Summary 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 Bibliography Includes bibliographical references and index Notes Print version record Subject Bacteriophages. Viral proteins. Microbial biotechnology. Peptide Library Viral Proteins Bacteriophages SCIENCE -- Life Sciences -- Biology. SCIENCE -- Life Sciences -- Microbiology. Bacteriophages Microbial biotechnology Viral proteins Form Electronic book Author Clackson, Tim. Lowman, Henry B. LC no. 2004299783 ISBN 9780191516450 0191516457 9786610845293 6610845298 9780199638734 019963873X

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