Directed Evolution Library Creation (eBook, PDF)

Methods and Protocols
Redaktion: Arnold, Frances H.; Georgiou, George

• Format: PDF

Produktbeschreibung

Directed protein evolution-a powerful technique for the discovery of new enzymes and therapeutic proteins-has matured to the point that it is now the centerpiece of enzyme engineering, where it has catalyzed the development of numerous experimental methods. In Directed Evolution Library Creation: Methods and Protocols, seasoned practitioners from many leading laboratories share their best experimental protocols for the generation of molecular diversity. Described in step-by-step detail to ensure experimental success, these protocols include readily reproducible methods for random mutagenesis of entire genes or segments of genes, for homologous and nonhomologous recombination, and for constructing in vivo libraries in bacteria and yeast. In addition to the various protocols for creating libraries, this volume also describes ways to analyze libraries, particularly those made by recombination. An accompanying volume, Directed Enzyme Evolution: Screening and Selection Methods (ISBN: 1-58829-286-X), is devoted entirely to selection and screening methods that can be applied to the directed evolution of enzymes. Taken together, Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods capture for newcomers and more experienced investigators alike all the key methods for using directed protein evolution to better understand protein structure-function relationships, to discover new enzymes and therapeutic proteins, and to design new assays suitable for specific applications.

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Produktdetails

• Verlag: Humana Press
• Seitenzahl: 226
• Erscheinungstermin: 3. Februar 2008
• Englisch
• ISBN-13: 9781592593958
• Artikelnr.: 64701465

Inhaltsangabe

Mutagenesis and Recombination Methods.- Generating Mutant Libraries Using Error-Prone PCR.- Preparing Libraries in Escherichia coli.- Preparing Libraries in Saccharomyces cerevisiae.- Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP).- Construction of Designed Protein Libraries Using Gene Assembly Mutagenesis.- Production of Randomly Mutated Plasmid Libraries Using Mutator Strains.- Evolution of Microorganisms Using Mutator Plasmids.- Random Insertion and Deletion Mutagenesis.- Random Oligonucleotide Mutagenesis.- Saturation Mutagenesis.- DNA Shuffling.- Family Shuffling with Single-Stranded DNA.- In Vitro DNA Recombination by Random Priming.- Staggered Extension Process (StEP) In Vitro Recombination.- RACHITT.- The Creation of ITCHY Hybrid Protein Libraries.- Preparation of SCRATCHY Hybrid Protein Libraries.- Sequence Homology-Independent Protein Recombination (SHIPREC).- Producing Chimeric Genes by CLERY.- Analysis of Library Diversity.- Analysis of Shuffled Libraries by Oligonucleotide Probe Hybridization.- Sequence Mapping of Combinatorial Libraries on Macro- or Microarrays.- Sequence Mapping of Combinatorial Libraries on Macro- and Microarrays.