Directed Evolution Library Creation

Directed Evolution Library Creation : Methods and Protocols

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Biological systems are very special substrates for engineering-uniquely the products of evolution, they are easily redesigned by similar approaches. A simple algorithm of iterative cycles of diversification and selection, evolution works at all scales, from single molecules to whole ecosystems. In the little more than a decade since the first reported applications of evolutionary design to enzyme engineering, directed evolution has matured to the point where it now represents the centerpiece of industrial biocatalyst development and is being practiced by thousands of academic and industrial scientists in com- nies and universities around the world. The appeal of directed evolution is easy to understand: it is conceptually straightforward, it can be practiced without any special instrumentation and, most important, it frequently yields useful solutions, many of which are totally unanticipated. Directed evolution has r- dered protein engineering readily accessible to a broad audience of scientists and engineers who wish to tailor a myriad of protein properties, including th- mal and solvent stability, enzyme selectivity, specific activity, protease s- ceptibility, allosteric control of protein function, ligand binding, transcriptional activation, and solubility. Furthermore, the range of applications has expanded to the engineering of more complex functions such as those performed by m- tiple proteins acting in concert (in biosynthetic pathways) or as part of mac- molecular complexes and biological more

Product details

  • Hardback | 226 pages
  • 157.5 x 236.2 x 20.3mm | 544.32g
  • Humana Press Inc.
  • Totowa, NJ, United States
  • English
  • 2003.
  • biography
  • 1588292851
  • 9781588292858

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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 more

Table of contents

Part I. Mutagenesis and Recombination Methods Generating Mutant Libraries Using Error-Prone PCR Patrick C. Cirino, Kimberly M. Mayer, and Daisuke Umeno Preparing Libraries in Escherichia coli Alexander V. Tobias Preparing Libraries in Saccharomyces cerevisiae Thomas Bulter and Miguel Alcalde Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP) Kentaro Miyazaki Construction of Designed Protein Libraries Using Gene Assembly Mutagenesis Paul H. Bessette, Marco A. Mena, Annalee W. Nguyen, and Patrick S. Daugherty Production of Randomly Mutated Plasmid Libraries Using Mutator Strains Annalee W. Nguyen and Patrick S. Daugherty Evolution of Microorganisms Using Mutator Plasmids Olga Selifonova and Volker Schellenberger Random Insertion and Deletion Mutagenesis Hiroshi Murakami, Takahiro Hohsaka, and Masahiko Sisido Random Oligonucleotide Mutagenesis Jessica L. Sneeden and Lawrence A. Loeb Saturation Mutagenesis Radu Georgescu, Geethani Bandara, and Lianhong Sun DNA Shuffling John M. Joern Family Shuffling with Single-Stranded DNA Wenjuan Zha, Tongbo Zhu, and Huimin Zhao In Vitro DNA Recombination by Random Priming Olga Esteban, Ryan D. Woodyer, and Huimin Zhao Staggered Extension Process (StEP) In Vitro Recombination Anna Marie Aguinaldo and Frances H. Arnold RACHITT: Gene Family Shuffling by Random Chimeragenesis on Transient Templates Wayne M. Coco The Creation of ITCHY Hybrid Protein Libraries Marc Ostermeier and Stefan Lutz Preparation of SCRATCHY Hybrid Protein Libraries: Size- and In-Frame Selection of Nucleic Acid Sequences Stefan Lutz and Marc Ostermeier Sequence Homology-Independent Protein Recombination (SHIPREC) Andrew K. Udit, Jonathan J. Silberg,and Volker Sieber Producing Chimeric Genes by CLERY: In Vitro and In Vivo Recombination Valerie Abecassis, Denis Pompon, and Gilles Truan Part II. Analysis of Library Diversity Analysis of Shuffled Libraries by Oligonucleotide Probe Hybridization Peter Meinhold, John M. Joern, and Jonathan J. Silberg Sequence Mapping of Combinatorial Libraries on Macro- or Microarrays: Experimental Design of DNA Arrays Valerie Abecassis, Gilles Truan, Loic Jaffrelo, and Denis Pompon Sequence Mapping of Combinatorial Libraries on Macro- and Microarrays: Bioinformatic Treatment of Data Denis Pompon, Gilles Truan, and Valerie Abecassis Indexshow more

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