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基本説明
A compendium of cutting-edge protocols for the generation of molecular diversity. An accompanying volume, Directed Enzyme Ecolution: (ISBN: 1-58829-286-X), is devoted entirely to selection and screening methods that can be applied to the directed evolution of enzymes.
Full Description
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 networks.
Contents
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.
