Overview

Seasoned practitioners from many leading laboratories describe their best readily reproducible screening strategies for isolating useful clones. These techniques have been optimized for sensitivity, high throughput, and robustness, and are of proven utility for directed evolution purposes. The assays presented use a variety of techniques, including genetic complementation, microtiter plates, solid-phase screens with colorimetric substrates, and flow cytometric screens. An accompanying volume, Directed Evolution Library Creation: Methods and Protocols (ISBN 1-58829-285-1), describes readily reproducible methods for the creation of mutated DNA molecules and DNA libraries. Copy for Both Volumes Directed Evolution Library Creation: Methods and Protocols and Directed Enzyme Evolution: Screening and Selection Methods constitute an extraordinary collection of all the key methods used today for directed evolution research. Described in step-by-step detail to ensure robust experimental results, these methods will enable both newcomers and more experienced investigators to design and implement directed evolution strategies for the engineering of novel proteins. The first volume describes methods for the creation of mutated DNA molecules, or DNA libraries, encoding variants of desired proteins. The second volume describes methods for screening DNA libraries to isolate mutant proteins that exhibit a specified function.

Full Product Details

Author:   Frances H. Arnold ,  George Georgiou
Publisher:   Humana Press Inc.
Imprint:   Humana Press Inc.
Edition:   2003 ed.
Volume:   230
Dimensions:   Width: 15.20cm , Height: 3.10cm , Length: 22.90cm
Weight:   0.844kg
ISBN:  

9781588292865

ISBN 10:   158829286
Pages:   383
Publication Date:   16 May 2003
Audience:   College/higher education ,  Professional and scholarly ,  Undergraduate ,  Professional & Vocational
Format:   Hardback
Publisher's Status:   Active
Availability:   In Print  
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Table of Contents

Genetic Selections.- Genetic Complementation Protocols.- Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase.- Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast.- Autogene Selections.- Selection for Soluble Proteins via Fusion with Chloramphenicol Acetyltransferase.- Proside.- Minimization of Proteins by Random Fragmentation and Selection.- Screens for Enzymes.- Evaluating a Screen and Analysis of Mutant Libraries.- Screening Mutant Libraries in Saccharomyces cerevisiae.- Solid-Phase Screening Using Digital Image Analysis.- Screening for Thermostability.- High-Throughput Screening of Mutant ?-Amylase Libraries for Increased Activity at 129°C.- High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450.- High-Throughput Screen for Aromatic Hydroxylation.- Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes.- High-Throughput Screens Based on NAD(P)H Depletion.- High-Throughput Tetramethylbenzidine (TMB) Screen for Peroxidases.- Screen for Oxidases by Detection of Hydrogen Peroxide with Horseradish Peroxidase.- Colorimetric Dehydrogenase Screen Based on NAD(P)H Generation.- Colorimetric Assays for Screening Laccases.- pH Sensing Agar Plate Assays for Esterolytic Enzyme Activity.- A pH-Indicator-Based Screen for Hydrolytic Haloalkane Dehalogenase.- Detection of Aromatic ?-Hydroxyketones with Tetrazolium Salts.- Selection of Heat-Stable Clostridium cellulovorans Cellulases After In Vitro Recombination.- Screening and Selection Strategies for Disulfide Isomerase Activity.- An Overview of High-Throughput Screening Systems for Enantioselective Enzymatic Transformations.- Select Protocols of High-Throughput ee-Screening Systems forAssaying Enantioselective Enzymes.- Directed Evolution of the Substrate Specificities of a Site-Specific Recombinase and an Aminoacyl-tRNA Synthetase Using Fluorescence-Activated Cell Sorting (FACS).- Calmodulin-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities.- High-Throughput FACS Method for Directed Evolution of Substrate Specificity.- Improving Protein Folding Efficiency by Directed Evolution Using the GFP Folding Reporter.

Reviews

"""...covers a considerable number of protocols for a broad range of enzymes...very useful..."" - ChemBioChem"

...covers a considerable number of protocols for a broad range of enzymes...very useful... - ChemBioChem

covers a considerable number of protocols for a broad range of enzymes very useful ChemBioChem

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