Directed Enzyme Evolution

Directed Enzyme Evolution : Screening and Selection Methods

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Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum "you get what you screen for. " The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective more

Product details

  • Hardback | 383 pages
  • 157.5 x 236.2 x 27.9mm | 771.12g
  • Humana Press Inc.
  • Totowa, NJ, United States
  • English
  • 2003 ed.
  • biography
  • 158829286X
  • 9781588292865

Review quote

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

Back cover copy

Directed evolution, the application of evolutionary design to enzyme engineering, requires effective screening strategies to isolate those proteins that perform a desired function from the libraries generated by the techniques. In Directed Enzyme Evolution: Screening and Selection Methods, seasoned practitioners from many leading laboratories describe their leading and 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. There are also representative examples of how phage libraries may be interrogated for enzymatic activity. Each protocol contains detailed step-by-step instructions and many notes on how best to deal with the problems that may occur. 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. Taken together, Directed Enzyme Evolution: Screening and Selection Methods and Directed Evolution Library Creation: Methods and Protocols 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. Genetic Selections Genetic Complementation Protocols Jessica L. Sneeden and Lawrence A. Loeb Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase Manel Camps and Lawrence A. Loeb Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast Ranga N. Venkatesan and Lawrence A. Loeb Autogene Selections Jijumon Chelliserrykattil and Andrew D. Ellington Selection for Soluble Proteins via Fusion with Chloramphenicol Acetyltransferase Volker Sieber Proside: A Phage-Based Method for Selecting Thermostable Proteins Andreas Martin, Franz X. Schmid, and Volker Sieber Minimization of Proteins by Random Fragmentation and Selection Gary W. Rudgers and Timothy Palzkill Part II. Screens for Enzymes Evaluating a Screen and Analysis of Mutant Libraries Oriana Salazar and Lianhong Sun Screening Mutant Libraries in Saccharomyces cerevisiae Thomas Bulter, Volker Sieber, and Miguel Alcalde Solid-Phase Screening Using Digital Image Analysis Alexander V. Tobias and John M. Joern Screening for Thermostability Patrick C. Cirino and Radu Georgescu High-Throughput Screening of Mutant a-Amylase Libraries for Increased Activity at 129 C Holger Berk and Robert J. Lebbink High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450 Christopher R. Otey High-Throughput Screen for Aromatic Hydroxylation Christopher R. Otey and John M. Joern Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes Edgardo T. Farinas High-Throughput Screens Based on NAD(P)H Depletion Anton Glieder and Peter Meinhold High-Throughput Tetramethylbenzidine (TMB) Screen for Peroxidases Radu Georgescu Screenfor Oxidases by Detection of Hydrogen Peroxide with Horseradish Peroxidase Lianhong Sun and Makoto Yagasaki Colorimetric Dehydrogenase Screen Based on NAD(P)H Generation Kimberly M. Mayer Colorimetric Assays for Screening Laccases Miguel Alcalde and Thomas Bulter pH Sensing Agar Plate Assays for Esterolytic Enzyme Activity Karl E. Griswold A pH-Indicator-Based Screen for Hydrolytic Haloalkane Dehalogenase Huimin Zhao Detection of Aromatic a-Hydroxyketones with Tetrazolium Salts Michael Breuer and Bernhard Hauer Selection of Heat-Stable Clostridium cellulovorans Cellulases After In Vitro Recombination Koichiro Murashima and Roy H. Doi Screening and Selection Strategies for Disulfide Isomerase Activity Ronald Lafond, Xiaoming Zhan, and George Georgiou An Overview of High-Throughput Screening Systems for Enantioselective Enzymatic Transformations Manfred T. Reetz Select Protocols of High-Throughput ee-Screening Systems for Assaying Enantioselective Enzymes Manfred T. Reetz Directed Evolution of the Substrate Specificities of a Site-Specific Recombinase and an Aminoacyl-tRNA Synthetase Using Fluorescence-Activated Cell Sorting (FACS) Stephen W. Santoro and Peter G. Schultz Calmodulin-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities Christian Heinis, Julian Bertschinger, and Dario Neri High-Throughput FACS Method for Directed Evolution of Substrate Specificity Mark J. Olsen, Jongsik Gam, Brent L. Iverson, and George Georgiou Improving Protein Folding Efficiency by Directed Evolution Using the GFP Folding Reporter Geoffrey S. Waldo Indexshow more