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 conditions.
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Product details

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

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 applications.
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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 DegreesC
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

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Review quote

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