Directed enzyme evolution : screening and selection methods / edited by Frances H. Arnold and George Georgiou.

Format:

Book

Contributor:

Arnold, Frances Hamilton.

Georgiou, George.

Series:

Methods in molecular biology (Clifton, N.J.) ; v. 230.

Methods in molecular biology ; 230

Language:

English

Subjects (All):

Directed Molecular Evolution--methods.

Enzymes.

Evolution, Molecular.

Gene Library.

Protein Engineering--methods.

Molecular evolution--Laboratory manuals.

Molecular evolution.

Gene libraries--Laboratory manuals.

Gene libraries.

Protein engineering--Laboratory manuals.

Protein engineering.

Genetic engineering--Laboratory manuals.

Genetic engineering.

Medical Subjects:

Directed Molecular Evolution--methods.

Enzymes.

Evolution, Molecular.

Gene Library.

Protein Engineering--methods.

Genre:

Laboratory manuals.

Physical Description:

xv, 383 pages : illustrations (some color) ; 24 cm.

Place of Publication:

Totowa, N.J. : Humana Press, [2003]

Summary:

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 1-58829285-1), 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.

Contents:

Part I Genetic Selections

1 Genetic Complementation Protocols / Jessica L. Sneeden, Lawrence A. Loeb 3

2 Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase / Manel Camps, Lawrence A. Loeb 11

3 Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast / Ranga N. Venkatesan, Lawrence A. Loeb 19

4 Autogene Selections / Jijumon Chelliserrykattil, Andrew D. Ellington 27

5 Selection for Soluble Proteins via Fusion with Chloramphenicol Acetyltransferase / Volker Sieber 45

6 Proside: A Phage-Based Method for Selecting Thermostable Proteins / Andreas Martin, Franz X. Schmid, Volker Sieber 57

7 Minimization of Proteins by Random Fragmentation and Selection / Gary W. Rudgers, Timothy Palzkill 71

Part II Screens for Enzymes

8 Evaluating a Screen and Analysis of Mutant Libraries / Oriana Salazar, Lianhong Sun 85

9 Screening Mutant Libraries in Saccharomyces cerevisiae / Thomas Bulter, Volker Sieber, Miguel Alcalde 99

10 Solid-Phase Screening Using Digital Image Analysis / Alexander V. Tobias, John M. Joern 109

11 Screening for Thermostability / Patrick C. Cirino, Radu Georgescu 117

12 High-Throughput Screening of Mutant [alpha]-Amylase Libraries for Increased Activity at 129[degree]C / Holger Berk, Robert J. Lebbink 127

13 High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450 / Christopher R. Otey 137

14 High-Througput Screen for Aromatic Hydroxylation / Christopher R. Otey, John M. Joern 141

15 Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes / Edgardo T. Farinas 149

16 High-Throughput Screens Based on NAD(P)H Depletion / Anton Glieder, Peter Meinhold 157

17 High-Throughput Tetramethylbenzidine (TMB) Screen for Peroxidases / Radu Georgescu 171

18 Screen for Oxidases by Detection of Hydrogen Peroxide with Horseradish Peroxidase / Lianhong Sun, Makoto Yagasaki 177

19 Colorimetric Dehydrogenase Screen Based on NAD(P)H Generation / Kimberly M. Mayer 183

20 Colorimetric Assays for Screening Laccases / Miguel Alcalde, Thomas Bulter 193

21 pH Sensing Agar Plate Assays for Esterolytic Enzyme Activity / Karl E. Griswold 203

22 A pH-Indicator-Based Screen for Hydrolytic Haloalkane Dehalogenase / Huimin Zhao 213

23 Detection of Aromatic [alpha]-Hydroxyketones with Tetrazolium Salts / Michael Breuer, Bernhard Hauer 223

24 Selection of Heat-Stable Clostridium cellulovorans Cellulases After In Vitro Recombination / Koichiro Murashima, Roy H. Doi 231

25 Screening and Selection Strategies for Disulfide Isomerase Activity / Ronald Lafond, Xiaoming Zhan, George Georgiou 239

26 An Overview of High-Throughput Screening Systems for Enantioselective Enzymatic Transformations / Manfred T. Reetz 259

27 Select Protocols of High-Throughput ee-Screening Systems for Assaying Enantioselective Enzymes / Manfred T. Reetz 283

28 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, Peter G. Schultz 291

29 Calmodulin-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities / Christian Heinis, Julian Bertschinger, Dario Neri 313

30 High-Throughput FACS Method for Directed Evolution of Substrate Specificity / Mark J. Olsen, Jongsik Gam, Brent L. Iverson, George Georgiou 329

31 Improving Protein Folding Efficiency by Directed Evolution Using the GFP Folding Reporter / Geoffrey S. Waldo 343.

Notes:

Includes bibliographic references and index.

ISBN:

158829286X

1592593968

OCLC:

52400248