Library Science

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.

Ed. Arnold, Frances H.; Georgiou, George

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Table of Contents

1. Genetic Complementation Protocols (pp. 3-10)
   Sneeden, Jessica L.; Loeb, Lawrence A.
2. Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase (pp. 11-18)
   Camps, Manel; Loeb, Lawrence A.
3. Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast (pp. 19-26)
   Venkatesan, Ranga N.; Loeb, Lawrence A.
4. Autogene Selections (pp. 27-44)
   Chelliserrykattil, Jijumon; Ellington, Andrew D.
5. Selection for Soluble Proteins via Fusion with Chloramphenicol Acetyltransferase (pp. 45-56)
   Sieber, Volker
6. Proside A Phage-Based Method for Selecting Thermostable Proteins (pp. 57-70)
   Martin, Andreas; Schmid, Franz X.; Sieber, Volker
7. Minimization of Proteins by Random Fragmentation and Selection (pp. 71-82)
   Rudgers, Gary W.; Palzkill, Timothy
8. Evaluating a Screen and Analysis of Mutant Libraries (pp. 85-98)
   Salazar, Oriana; Sun, Lianhong
9. Screening Mutant Libraries in Saccharomyces cerevisiae (pp. 99-108)
   Bulter, Thomas; Sieber, Volker; Alcalde, Miguel
10. Solid-Phase Screening Using Digital Image Analysis (pp. 109-116)
    Tobias, Alexander V.; Joern, John M.
11. Screening for Thermostability (pp. 117-126)
    Cirino, Patrick C.; Georgescu, Radu
12. High-Throughput Screening of Mutant a-Amylase Libraries for Increased Activity at 129°C (pp. 127-136)
    Berk, Holger; Lebbink, Robert J.
13. High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450 (pp. 137-140)
    Otey, Christopher R.
14. High-Throughput Screen for Aromatic Hydroxylation (pp. 141-148)
    Otey, Christopher R.; Joern, John M.
15. Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes (pp. 149-156)
    Farinas, Edgardo T.
16. High-Throughput Screens Based on NAD(P)H Depletion (pp. 157-170)
    Glieder, Anton; Meinhold, Peter
17. High-Throughput Tetramethylbenzidine (TMB) Screen for Peroxidases (pp. 171-176)
    Georgescu, Radu
18. Screen for Oxidases by Detection of Hydrogen Peroxide with Horseradish Peroxidase (pp. 177-182)
    Sun, Lianhong; Yagasaki, Makoto
19. Colorimetric Dehydrogenase Screen Based on NAD(P)H Generation (pp. 183-192)
    Mayer, Kimberly M.
20. Colorimetric Assays for Screening Laccases (pp. 193-202)
    Alcalde, Miguel; Bulter, Thomas
21. pH Sensing Agar Plate Assays for Esterolytic Enzyme Activity (pp. 203-212)
    Griswold, Karl E.
22. A pH-Indicator-Based Screen for Hydrolytic Haloalkane Dehalogenase (pp. 213-222)
    Zhao, Huimin
23. Detection of Aromatic a-Hydroxyketones with Tetrazolium Salts (pp. 223-230)
    Breuer, Michael; Hauer, Bernhard
24. Selection of Heat-Stable Clostridium cellulovorans Cellulases After In Vitro Recombination (pp. 231-238)
    Murashima, Koichiro; Doi, Roy H.
25. Screening and Selection Strategies for Disulfide Isomerase Activity (pp. 239-258)
    Lafond, Ronald; Zhan, Xiaoming; Georgiou, George
26. An Overview of High-Throughput Screening Systems for Enantioselective Enzymatic Transformations (pp. 259-282)
    Reetz, Manfred T.
27. Select Protocols of High-Throughput ee-Screening Systems for Assaying Enantioselective Enzymes (pp. 283-290)
    Reetz, Manfred T.
28. Directed Evolution of the Substrate Specificities of a Site-Specific Recombinase and an Aminoacyl-tRNA Synthetase Using Fluorescence-Activated Cell Sorting (FACS) (pp. 291-312)
    Santoro, Stephen W.; Schultz, Peter G.
29. Calmodulin-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities (pp. 313-328)
    Heinis, Christian; Bertschinger, Julian; Neri, Dario
30. High-Throughput FACS Method for Directed Evolution of Substrate Specificity (pp. 329-342)
    Olsen, Mark J.; Gam, Jongsik; Iverson, Brent L.; Georgiou, George
31. Improving Protein Folding Efficiency by Directed Evolution Using the GFP Folding Reporter (pp. 343-360)
    Waldo, Geoffrey S.

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