- ホーム
- > 洋書
- > 英文書
- > Science / Mathematics
Full Description
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.
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 129oC.- 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.
