2002 г
Introduction [1]
Evolutionary Biotechnology - From Ideas and Concepts to Experiments and Computer Simulations [5]
Evolution in vivo - From Natural Selection to Population Genetics [5]
Evolution in vitro - From Kinetic Equations to Magic Molecules [8]
Evolution in silico - From Neutral Networks to Multi-stable Molecules [16]
Sequence Structure Mappings of Proteins [25]
Concluding Remarks [26]
Using Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases [29]
ntroduction [29]
Selection versus Screening [30]
Classical solutions to the sorting problem [31]
Advantages and limitations of selection [32]
Genetic Selection of Novel Chorismate Mutases [33]
The selection system [35]
Mechanistic studies [37]
Active site residues [37]
Random protein truncation [42]
Structural studies [44]
Constraints on interhelical loops [44]
Altering protein topology [46]
New quateary structures [47]
Stable monomeric mutases [49]
Augmenting weak enzyme activity [51]
Protein design [53]
Summary and General Perspectives [57]
Construction of Environmental Libraries for Functional Screening of Enzyme Activity [63]
Sample Collection and DNA Isolation from Environmental Samples [65]
Construction of Environmental Libraries [68]
Screening of Environmental Libraries [71]
Conclusions [76]
nvestigation of Phage Display for the Directed Evolution of Enzymes [79]
ntroduction [79]
The Phage Display [79]
Phage Display of Enzymes [81]
The expression vectors [81]
Filamentous bacteriophages [81]
Other phages [83]
Phage-enzymes [84]
Creating Libraries of Mutants [87]
Selection of Phage-enzymes [89]
Selection for binding [89]
Selection for catalytic activity [90]
Selection with substrate or product analogues [90]
Selection with transition-state analogues [92]
Selection of reactive active site residues by affinity labeling [96]
Selection with suicide substrates [98]
Selections based directly on substrate transformations [102]
Conclusions [108]
Directed Evolution of Binding Proteins by Cell Surface Display: Analysis of the Screening Process [111]
ntroduction [111]
Library Construction [113]
Mutagenesis [113]
Expression [114]
Mutant Isolation [115]
Differential labeling [115]
Screening [119]
Summary [124]
Acknowledgments [124]
Yeast n-Hybrid Systems for Molecular Evolution [127]
ntroduction [127]
Technical Considerations [130]
Yeast two-hybrid assay [130]
Alteative assays [141]
Applications [147]
Protein-protein interactions [147]
Protein-DNA interactions [149]
Protein-RNA interactions [150]
Protein-small molecule interactions [153]
Conclusion [155]
Advanced Screening Strategies for Biocatalyst Discovery [159]
ntroduction [159]
Semi-quantitative Screening in Agar-plate Formats [161]
Solution-based Screening in Microplate Formats [164]
Robotics and Automation [169]
Engineering Protein Evolution [177]
ntroduction [177]
Mechanisms of Protein Evolution in Nature [178]
Gene duplication [179]
Tandem duplication [180]
(?)-barrels [181]
Circular permutation [182]
Oligomerization [183]
Gene fusion [184]
Domain recruitment [184]
Exon shuffling [186]
Engineering Genes and Gene Fragments [187]
Protein fragmentation [188]
Rational swapping of secondary structure elements and domains [189]
Combinatorial gene fragment shuffling [190]
Modular recombination and protein folding [194]
Rational domain assembly - engineering zinc fingers [199]
Combinatorial domain recombination - exon shuffling [200]
Gene Fusion - From Bi- to Multifunctional Enzymes [203]
End-to-end gene fusions [203]
Gene insertions [203]
Modular design in multifunctional enzymes [204]
Perspectives [208]
Exploring the Diversity of Heme Enzymes through Directed Evolution [215]
ntroduction [215]
Heme Proteins [216]
Cytochromes P450 [218]
ntroduction [218]
Mechanism [220]
The catalytic cycle [220]
Uncoupling [222]
Peroxide shunt pathway [222]
Peroxidases [223]
ntroduction [223]
Mechanism [223]
Compound I formation [223]
Oxidative dehydrogenation [226]
Oxidative halogenation [226]
Peroxide disproportionation [226]
Oxygen transfer [227]
Comparison of P450s and Peroxidases [227]
Chloroperoxidase [228]
Mutagenesis Studies [229]
P450s [230]
P450cam [230]
Eukaryotic P450s [230]
HRP [231]
CPO [231]
Myoglobin (Mb) [232]
Directed Evolution of Heme Enzymes [233]
P450s [233]
Peroxidases [234]
CPO [236]
Catalase I [236]
Myoglobin [237]
Methods for recombination of P450s [237]
Conclusions [238]
Directed Evolution as a Means to Create Enantioselective Enzymes for Use in Organic Chemistry [245]
ntroduction [245]
Mutagenesis Methods [247]
Overexpression of Genes and Secretion of Enzymes [248]
High-Throughput Screening Systems for Enantioselectivity [250]
Examples of Directed Evolution of Enantioselective Enzymes [257]
Kinetic resolution of a chiral ester catalyzed by mutant Upases [257]
Evolution of a lipase for the stereoselective hydrolysis of a meso-compound [268]
Kinetic resolution of a chiral ester catalyzed by a mutant esterase [269]
mproving the enantioselectivity of a transaminase [270]
nversion of the enantioselectivity of a hydantoinase [270]
Evolving aldolases which accept both D- and L-glyceraldehydes [271]
Conclusions [273]
Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DMA [281]
ntroduction [281]
Directed Evolution of Enzymes [282]
Site-directed mutagenesis [283]
Directed evolution [284]
Genetic damage [285]
PCR mutagenesis [286]
DNA shuffling [287]
Substitution by oligonucleotides containing random mutations (random mutagenesis) [288]
Directed Evolution of DNA polymerases [289]
Random mutagenesis of Thermus aquaticus DNA Pol I [291]
Determination of structural components for Taq DNA polymerase fidelity [292]
Directed evolution of a RNA polymerase from Taq DNA polymerase [293]
Mutability of the Taq polymerase active site [294]
Random oligonucleotide mutagenesis of Escherichia coli Pol I [294]
Directed Evolution of Thymidine Kinase [295]
Directed Evolution of Thymidylate Synthase [297]
O6-Alkylguanine-DNA Alkyltransferase [300]
Discussion [302]
Evolutionary Generation versus Rational Design of Restriction Endonucleases with Novel Specificity [309]
ntroduction [309]
Biology of restriction/modification systems [309]
Biochemical properties of type II restriction endonucleases [310]
Applications for type II restriction endonucleases [311]
Setting the stage for protein engineering of type II restriction endonucleases [313]
Design of Restriction Endonucleases with New Specificities [313]
Rational design [313]
Attempts to employ rational design to change the specificity of restriction enzymes [313]
Changing the substrate specificity of type Us restriction enzymes by domain fusion [316]
Rational design to extend specificities of type II restriction enzymes [316]
Evolutionary design of extended specificities [318]
Summary and Outlook [324]
Evolutionary Generation of Enzymes with Novel Substrate Specificities [329]
ntroduction [329]
General Considerations [331]
Examples [333]
Group 1 [333]
Group 2 [337]
Group 3 [338]
Conclusions [339]
Introduction [1]
Evolutionary Biotechnology - From Ideas and Concepts to Experiments and Computer Simulations [5]
Evolution in vivo - From Natural Selection to Population Genetics [5]
Evolution in vitro - From Kinetic Equations to Magic Molecules [8]
Evolution in silico - From Neutral Networks to Multi-stable Molecules [16]
Sequence Structure Mappings of Proteins [25]
Concluding Remarks [26]
Using Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases [29]
ntroduction [29]
Selection versus Screening [30]
Classical solutions to the sorting problem [31]
Advantages and limitations of selection [32]
Genetic Selection of Novel Chorismate Mutases [33]
The selection system [35]
Mechanistic studies [37]
Active site residues [37]
Random protein truncation [42]
Structural studies [44]
Constraints on interhelical loops [44]
Altering protein topology [46]
New quateary structures [47]
Stable monomeric mutases [49]
Augmenting weak enzyme activity [51]
Protein design [53]
Summary and General Perspectives [57]
Construction of Environmental Libraries for Functional Screening of Enzyme Activity [63]
Sample Collection and DNA Isolation from Environmental Samples [65]
Construction of Environmental Libraries [68]
Screening of Environmental Libraries [71]
Conclusions [76]
nvestigation of Phage Display for the Directed Evolution of Enzymes [79]
ntroduction [79]
The Phage Display [79]
Phage Display of Enzymes [81]
The expression vectors [81]
Filamentous bacteriophages [81]
Other phages [83]
Phage-enzymes [84]
Creating Libraries of Mutants [87]
Selection of Phage-enzymes [89]
Selection for binding [89]
Selection for catalytic activity [90]
Selection with substrate or product analogues [90]
Selection with transition-state analogues [92]
Selection of reactive active site residues by affinity labeling [96]
Selection with suicide substrates [98]
Selections based directly on substrate transformations [102]
Conclusions [108]
Directed Evolution of Binding Proteins by Cell Surface Display: Analysis of the Screening Process [111]
ntroduction [111]
Library Construction [113]
Mutagenesis [113]
Expression [114]
Mutant Isolation [115]
Differential labeling [115]
Screening [119]
Summary [124]
Acknowledgments [124]
Yeast n-Hybrid Systems for Molecular Evolution [127]
ntroduction [127]
Technical Considerations [130]
Yeast two-hybrid assay [130]
Alteative assays [141]
Applications [147]
Protein-protein interactions [147]
Protein-DNA interactions [149]
Protein-RNA interactions [150]
Protein-small molecule interactions [153]
Conclusion [155]
Advanced Screening Strategies for Biocatalyst Discovery [159]
ntroduction [159]
Semi-quantitative Screening in Agar-plate Formats [161]
Solution-based Screening in Microplate Formats [164]
Robotics and Automation [169]
Engineering Protein Evolution [177]
ntroduction [177]
Mechanisms of Protein Evolution in Nature [178]
Gene duplication [179]
Tandem duplication [180]
(?)-barrels [181]
Circular permutation [182]
Oligomerization [183]
Gene fusion [184]
Domain recruitment [184]
Exon shuffling [186]
Engineering Genes and Gene Fragments [187]
Protein fragmentation [188]
Rational swapping of secondary structure elements and domains [189]
Combinatorial gene fragment shuffling [190]
Modular recombination and protein folding [194]
Rational domain assembly - engineering zinc fingers [199]
Combinatorial domain recombination - exon shuffling [200]
Gene Fusion - From Bi- to Multifunctional Enzymes [203]
End-to-end gene fusions [203]
Gene insertions [203]
Modular design in multifunctional enzymes [204]
Perspectives [208]
Exploring the Diversity of Heme Enzymes through Directed Evolution [215]
ntroduction [215]
Heme Proteins [216]
Cytochromes P450 [218]
ntroduction [218]
Mechanism [220]
The catalytic cycle [220]
Uncoupling [222]
Peroxide shunt pathway [222]
Peroxidases [223]
ntroduction [223]
Mechanism [223]
Compound I formation [223]
Oxidative dehydrogenation [226]
Oxidative halogenation [226]
Peroxide disproportionation [226]
Oxygen transfer [227]
Comparison of P450s and Peroxidases [227]
Chloroperoxidase [228]
Mutagenesis Studies [229]
P450s [230]
P450cam [230]
Eukaryotic P450s [230]
HRP [231]
CPO [231]
Myoglobin (Mb) [232]
Directed Evolution of Heme Enzymes [233]
P450s [233]
Peroxidases [234]
CPO [236]
Catalase I [236]
Myoglobin [237]
Methods for recombination of P450s [237]
Conclusions [238]
Directed Evolution as a Means to Create Enantioselective Enzymes for Use in Organic Chemistry [245]
ntroduction [245]
Mutagenesis Methods [247]
Overexpression of Genes and Secretion of Enzymes [248]
High-Throughput Screening Systems for Enantioselectivity [250]
Examples of Directed Evolution of Enantioselective Enzymes [257]
Kinetic resolution of a chiral ester catalyzed by mutant Upases [257]
Evolution of a lipase for the stereoselective hydrolysis of a meso-compound [268]
Kinetic resolution of a chiral ester catalyzed by a mutant esterase [269]
mproving the enantioselectivity of a transaminase [270]
nversion of the enantioselectivity of a hydantoinase [270]
Evolving aldolases which accept both D- and L-glyceraldehydes [271]
Conclusions [273]
Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DMA [281]
ntroduction [281]
Directed Evolution of Enzymes [282]
Site-directed mutagenesis [283]
Directed evolution [284]
Genetic damage [285]
PCR mutagenesis [286]
DNA shuffling [287]
Substitution by oligonucleotides containing random mutations (random mutagenesis) [288]
Directed Evolution of DNA polymerases [289]
Random mutagenesis of Thermus aquaticus DNA Pol I [291]
Determination of structural components for Taq DNA polymerase fidelity [292]
Directed evolution of a RNA polymerase from Taq DNA polymerase [293]
Mutability of the Taq polymerase active site [294]
Random oligonucleotide mutagenesis of Escherichia coli Pol I [294]
Directed Evolution of Thymidine Kinase [295]
Directed Evolution of Thymidylate Synthase [297]
O6-Alkylguanine-DNA Alkyltransferase [300]
Discussion [302]
Evolutionary Generation versus Rational Design of Restriction Endonucleases with Novel Specificity [309]
ntroduction [309]
Biology of restriction/modification systems [309]
Biochemical properties of type II restriction endonucleases [310]
Applications for type II restriction endonucleases [311]
Setting the stage for protein engineering of type II restriction endonucleases [313]
Design of Restriction Endonucleases with New Specificities [313]
Rational design [313]
Attempts to employ rational design to change the specificity of restriction enzymes [313]
Changing the substrate specificity of type Us restriction enzymes by domain fusion [316]
Rational design to extend specificities of type II restriction enzymes [316]
Evolutionary design of extended specificities [318]
Summary and Outlook [324]
Evolutionary Generation of Enzymes with Novel Substrate Specificities [329]
ntroduction [329]
General Considerations [331]
Examples [333]
Group 1 [333]
Group 2 [337]
Group 3 [338]
Conclusions [339]