directed molecular evolution of proteins

safecover (100x141x16M jpeg) Directed Molecular Evolution of Proteins Edited by S. Brakmann and K. Johnsson Directed Molecular Evolution of Proteins:orHow to Improve Enzymes for Biocatalysis. Edited by Susanne Brakmann and Kai Johnsson Copyright ã 2002 Wiley-VCH Verlag GmbH & Co. KGaA ISBNs: 3-527-30423-1 (Hardback); 3-527-60064-7 (Electronic) Related Titles from Wiley-VCH Kellner,R.; Lottspeich, F.; Meyer, H. E. Microcharacterization of Proteins 1999 ISBN 3-527-30084-8 Bannwarth,W.; Felder, E.; Mannhold, R.; Kubinyi, H.; Timmermann, H. Combinatorial Chemistry. A Practical Approach 2000 ISBN 3-527-30186-0 Gualtieri,F.; Mannhold, R.; Kubinyi, H.; Timmermann, H. New Trends in Synthetic Medicinal Chemistry 2001 ISBN 3-527-29799-5 Clark,D. E.; Mannhold, R.; Kubinyi, H.; Timmermann, H. Evolutionary Algorithms in Molecular Design 2000 ISBN 3-527-30155-0 Directed Molecular Evolution of Proteins or How to Improve Enzymes for Biocatalysis Edited by Susanne Brakmann and Kai Johnsson The Editor of this volume Dr. Susanne Brakmann AG ¹Angewandte Molekulare Evolutionª Institut fuÈr Spezielle Zoologie UniversitaÈt Leipzig Talstraûe 33 04103 Leipzig, Germany Prof. Dr. Kai Johnsson Institute of Molecular and Biological Chemistry Swiss Federal Institute of Technology Lausanne CH-1015 Lausanne, Switzerland Cover Illustration Recent advances in automation and robotics have greatly facilitated the high ± throughput screening for proteins with desired functions. Among other devices liquid handling tools are integral parts of most screening robots. Depicted are 96-channel pipettors for the microliter- and submicroliter range (illustrations kindly provided by Cybio AG, Jena). This book was carefully produced. Nevertheless, editors, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that state- ments, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek ± CIP Cataloguing-in-Pub- lication Data A catalogue record for this publication is available from Die Deutsche Bibliothek. ã Wiley-VCH Verlag GmbH, Weinheim 2002 All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form ± by photoprinting, mi- crofilm, or any other means ± nor transmitted or translated into machine language without written permission from the publishers. In this publication, even without specific indi- cation, use of registered names, trademarks, etc., and reference to patents or utility models does not imply that such names or any such information are exempt from the relevant protective laws and reg- ulations and, therefore, free for general use, nor does mention of suppliers or of particular com- mercial products constitute endorsement or recommendation for use. Printed on acid-free paper. Printed in the Federal Republic of Germany. Composition Mitterweger & Partner Kommunikationsgesellschaft mbH, Plankstadt Printing betz-druck GmbH, Darmstadt Bookbinding Groûbuchbinderei J. SchaÈffer GmbH & Co. KG, GruÈnstadt ISBN 3-527-30423-1 Contents List of Contributors XI 1 Introduction 1 2Evolutionary Biotechnology ± From Ideas and Concepts to Experiments and Computer Simulations 5 2.1 Evolution in vivo ± From Natural Selection to Population Genetics 5 2.2 Evolution in vitro ± From Kinetic Equations to Magic Molecules 8 2.3 Evolution in silico ± From Neutral Networks to Multi-stable Molecules 16 2.4 Sequence Structure Mappings of Proteins 25 2.5 Concluding Remarks 26 3 Using Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases 29 3.1 Introduction 29 3.2 Selection versus Screening 30 3.2.1 Classical solutions to the sorting problem 31 3.2.2 Advantages and limitations of selection 32 3.3 Genetic Selection of Novel Chorismate Mutases 33 3.3.1 The selection system 35 3.3.2 Mechanistic studies 37 3.3.2.1 Active site residues 37 3.3.2.2 Random protein truncation 42 3.3.3 Structural studies 44 3.3.3.1 Constraints on interhelical loops 44 3.3.4 Altering protein topology 46 3.3.4.1 New quaternary structures 47 3.3.4.2 Stable monomeric mutases 49 3.3.5 Augmenting weak enzyme activity 51 3.3.6 Protein design 53 3.4 Summary and General Perspectives 57 Directed Molecular Evolution of Proteins:orHow to Improve Enzymes for Biocatalysis. Edited by Susanne Brakmann and Kai Johnsson Copyright ã 2002 Wiley-VCH Verlag GmbH & Co. KGaA ISBNs: 3-527-30423-1 (Hardback); 3-527-60064-7 (Electronic) 4Construction of Environmental Libraries for Functional Screening of Enzyme Activity 63 4.1 Sample Collection and DNA Isolation from Environmental Samples 65 4.2 Construction of Environmental Libraries 68 4.3 Screening of Environmental Libraries 71 4.4 Conclusions 76 5 Investigation of Phage Display for the Directed Evolution of Enzymes 79 5.1 Introduction 79 5.2 The Phage Display 79 5.3 Phage Display of Enzymes 81 5.3.1 The expression vectors 81 5.3.1.1 Filamentous bacteriophages 81 5.3.1.2 Other phages 83 5.3.2 Phage-enzymes 84 5.4 Creating Libraries of Mutants 87 5.5 Selection of Phage-enzymes 89 5.5.1 Selection for binding 89 5.5.2 Selection for catalytic activity 90 5.5.2.1 Selection with substrate or product analogues 90 5.5.2.2 Selection with transition-state analogues 92 5.5.2.3 Selection of reactive active site residues by affinity labeling 96 5.5.2.4 Selection with suicide substrates 98 5.5.2.5 Selections based directly on substrate transformations 102 5.6 Conclusions 108 6 Directed Evolution of Binding Proteins by Cell Surface Display: Analysis of the Screening Process 111 6.1 Introduction 111 6.2 Library Construction 113 6.2.1 Mutagenesis 113 6.2.2 Expression 114 6.3 Mutant Isolation 115 6.3.1 Differential labeling 115 6.3.2 Screening 119 6.4 Summary 124 Acknowledgments 124 7Yeast n-Hybrid Systems for Molecular Evolution 127 7.1 Introduction 127 7.2 Technical Considerations 130 7.2.1 Yeast two-hybrid assay 130 7.2.2 Alternative assays 141 7.3 Applications 147 7.3.1 Protein-protein interactions 147 7.3.2 Protein-DNA interactions 149 ContentsVI 7.3.3 Protein-RNA interactions 150 7.3.4 Protein-small molecule interactions 153 7.4 Conclusion 155 8 Advanced Screening Strategies for Biocatalyst Discovery 159 8.1 Introduction 159 8.2 Semi-quantitative Screening in Agar-plate Formats 161 8.3 Solution-based Screening in Microplate Formats 164 8.4 Robotics and Automation 169 9 Engineering Protein Evolution 177 9.1 Introduction 177 9.2 Mechanisms of Protein Evolution in Nature 178 9.2.1 Gene duplication 179 9.2.2 Tandem duplication 180 ba-barrels 181 9.2.3 Circular permutation 182 9.2.4 Oligomerization 183 9.2.5 Gene fusion 184 9.2.6 Domain recruitment 184 9.2.7 Exon shuffling 186 9.3 Engineering Genes and Gene Fragments 187 9.3.1 Protein fragmentation 188 9.3.2 Rational swapping of secondary structure elements and domains 189 9.3.3 Combinatorial gene fragment shuffling 190 9.3.4 Modular recombination and protein folding 194 9.3.5 Rational domain assembly ± engineering zinc fingers 199 9.3.6 Combinatorial domain recombination ± exon shuffling 200 9.4 Gene Fusion ± From Bi- to Multifunctional Enzymes 203 9.4.1 End-to-end gene fusions 203 9.4.2 Gene insertions 203 9.4.3 Modular design in multifunctional enzymes 204 9.5 Perspectives 208 10 Exploring the Diversity of Heme Enzymes through Directed Evolution 215 10.1 Introduction 215 10.2 Heme Proteins 216 10.3 Cytochromes P450 218 10.3.1 Introduction 218 10.3.1 Mechanism 220 10.3.2.1 The catalytic cycle 220 10.3.2.2 Uncoupling 222 10.3.2.3 Peroxide shunt pathway 222 10.4 Peroxidases 223 10.4.1 Introduction 223 10.4.2 Mechanism 223 VII 10.4.2.1 Compound I formation 223 10.4.2.2 Oxidative dehydrogenation 226 10.4.2.3 Oxidative halogenation 226 10.4.2.4 Peroxide disproportionation 226 10.4.2.5 Oxygen transfer 227 10.5 Comparison of P450s and Peroxidases 227 10.6 Chloroperoxidase 228 10.7 Mutagenesis Studies 229 10.7.1 P450s 230 10.7.1.1 P450 cam 230 10.7.1.2 Eukaryotic P450s 230 10.7.2 HRP 231 10.7.3 CPO 231 10.7.4 Myoglobin (Mb) 232 10.8 Directed Evolution of Heme Enzymes 233 10.8.1 P450s 233 10.8.2 Peroxidases 234 10.8.3 CPO 236 10.8.4 Catalase I 236 10.8.5 Myoglobin 237 10.8.6 Methods for recombination of P450s 237 10.9 Conclusions 238 11 Directed Evolution as a Means to Create Enantioselective Enzymes for Use in Organic Chemistry 245 11.1 Introduction 245 11.2 Mutagenesis Methods 247 11.3 Overexpression of Genes and Secretion of Enzymes 248 11.4 High-Throughput Screening Systems for Enantioselectivity 250 11.5 Examples of Directed Evolution of Enantioselective Enzymes 257 11.5.1 Kinetic resolution of a chiral ester catalyzed by mutant lipases 257 11.5.2 Evolution of a lipase for the stereoselective hydrolysis of a meso-compound 268 11.5.3 Kinetic resolution of a chiral ester catalyzed by a mutant esterase 269 11.5.4 Improving the enantioselectivity of a transaminase 270 11.5.5 Inversion of the enantioselectivity of a hydantoinase 270 11.5.6 Evolving aldolases which accept both D- and L-glyceraldehydes 271 11.6 Conclusions 273 12 Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DNA 281 12.1 Introduction 281 12.2 Directed Evolution of Enzymes 282 12.2.1 Site-directed mutagenesis 283 12.2.2 Directed evolution 284 ContentsVIII 12.2.3 Genetic damage 285 12.2.4 PCR mutagenesis 286 12.2.5 DNA shuffling 287 12.2.6 Substitution by oligonucleotides containing random mutations (random mutagenesis) 288 12.3 Directed Evolution of DNA polymerases 289 12.3.1 Random mutagenesis of Thermus aquaticus DNA Pol I 291 12.3.1.1 Determination of structural components for Taq DNA polymerase fidelity 292 12.3.1.2 Directed evolution of a RNA polymerase from Taq DNA polymerase 293 12.3.1.3 Mutability of the Taq polymerase active site 294 12.3.2 Random oligonucleotide mutagenesis of Escherichia coli Pol I 294 12.4 Directed Evolution of Thymidine Kinase 295 12.5 Directed Evolution of Thymidylate Synthase 297 12.6 O 6 -Alkylguanine-DNA Alkyltransferase 300 12.7 Discussion 302 13 Evolutionary Generation versus Rational Design of Restriction Endonucleases with Novel Specificity 309 13.1 Introduction 309 13.1.1 Biology of restriction/modification systems 309 13.1.2 Biochemical properties of type II restriction endonucleases 310 13.1.3 Applications for type II restriction endonucleases 311 13.1.4 Setting the stage for protein engineering of type II restriction endonucleases 313 13.2 Design of Restriction Endonucleases with New Specificities 313 13.2.1 Rational design 313 13.2.1.1 Attempts to employ rational design to change the specificity of restriction enzymes 313 14.2.1.1 Changing the substrate specificity of type IIs restriction enzymes by domain fusion 316 13.2.1.3 Rational design to extend specificities of type II restriction enzymes 316 13.2.2 Evolutionary design of extended specificities 318 13.3 Summary and Outlook 324 14 Evolutionary Generation of Enzymes with Novel Substrate Specificities 329 14.1 Introduction 329 14.2 General Considerations 331 14.3 Examples 333 14.3.1 Group 1 333 14.3.2 Group 2 337 14.3.3 Group 3 338 14.4 Conclusions 339 Index 343 IX [...]... one of the most dynamic fields in chemistry and biology The book presented here is a loose collection of articles aiming to provide an overview of the current state of the art of the directed evolution of proteins as well as highlighting the challenges and possibilities in the field that lie ahead Although the first examples of directed molecular evolution date back to the pioneering experiments of. .. attention of the general scientific community [3, 4] In the last decade, directed evolution has become a key technology for biomolecule engineering The success of the evolutionary approach, however, not only depends on the potency of the method itself but is also a result of the limitations of alternative approaches, as our lack of understanding of the structure-function relationship of proteins in... Mendel Molecular models for evolution under controlled conditions became available only in the second half of the twentieth century after the initiation of molecular biology This chapter presents an account of the origins of molecular evolution and develops the concepts that have led to successful applications in the evolutionary design of biopolymers with predefined properties and functions 2.1 Evolution. .. of biomolecules with new functions What are the prerequisites for a successful directed evolution experiment? In its broadest sense, (directed) evolution can be considered as repeated cycles of variation followed by selection In the first chapter of the book, the underlying principles of this concept and their application to the evolutionary design of biomolecules are reviewed by P Schuster ± one of. .. P Schuster ± one of the pioneers in the field of molecular evolution Naturally, the first step of each evolutionary project is the creation of diversity The most straightforward approach to create a library of proteins is to introduce random mutations into the gene of interest by techniques such as error-prone PCR or saturation mutagenesis The success of random mutagenesis strategies is witnessed by... enantioselectivity of enzymes; T Lanio et al present their approaches for the evolutionary generation of restriction endonucleases, U T Bornscheuer reports on the functional optimization of lipases, and last but not least, P C Cirino and F H Arnold give an overview of directed evolution experiments with heme enzymes Clearly, there are various developments and applications in the field of directed evolution. .. et al and of M Eigen and W Gardiner, who proposed that evolutionary approaches be adapted for the engineering of biomolecules [1, 2], it was the success of methods such as phage display for in vitro selection of peptides and proteins as well the selection of functional nucleic acids using the SELEX procedure (Systematic Evolution of Ligands by Exponential enrichment) that brought the power of this concept... predominant role of evolution was the elimination of deleterious variants Kimura's view was strongly supported by the data obtained from comparative sequence analysis of proteins and nucleic acids [6], which became the basis of current molecular phylogeny Genotypes are changing steadily and this also during epochs of phenotypic stasis Despite overwhelming indirect hints for neutral evolution from molecular. .. factor of three within only six transfer steps (no.8 ± no.13), and we notice a clear indication of stepwise optimization of replication rate Thus, occurrence of evolution in the Darwinian sense as the interplay of variation and natural selection is not bound to the existence of cellular life Molecular biologists have discovered and are currently still revealing a true wealth of data on the nature of the... overview of advanced screening strategies is given in the article of A Schwienhost In the chapter written by K D Wittrup a discussion of the prerequisites for a successful screening process is given, analyzing the outcome of the directed evolution of proteins displayed on cell surfaces as a function of the screening conditions The power of intelligently designed screening processes is demonstrated in . safecover (100x141x16M jpeg) Directed Molecular Evolution of Proteins Edited by S. Brakmann and K. Johnsson Directed Molecular Evolution of Proteins: orHow to Improve Enzymes for Biocatalysis. Edited. Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DNA 281 12.1 Introduction 281 12.2 Directed Evolution of Enzymes 282 12.2.1 Site -directed mutagenesis 283 12.2.2 Directed evolution. different chapters of this book describing case studies of particular classes of proteins and enzymes. In addition, recombination of mutant Directed Molecular Evolution of Proteins: orHow to Improve