Opis: Protein Engineering Handbook: v. 3

An introduction to 17 innovative and commercially important approaches to engineering novel and improved proteins for diverse applications in biotechnology, chemistry, bioanalytics and medicine. As such, key developments covered in this reference and handbook include de novo enzyme design, cofactor design and metalloenzymes, extremophile proteins, and chemically resistant proteins for industrial processes. The editors integrate academic innovations and industrial applications so as to arrive at a balanced view of this multi-faceted topic. Throughout, the content is chosen to complement and extend the previously published two-volume handbook by the same authors, resulting in a superb overview of this burgeoning field.Preface XV List of Contributors XVII 1 Dirigent Effects in Biocatalysis 1 Bettina M. Nestl, Bernd A. Nebel, and Bernhard Hauer 1.1 Introduction 1 1.2 Dirigent Proteins 3 1.3 Solvents and Unconventional Reaction Media 4 1.3.1 Ionic Liquids 7 1.3.2 Microemulsions and Reversed Micelles Systems 10 1.4 Structure and Folding 12 1.5 Structured and Unstructured Domains 14 1.6 Isozymes, Moonlighting Proteins, and Promiscuity: Supertalented Enzymes 19 1.7 Conclusions 22 Acknowledgment 23 References 23 2 Protein Engineering Guided by Natural Diversity 29 James T. Kratzer, Megan F. Cole, and Eric A. Gaucher 2.1 Approaches 29 2.1.1 Ancestral Sequence Reconstruction (ASR) 30 2.1.2 Ancestral Mutation Method 31 2.1.3 Reconstructing Evolutionary Adaptive Paths (REAP) 32 2.2 Protocols 34 2.2.1 Practical Steps to Using ASR 34 2.2.2 Reconstructing Evolutionary Adaptive Paths: A Focused Application of ASR 36 2.3 Future Directions 38 2.3.1 Industrial Applications 40 2.3.2 Biomedical 41 2.3.3 Drug Discovery 41 2.3.4 Paleobiology 42 2.3.5 Synthetic Biology 43 2.3.6 Experimental Validation of ASR 43 2.4 Conclusions 44 References 44 3 Protein Engineering Using Eukaryotic Expression Systems 47 Martina Geier and Anton Glieder 3.1 Introduction 47 3.2 Eukaryotic Expression Systems 48 3.2.1 Yeast Expression Platforms 48 3.2.1.1 Saccharomyces cerevisiae 48 3.2.1.2 Pichia pastoris 51 3.2.1.3 Pichia angusta 54 3.2.1.4 Alternative Yeasts 55 3.2.2 Filamentous Fungi 56 3.2.3 Insect Cells 58 3.2.4 Mammalian Cell Cultures 59 3.2.5 Transgenic Animals and Plants 61 3.2.6 Cell-Free Expression Systems 61 3.3 Conclusions 63 References 65 4 Protein Engineering in Microdroplets 73 Yolanda Schaerli, Balint Kintses, and Florian Hollfelder 4.1 Introduction 73 4.2 Droplet Formats 75 4.2.1 "Bulk" Emulsions 75 4.2.1.1 Catalytic Selections Involving DNA Substrates 76 4.2.1.2 Using the Droplet Compartment to Form a Permanent Genotype-Phenotype Linkage for Selections of Binders 77 4.2.2 Double "Bulk" Emulsions 78 4.2.3 Microfl uidic Droplets 79 4.3 Perspectives 83 Acknowledgments 84 References 84 5 Folding and Dynamics of Engineered Proteins 89 Michelle E. McCully and Valerie Daggett 5.1 Introduction 89 5.2 Proof-of-Principle Protein Designs 90 5.2.1 FSD-1, a Heterogeneous Native State and Complicated Folding Pathway 91 5.2.2 alpha3D, a Dynamic Core Leads to Fast Folding and Thermal Stability 94 5.2.3 Three-Helix Bundle Thermostabilized Proteins 96 5.2.4 Top7, a Novel Fold Topology 97 5.2.5 Other Rosetta Designs 100 5.3 Proteins Designed for Function 102 5.3.1 Ligands 103 5.3.1.1 Metal-Binding Four-Helix Bundles, the Effectiveness of Negative Design 103 5.3.1.2 Peptide Binding 105 5.3.2 Enzymes 106 5.3.2.1 Retro-Aldol Enzyme, Accommodating a Two-Step Reaction 106 5.3.2.2 Kemp Elimination Enzyme, Rigid Active Site Geometry Promotes Catalysis 108 5.4 Conclusions and Outlook 110 Acknowledgments 111 References 112 6 Engineering Protein Stability 115 Ciaran O'Fagain 6.1 Introduction 115 6.2 Power and Scope of Protein Engineering to Enhance Stability 116 6.2.1 Thermal Stabilizations 116 6.2.1.1 Potential Therapeutics: Rational Design with Computational Support 116 6.2.1.2 Analytical Tools: Green Fluorescent Protein and Luciferase 128 6.2.1.3 "Stiffening" a Protein by Gly-to-Pro Replacement: Methyl Parathion Hydrolase 128 6.2.2 Thermal Is Not the Only Stability: Oxidative and Other Chemical Stabilities 129 6.2.2.1 Oxidative Stability 129 6.2.2.2 Stabilization against Aldehydes and Solvents 130 6.2.2.3 Alkaline Tolerance 131 6.3 Measurement of a Protein's Kinetic Stability 132 6.3.1 Materials and General Hints 132 6.3.2 Thermal Stability 132 6.3.2.1 Thermal Profi le 132 6.3.2.2 Thermal Inactivation 133 6.3.3 Measurement of Oxidative Stability 134 6.3.4 Stability Analysis and Accelerated Degradation Testing 135 6.3.4.1 Set-Up 136 6.3.4.2 Analysis of Results 137 6.4 Developments in Protein Stabilization 137 References 139 7 Enzymes from Thermophilic Organisms 145 Tamotsu Kanai and Haruyuki Atomi 7.1 Introduction 145 7.2 Hyperthermophiles 146 7.3 Enzymes from Thermophiles and Their Reactions 146 7.4 Production of Proteins from (Hyper)Thermophiles 148 7.5 Protein Engineering of Thermophilic Proteins 154 7.6 Cell Engineering in Hyperthermophiles 156 7.7 Future Perspectives 157 References 157 8 Enzyme Engineering by Cofactor Redesign 163 Malgorzata M. Kopacz, Frank. Hollmann, and Marco W. Fraaije 8.1 Introduction 163 8.2 Natural Cofactors: Types, Occurrence, and Chemistry 164 8.3 Inorganic Cofactors 165 8.4 Organic Cofactors 168 8.5 Redox Cofactors 169 8.5.1 Nicotinamide Cofactor Engineering 170 8.5.2 Heme Cofactor Engineering 173 8.5.2.1 Reconstitution of Myoglobin 174 8.5.2.2 Artificial Metalloproteins Based on Serum Albumins 175 8.5.3 Flavin Cofactor Engineering 176 8.6 Concluding Remarks 180 References 181 9 Biocatalyst Identifi cation by Anaerobic High-Throughput Screening of Enzyme Libraries and Anaerobic Microorganisms 193 Helen S. Toogood and Nigel S. Scrutton 9.1 Introduction 193 9.2 Oxygen-Sensitive Biocatalysts 194 9.2.1 Flavoproteins 194 9.2.2 Iron-Sulfur-Containing Proteins 195 9.2.3 Other Causes of Oxygen Sensitivity 197 9.3 Biocatalytic Potential of Oxygen-Sensitive Enzymes and Microorganisms 198 9.3.1 Old Yellow Enzymes (OYEs) 198 9.3.2 Enoate Reductases 200 9.3.3 Other Enzymes 202 9.3.4 Whole-Cell Anaerobic Fermentations 202 9.4 Anaerobic High-Throughput Screening 203 9.4.1 Semi-Anaerobic Screening Protocols 204 9.4.2 Anaerobic Robotic High-Throughput Screening 205 9.4.2.1 Purifi ed Enzyme versus Whole-Cell Extracts 207 9.4.2.2 Indirect Kinetic Screening versus Direct Product Determination 208 9.4.3 Potential Extensions of Robotic Anaerobic High-Throughput Screening 209 9.5 Conclusions and Outlook 210 References 210 10 Organometallic Chemistry in Protein Scaffolds 215 Yvonne M. Wilson, Marc Durrenberger, and Thomas R. Ward 10.1 Introduction 215 10.1.1 Concept 215 10.1.2 Considerations for Designing an Artifi cial Metalloenzyme 216 10.1.2.1 Organometallic Complex 216 10.1.2.2 Biomolecular Scaffold 218 10.1.2.3 Anchoring Strategy 219 10.1.2.4 Advantages and Disadvantages of the Different Anchoring Modes 221 10.1.2.5 Spacer 222 10.1.3 Other Key Developments in the Field 223 10.1.4 Why Develop Artifi cial Metalloenzymes? 223 10.2 Protocol/Practical Considerations 226 10.2.1 Protein Scaffold 226 10.2.1.1 Determination of Free Binding Sites 226 10.2.2 Organometallic Catalyst 228 10.2.2.1 Synthesis of [CpIr(biot-p-L)Cl] 229 10.2.2.2 N'-(4-Biotinamidophenylsulfonyl)-Ethylenediamine TFA Salt 230 10.2.3 Combination of Biotinylated Metal Catalyst and Streptavidin Host 231 10.2.3.1 Binding Affi nity of the Biotinylated Complex to Streptavidin 231 10.2.4 Catalysis 232 10.2.4.1 Catalysis Controls 232 10.3 Goals 234 10.3.1 Rate Acceleration 234 10.3.2 High-Throughput Screening 234 10.3.2.1 Considerations for Screening of Artificial Metalloenzymes 235 10.3.3 Expansion of Substrate Scope 236 10.3.4 Upscaling 236 10.3.5 Potential Applications 237 10.4 Summary 237 Acknowledgments 237 References 238 11 Engineering Protease Specificity 243 Philip N. Bryan 11.1 Introduction 243 11.1.1 Overview 243 11.1.2 Some Basic Points 244 11.1.2.1 Mechanism for a Serine Protease 244 11.1.2.2 Measuring Specifi city 244 11.1.2.3 Binding Interactions 245 11.1.3 Nature versus Researcher 247 11.1.3.1 P1 Specifi city of Chymotrypsin-like Proteases 247 11.1.3.2 The S1 Site of Subtilisin 247 11.1.3.3 The S4 Site of Subtilisin 250 11.1.3.4 Other Subsites in Subtilisin 250 11.1.3.5 Kinetic Coupling and Specifi city 251 11.2 Protocol and Practical Considerations 251 11.2.1 Remove and Regenerate 251 11.2.2 Engineering Highly Stable and Independently Folding Subtilisins 252 11.2.3 Engineering of P4 Pocket to Increase Substrate Specifi city 253 11.2.4 Destroying the Active Site in Order to Save It 254 11.2.5 Identifying a Cognate Sequence for Anion-Triggered Proteases Using the Subtilisin Prodomain 255 11.2.6 Tunable Chemistry and Specifi city 257 11.2.7 Purification Proteases Based on Prodomain--Subtilisin Interactions and Triggered Catalysis 258 11.2.8 Design of a Mechanism-Based Selection System 259 11.2.8.1 Step 1: Ternary Complex Formation 259 11.2.8.2 Step 2: Acylation 263 11.2.8.3 Steps 3 and 4: Deacylation and Product Release 265 11.2.9 Evolving Protease Specifi city Regulated with Anion Cofactors by Phage Display 266 11.2.9.1 Construction and Testing of Subtilisin Phage 266 11.2.9.2 Random Mutagenesis and Transformation 267 11.2.9.3 Selection of Anions 267 11.2.9.4 Evolving the Anion Site 267 11.2.9.5 Catch-and-Release Phage Display 267 11.2.9.6 Conclusions 269 11.2.10 Evolving New Specifi cities at P4 269 11.3 Concepts, Challenges, and Visions on Future Developments 270 11.3.1 Design Challenges 270 11.3.2 Challenges in Directed Evolution 271 11.3.2.1 One Must Go Deep into Sequence Space 271 11.3.2.2 Methods Which Maximize Substrate Binding Affinity Are Not Productive 272 11.3.2.3 The Desired Protease May Be Toxic to Cells 272 11.3.3 The Quest for Restriction Proteases 272 11.3.3.1 Not All Substrate Sequences Are Created Equal 273 11.3.4 Final Thoughts: Gilded or Golden? 273 Acknowledgments 274 References 274 12 Polymerase Engineering: From PCR and Sequencing to Synthetic Biology 279 Vitor B. Pinheiro, Jennifer L. Ong, and Philipp Holliger 12.1 Introduction 279 12.2 PCR 281 12.3 Sequencing 281 12.3.1 First-Generation Sequencing 282 12.3.2 Next-Generation Sequencing Technologies 284 12.4 Polymerase Engineering Strategies 288 12.5 Synthetic Informational Polymers 291 References 295 13 Engineering Glycosyltransferases 303 John McArthur and Gavin J. Williams 13.1 Introduction to Glycosyltransferases 303 13.2 Glycosyltransferase Sequence, Structure, and Mechanism 304 13.3 Examples of Glycosyltransferase Engineering 307 13.3.1 Chimeragenesis and Rational Design 307 13.3.2 Directed Evolution 310 13.3.2.1 Fluorescence-Based Screening 311 13.3.2.2 Reverse Glycosylation Reactions 312 13.3.2.3 ELISA-Based Screens 313 13.3.2.4 pH Indicator Assays 314 13.3.2.5 Chemical Complementation 314 13.3.2.6 Low-Throughput Assays 314 13.4 Practical Considerations for Screening Glycosyltransferases 315 13.4.1 Enzyme Expression and Choice of Expression Vector 315 13.4.2 Provision of Acceptor and NDP-donor Substrate 315 13.4.3 General Considerations for Microplate-Based Screens 317 13.4.4 Promiscuity, Profi ciency, and Specifi city 317 13.5 Future Directions and Outlook 318 References 319 14 Protein Engineering of Cytochrome P450 Monooxygenases 327 Katja Koschorreck, Clemens J. von Buhler, Sebastian Schulz, and Vlada B. Urlacher 14.1 Cytochrome P450 Monooxygenases 327 14.1.1 Introduction 327 14.1.2 Catalytic Cycle of Cytochrome P450 Monooxygenases 328 14.1.3 Redox Partner Proteins 329 14.2 Engineering of P450 Monooxygenases 330 14.2.1 Molecular Background for P450 Engineering 330 14.2.2 Altering Substrate Selectivity and Improving Enzyme Activity 332 14.2.2.1 Rational and Semi-Rational Design 332 14.2.2.2 Directed Evolution and Its Combination with Computational Design 336 14.2.2.3 Decoy Molecules 338 14.2.3 Improving Solvent and Temperature Stability of P450 Monooxygenases 340 14.2.3.1 Solvent Stability 341 14.2.3.2 Thermostability 342 14.2.4 Improving Recombinant Expression and Solubility of P450 Monooxygenases 343 14.2.4.1 N-Terminal Modifi cations 344 14.2.4.2 Modifi cations within the F-G Loop 346 14.2.4.3 Improving Expression by Rational Protein Design and Directed Evolution 348 14.2.5 Engineering the Electron Transport Chain and Cofactors of P450s 349 14.2.5.1 Genetic Fusion of Proteins 349 14.2.5.2 Enzymatic Fusion and Self-Assembling Oligomers 352 14.3 Conclusions 354 References 355 15 Progress and Challenges in Computational Protein Design 363 Yih-En Andrew Ban, Daniela Rothlisberger-Grabs, Eric A. Althoff, and Alexandre Zanghellini 15.1 Introduction 363 15.2 The Technique of Computational Protein Design 363 15.2.1 Principles of Protein Design 363 15.2.2 A Brief Review of Force-Fields for CPD 364 15.2.3 Optimization Algorithms for Fixed-Backbone Protein Design (P1') 368 15.3 Protein Core Redesign, Structural Alterations, and Thermostabilization 371 15.3.1 Protein Core Redesign and de novo Fold Design 371 15.3.2 Computational Alteration of Protein Folds 373 15.3.2.1 Loop Grafting 374 15.3.2.2 de novo Loop Design 375 15.3.2.3 Fold Switching 376 15.3.2.4 Fold Alteration: Looking Ahead 377 15.3.2.5 Computational Optimization of the Thermostability of Proteins 377 15.4 Computational Enzyme Design 380 15.4.1 de novo Enzyme Design 380 15.4.1.1 Initial Proofs-of-Concept 380 15.4.1.2 Review of Recent Developments 382 15.4.2 Computational Redesign of the Substrate Specifi city of Enzymes 383 15.4.2.1 Fixed-Backbone and Flexible-Backbone Substrate Specifi city Switches 383 15.4.2.2 Limitations and Feedback Obtained from Experimental Optimization Attempts 385 15.4.3 Frontiers in Computational Enzyme Design 386 15.5 Computational Protein--Protein Interface Design 388 15.5.1 Natural Protein--Protein Interfaces Redesign 389 15.5.2 Two-Sided de novo Design of Protein Interfaces 390 15.5.3 One-Sided de novo Design of Protein Interfaces 392 15.5.4 Frontiers in Protein--Protein Interaction Design 393 15.6 Computational Redesign of DNA Binding and Specifi city 394 15.7 Conclusions 396 References 396 16 Simulation of Enzymes in Organic Solvents 407 Tobias Kulschewski and Jurgen Pleiss 16.1 Enzymes in Organic Solvents 407 16.2 Molecular Dynamics Simulations of Proteins and Solvents 408 16.3 The Role of the Solvent 410 16.4 Simulation of Protein Structure and Flexibility 411 16.5 Simulation of Catalytic Activity and Enantioselectivity 413 16.6 Simulation of Solvent-Induced Conformational Transitions 414 16.7 Challenges 415 16.8 The Future of Biocatalyst Design 416 References 417 17 Engineering of Protein Tunnels: The Keyhole--Lock--Key Model for Catalysis by Enzymes with Buried Active Sites 421 Zbynek Prokop, Artur Gora, Jan Brezovsky, Radka Chaloupkova, Veronika Stepankova, and Jiri Damborsky 17.1 Traditional Models of Enzymatic Catalysis 421 17.2 Defi nition of the Keyhole--Lock--Key Model 422 17.3 Robustness and Applicability of the Keyhole--Lock--Key Model 424 17.3.1 Enzymes with One Tunnel Connecting a Buried Active Site to the Protein Surface 424 17.3.2 Enzymes with More than One Tunnel Connecting a Buried Active Site to the Protein Surface 433 17.3.3 Enzymes with One Tunnel Between Two Distinct Active Sites 436 17.4 Evolutionary and Functional Implications of the Keyhole--Lock--Key Model 437 17.5 Engineering Implications of the Keyhole--Lock--Key Model 438 17.5.1 Engineering Activity 442 17.5.2 Engineering Specifi city 443 17.5.3 Engineering Stereoselectivity 443 17.5.4 Engineering Stability 443 17.6 Software Tools for the Rational Engineering of Keyholes 444 17.6.1 Analysis of Tunnels in a Single Protein Structure 445 17.6.2 Analysis of Tunnels in the Ensemble of Protein Structures 445 17.6.3 Analysis of Tunnels in the Ensemble of Protein--Ligand Complexes 447 17.7 Case Studies with Haloalkane Dehalogenases 448 17.8 Conclusions 450 References 452 Index 465

Szczegóły: Protein Engineering Handbook: v. 3

Tytuł: Protein Engineering Handbook: v. 3
Producent: VCH
ISBN: 9783527331239
Rok produkcji: 2012
Ilość stron: 502
Oprawa: Twarda
Waga: 1.07 kg

Recenzje: Protein Engineering Handbook: v. 3