Protein Engineering Handbook, Volume 3

by Lutz, Stefan; Bornscheuer, Uwe Theo

• ISBN13: 9783527331239
• ISBN10: 3527331239
• Edition: 1st
• Format: Hardcover
• Copyright: 2012-11-28
• Publisher: Wiley-VCH

Summary

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.

Author Biography

Stefan Lutz holds a B. S. degree from the Zurich University of Applied Sciences (Switzerland), and a M.S. degree from the University of Teesside (UK). He then obtained a Ph.D. from the University of Florida and spent three years as a Postdoc with Stephen Benkovic at Pennsylvania State University under a fellowship of the Swiss National Science Foundation. Since 2002 he has been a Chemistry professor at Emory University in Atlanta, Georgia (USA). The research in the Lutz laboratory focuses on the structure-function relationship of proteins through combinatorial protein engineering and design.

Uwe Bornscheuer studied Chemistry at the University of Hannover (Germany), where he obtained a Ph. D. at the Institute of Technical Chemistry. He then spent a postdoctoral year at the University of Nagoya, Japan, before returning to Germany to join the Institute of Technical Biochemistry at the University of Stuttgart. Since 1999 he has been Professor for Biotechnology and Enzyme Catalysis at the University of Greifswald. His main research interest is the application of engineered enzymes in the synthesis of optically active compounds and in lipid modification.

Table of Contents

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 α3D, 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
Ciarán Ó’Fágáin

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 Dürrenberger, 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 [Cp*Ir(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 Bühler, 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 Röthlisberger-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 Jürgen 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