Table Of ContentEdited by
Stefan Lutz and
Uwe T. Bornscheuer
Protein Engineering Handbook
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Edited by Stefan Lutz and Uwe T. Bornscheuer
Protein Engineering Handbook
Volume 3
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V
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
VI Contents
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 Microfluidic 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 αD, a Dynamic Core Leads to Fast Folding and Thermal Stability 94
3
5.2.3 Three-Helix Bundle Thermostabilized Proteins 96
5.2.4 Top7, a Novel Fold Topology 97
Contents VII
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 Profile 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
VIII Contents
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 Identification 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 Purified 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
