Protein engineering handbook / edited by Stefan Lutz and Uwe T. Bornscheuer

Incluye bibliografía e índice: páginas 465-479

Preface.. List of Contributors.. 1 Dirigent Effects in Biocatalysis.. 1.1 Introduction.. 1.2 Dirigent Proteins.. 1.3 Solvents and Unconventional Reaction Media.. 1.3.1 Ionic Liquids.. 1.3.2 Microemulsions and Reversed Micelles Systems.. 1.4 Structure and Folding.. 1.5 Structured and Unstructured Domains.. 1.6 Isozymes, Moonlighting Proteins, and Promiscuity: Supertalented Enzymes.. 1.7 Conclusions.. Acknowledgment.. References.. 2 Protein Engineering Guided by Natural Diversity.. 2.1 Approaches.. 2.1.1 Ancestral Sequence Reconstruction (ASR.. 2.1.2 Ancestral Mutation Method.. 2.1.3 Reconstructing Evolutionary Adaptive Paths (REAP.. 2.2 Protocols.. 2.2.1 Practical Steps to Using ASR.. 2.2.2 Reconstructing Evolutionary Adaptive Paths: A Focused Application of ASR.. 2.3 Future Directions.. 2.3.1 Industrial Applications.. 2.3.2 Biomedical.. 2.3.3 Drug Discovery.. 2.3.4 Paleobiology.. 2.3.5 Synthetic Biology.. 2.3.6 Experimental Validation of ASR.. 2.4 Conclusions.. References.. 3 Protein Engineering Using Eukaryotic Expression Systems.. 3.1 Introduction.. 3.2 Eukaryotic Expression Systems.. 3.2.1 Yeast Expression Platforms.. 3.2.1.1 Saccharomyces cerevisiae.. 3.2.1.2 Pichia pastoris.. 3.2.1.3 Pichia angusta.. 3.2.1.4 Alternative Yeasts.. 3.2.2 Filamentous Fungi.. 3.2.3 Insect Cells.. 3.2.4 Mammalian Cell Cultures.. 3.2.5 Transgenic Animals and Plants.. 3.2.6 Cell-Free Expression Systems.. 3.3 Conclusions.. References.. 4 Protein Engineering in Microdroplets.. 4.1 Introduction.. 4.2 Droplet Formats.. 4.2.1 "Bulk" Emulsions.. 4.2.1.1 Catalytic Selections Involving DNA Substrates.. 4.2.1.2 Using the Droplet Compartment to Form a Permanent Genotype-Phenotype Linkage for Selections of Binders.. 4.2.2 Double "Bulk" Emulsions.. 4.2.3 Microfluidic Droplets.. 4.3 Perspectives.. Acknowledgments.. References

5 Folding and Dynamics of Engineered Proteins.. 5.1 Introduction.. 5.2 Proof-of-Principle Protein Designs.. 5.2.1 FSD-1, a Heterogeneous Native State and Complicated Folding Pathway.. 5.2.2 α3D, a Dynamic Core Leads to Fast Folding and Thermal Stability.. 5.2.3 Three-Helix Bundle Thermostabilized Proteins.. 5.2.4 Top7, a Novel Fold Topology.. 5.2.5 Other Rosetta Designs.. 5.3 Proteins Designed for Function.. 5.3.1 Ligands.. 5.3.1.1 Metal-Binding Four-Helix Bundles, the Effectiveness of Negative Design.. 5.3.1.2 Peptide Binding.. 5.3.2 Enzymes.. 5.3.2.1 Retro-Aldol Enzyme, Accommodating a Two-Step Reaction.. 5.3.2.2 Kemp Elimination Enzyme, Rigid Active Site Geometry Promotes Catalysis.. 5.4 Conclusions and Outlook.. Acknowledgments.. References.. 6 Engineering Protein Stability.. 6.1 Introduction.. 6.2 Power and Scope of Protein Engineering to Enhance Stability.. 6.2.1 Thermal Stabilizations.. 6.2.1.1 Potential Therapeutics: Rational Design with Computational Support.. 6.2.1.2 Analytical Tools: Green Fluorescent Protein and Luciferase.. 6.2.1.3 "Stiffening" a Protein by Gly-to-Pro Replacement: Methyl Parathion Hydrolase.. 6.2.2 Thermal Is Not the Only Stability: Oxidative and Other Chemical Stabilities.. 6.2.2.1 Oxidative Stability.. 6.2.2.2 Stabilization against Aldehydes and Solvents.. 6.2.2.3 Alkaline Tolerance.. 6.3 Measurement of a Protein's Kinetic Stability.. 6.3.1 Materials and General Hints.. 6.3.2 Thermal Stability.. 6.3.2.1 Thermal Profile.. 6.3.2.2 Thermal Inactivation.. 6.3.3 Measurement of Oxidative Stability.. 6.3.4 Stability Analysis and Accelerated Degradation Testing.. 6.3.4.1 Set-Up.. 6.3.4.2 Analysis of Results.. 6.4 Developments in Protein Stabilization.. References.. 7 Enzymes from Thermophilic Organisms.. 7.1 Introduction.. 7.2 Hyperthermophiles.. 7.3 Enzymes from Thermophiles and Their Reactions.. 7.4 Production of Proteins from (Hyper Thermophiles

7.5 Protein Engineering of Thermophilic Proteins.. 7.6 Cell Engineering in Hyperthermophiles.. 7.7 Future Perspectives.. References.. 8 Enzyme Engineering by Cofactor Redesign.. 8.1 Introduction.. 8.2 Natural Cofactors: Types, Occurrence, and Chemistry.. 8.3 Inorganic Cofactors.. 8.4 Organic Cofactors.. 8.5 Redox Cofactors.. 8.5.1 Nicotinamide Cofactor Engineering.. 8.5.2 Heme Cofactor Engineering.. 8.5.2.1 Reconstitution of Myoglobin.. 8.5.2.2 Artificial Metalloproteins Based on Serum Albumins.. 8.5.3 Flavin Cofactor Engineering.. 8.6 Concluding Remarks.. References.. 9 Biocatalyst Identification by Anaerobic High-Throughput Screening of Enzyme Libraries and Anaerobic Microorganisms.. 9.1 Introduction.. 9.2 Oxygen-Sensitive Biocatalysts.. 9.2.1 Flavoproteins.. 9.2.2 Iron-Sulfur-Containing Proteins.. 9.2.3 Other Causes of Oxygen Sensitivity.. 9.3 Biocatalytic Potential of Oxygen-Sensitive Enzymes and Microorganisms.. 9.3.1 Old Yellow Enzymes (OYEs.. 9.3.2 Enoate Reductases.. 9.3.3 Other Enzymes.. 9.3.4 Whole-Cell Anaerobic Fermentations.. 9.4 Anaerobic High-Throughput Screening.. 9.4.1 Semi-Anaerobic Screening Protocols.. 9.4.2 Anaerobic Robotic High-Throughput Screening.. 9.4.2.1 Purified Enzyme versus Whole-Cell Extracts.. 9.4.2.2 Indirect Kinetic Screening versus Direct Product Determination.. 9.4.3 Potential Extensions of Robotic Anaerobic High-Throughput Screening.. 9.5 Conclusions and Outlook.. References.. 10 Organometallic Chemistry in Protein Scaffolds.. 10.1 Introduction.. 10.1.1 Concept.. 10.1.2 Considerations for Designing an Artificial Metalloenzyme.. 10.1.2.1 Organometallic Complex.. 10.1.2.2 Biomolecular Scaffold.. 10.1.2.3 Anchoring Strategy.. 10.1.2.4 Advantages and Disadvantages of the Different Anchoring Modes.. 10.1.2.5 Spacer.. 10.1.3 Other Key Developments in the Field.. 10.1.4 Why Develop Artificial Metalloenzymes?.. 10.2 Protocol/Practical Considerations.. 10.2.1 Protein Scaffold

10.2.1.1 Determination of Free Binding Sites.. 10.2.2 Organometallic Catalyst.. 10.2.2.1 Synthesis of [Cp*Ir(biot-p-LCl].. 10.2.2.2 N'-: p. -Biotinamidophenylsulfonyl-Ethylenediamine TFA Salt 10.2.3 Combination of Biotinylated Metal Catalyst and Streptavidin.. Host.. 10.2.3.1 Binding Affinity of the Biotinylated Complex to Streptavidin.. 10.2.4 Catalysis.. 10.2.4.1 Catalysis Controls.. 10.3 Goals.. 10.3.1 Rate Acceleration.. 10.3.2 High-Throughput Screening.. 10.3.2.1 Considerations for Screening of Artificial Metalloenzymes.. 10.3.3 Expansion of Substrate Scope.. 10.3.4 Upscaling.. 10.3.5 Potential Applications.. 10.4 Summary.. Acknowledgments.. References.. 11 Engineering Protease Specificity.. 11.1 Introduction.. 11.1.1 Overview.. 11.1.2 Some Basic Points.. 11.1.2.1 Mechanism for a Serine Protease.. 11.1.2.2 Measuring Specificity.. 11.1.2.3 Binding Interactions.. 11.1.3 Nature versus Researcher.. 11.1.3.1 P1 Specificity of Chymotrypsin-like Proteases.. 11.1.3.2 The S1 Site of Subtilisin.. 11.1.3.3 The S4 Site of Subtilisin.. 11.1.3.4 Other Subsites in Subtilisin.. 11.1.3.5 Kinetic Coupling and Specificity.. 11.2 Protocol and Practical Considerations.. 11.2.1 Remove and Regenerate.. 11.2.2 Engineering Highly Stable and Independently Folding Subtilisins.. 11.2.3 Engineering of P4 Pocket to Increase Substrate Specificity.. 11.2.4 Destroying the Active Site in Order to Save It.. 11.2.5 Identifying a Cognate Sequence for Anion-Triggered Proteases Using the Subtilisin Prodomain.. 11.2.6 Tunable Chemistry and Specificity.. 11.2.7 Purifi cation Proteases Based on Prodomain-Subtilisin Interactions and Triggered Catalysis.. 11.2.8 Design of a Mechanism-Based Selection System.. 11.2.8.1 Step 1: Ternary Complex Formation.. 11.2.8.2 Step 2: Acylation.. 11.2.8.3 Steps 3 and 4: Deacylation and Product Release.. 11.2.9 Evolving Protease Specificity Regulated with Anion Cofactors by Phage Display

11.2.9.1 Construction and Testing of Subtilisin Phage.. 11.2.9.2 Random Mutagenesis and Transformation.. 11.2.9.3 Selection of Anions.. 11.2.9.4 Evolving the Anion Site.. 11.2.9.5 Catch-and-Release Phage Display.. 11.2.9.6 Conclusions.. 11.2.10 Evolving New Specificities at P4.. 11.3 Concepts, Challenges, and Visions on Future Developments.. 11.3.1 Design Challenges.. 11.3.2 Challenges in Directed Evolution.. 11.3.2.1 One Must Go Deep into Sequence Space.. 11.3.2.2 Methods Which Maximize Substrate Binding Affinity Are Not Productive.. 11.3.2.3 The Desired Protease May Be Toxic to Cells.. 11.3.3 The Quest for Restriction Proteases.. 11.3.3.1 Not All Substrate Sequences Are Created Equal.. 11.3.4 Final Thoughts: Gilded or Golden?.. Acknowledgments.. References.. 12 Polymerase Engineering: From PCR and Sequencing to Synthetic Biology.. 12.1 Introduction.. 12.2 PCR.. 12.3 Sequencing.. 12.3.1 First-Generation Sequencing.. 12.3.2 Next-Generation Sequencing Technologies.. 12.4 Polymerase Engineering Strategies.. 12.5 Synthetic Informational Polymers.. References.. 13 Engineering Glycosyltransferases.. 13.1 Introduction to Glycosyltransferases.. 13.2 Glycosyltransferase Sequence, Structure, and Mechanism.. 13.3 Examples of Glycosyltransferase Engineering.. 13.3.1 Chimeragenesis and Rational Design.. 13.3.2 Directed Evolution.. 13.3.2.1 Fluorescence-Based Screening.. 13.3.2.2 Reverse Glycosylation Reactions.. 13.3.2.3 ELISA-Based Screens.. 13.3.2.4 pH Indicator Assays.. 13.3.2.5 Chemical Complementation.. 13.3.2.6 Low-Throughput Assays.. 13.4 Practical Considerations for Screening Glycosyltransferases.. 13.4.1 Enzyme Expression and Choice of Expression Vector.. 13.4.2 Provision of Acceptor and NDP-donor Substrate.. 13.4.3 General Considerations for Microplate-Based Screens.. 13.4.4 Promiscuity, Proficiency, and Specificity

13.5 Future Directions and Outlook.. References.. 14 Protein Engineering of Cytochrome P450 Monooxygenases.. 14.1 Cytochrome P450 Monooxygenases.. 14.1.1 Introduction.. 14.1.2 Catalytic Cycle of Cytochrome P450 Monooxygenases.. 14.1.3 Redox Partner Proteins.. 14.2 Engineering of P450 Monooxygenases.. 14.2.1 Molecular Background for P450 Engineering.. 14.2.2 Altering Substrate Selectivity and Improving Enzyme Activity.. 14.2.2.1 Rational and Semi-Rational Design.. 14.2.2.2 Directed Evolution and Its Combination with Computational Design.. 14.2.2.3 Decoy Molecules.. 14.2.3 Improving Solvent and Temperature Stability of P450 Monooxygenases.. 14.2.3.1 Solvent Stability.. 14.2.3.2 Thermostability.. 14.2.4 Improving Recombinant Expression and Solubility of P450 Monooxygenases.. 14.2.4.1 N-Terminal Modifications.. 14.2.4.2 Modifications within the F-G Loop.. 14.2.4.3 Improving Expression by Rational Protein Design and Directed Evolution.. 14.2.5 Engineering the Electron Transport Chain and Cofactors of P450s.. 14.2.5.1 Genetic Fusion of Proteins.. 14.2.5.2 Enzymatic Fusion and Self-Assembling Oligomers.. 14.3 Conclusions.. References.. 15 Progress and Challenges in Computational Protein Design.. 15.1 Introduction.. 15.2 The Technique of Computational Protein Design.. 15.2.1 Principles of Protein Design.. 15.2.2 A Brief Review of Force-Fields for CPD.. 15.2.3 Optimization Algorithms for Fixed-Backbone Protein Design (P1'.. 15.3 Protein Core Redesign, Structural Alterations, and Thermostabilization.. 15.3.1 Protein Core Redesign and de novo Fold Design.. 15.3.2 Computational Alteration of Protein Folds.. 15.3.2.1 Loop Grafting.. 15.3.2.2 de novo Loop Design.. 15.3.2.3 Fold Switching.. 15.3.2.4 Fold Alteration: Looking Ahead.. 15.3.2.5 Computational Optimization of the Thermostability of Proteins.. 15.4 Computational Enzyme Design.. 15.4.1 de novo Enzyme Design.. 15.4.1.1 Initial Proofs-of-Concept

15.4.1.2 Review of Recent Developments.. 15.4.2 Computational Redesign of the Substrate Specificity of Enzymes.. 15.4.2.1 Fixed-Backbone and Flexible-Backbone Substrate Specificity Switches.. 15.4.2.2 Limitations and Feedback Obtained from Experimental Optimization Attempts.. 15.4.3 Frontiers in Computational Enzyme Design.. 15.5 Computational Protein-Protein Interface Design.. 15.5.1 Natural Protein-Protein Interfaces Redesign.. 15.5.2 Two-Sided de novo Design of Protein Interfaces.. 15.5.3 One-Sided de novo Design of Protein Interfaces.. 15.5.4 Frontiers in Protein-Protein Interaction Design.. 15.6 Computational Redesign of DNA Binding and Specificity.. 15.7 Conclusions.. References.. 16 Simulation of Enzymes in Organic Solvents.. 16.1 Enzymes in Organic Solvents.. 16.2 Molecular Dynamics Simulations of Proteins and Solvents.. 16.3 The Role of the Solvent.. 16.4 Simulation of Protein Structure and Flexibility.. 16.5 Simulation of Catalytic Activity and Enantioselectivity.. 16.6 Simulation of Solvent-Induced Conformational Transitions.. 16.7 Challenges.. 16.8 The Future of Biocatalyst Design.. References.. 17 Engineering of Protein Tunnels: The Keyhole-Lock-Key Model for Catalysis by Enzymes with Buried Active Sites.. 17.1 Traditional Models of Enzymatic Catalysis.. 17.2 Definition of the Keyhole-Lock-Key Model.. 17.3 Robustness and Applicability of the Keyhole-Lock-Key Model.. 17.3.1 Enzymes with One Tunnel Connecting a Buried Active Site to the Protein Surface.. 17.3.2 Enzymes with More than One Tunnel Connecting a Buried Active Site to the Protein Surface.. 17.3.3 Enzymes with One Tunnel Between Two Distinct Active Sites.. 17.4 Evolutionary and Functional Implications of the Keyhole-Lock-Key Model

17.5 Engineering Implications of the Keyhole-Lock-Key Model.. 17.5.1 Engineering Activity.. 17.5.2 Engineering Specificity.. 17.5.3 Engineering Stereoselectivity.. 17.5.4 Engineering Stability.. 17.6 Software Tools for the Rational Engineering of Keyholes.. 17.6.1 Analysis of Tunnels in a Single Protein Structure.. 17.6.2 Analysis of Tunnels in the Ensemble of Protein Structures.. 17.6.3 Analysis of Tunnels in the Ensemble of Protein-Ligand Complexes.. 17.7 Case Studies with Haloalkane Dehalogenases.. 17.8 Conclusions.. References.. Index..

This introduction collects 17 innovative approaches to engineer 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 editors, resulting in a superb overview of this burgeoning field. eng