Yeast surface display : methods and protocols

This detailed volume explores a wide variety of applications of yeast surface display, an extensively used protein engineering technology. Beginning with detailed protocols for the construction and efficient selection/screening of yeast surface display libraries, as well as for the analysis of individual yeast-displayed protein variants, the book continues with protocols describing the selection of yeast surface display libraries for binding to mammalian cells or to extracellular matrix as well as protocols for a broad spectrum of specialized yeast surface display applications, demonstrating the versatility of this display platform. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible methodologies, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Yeast Surface Display serves as a comprehensive resource that enables the implementation of this powerful and versatile technique in virtually any molecular biology laboratory, even in the absence of any prior yeast surface display experience. Preface Contents Contributors Part I: Introduction to Yeast Surface Display and Its Applications Chapter 1: Yeast Surface Display: New Opportunities for a Time-Tested Protein Engineering System 1 Overview of the Yeast Display Approach 2 Methodological Extensions and Developments 3 Application of Yeast Display to Antibody Engineering 4 Engineering Thermostability and Secretion Efficiency 5 YSD to Engineer Non-antibody Proteins 6 Enzyme Engineering by YSD 7 Applications in T Cell Antigen Presentation and Yeast-Based Vaccines 8 Screening cDNA or Natural Protein Libraries 9 Combining YSD and Deep Sequencing 10 Yeast Display in the Rapid Response to the SARS-CoV-2 Pandemic 11 Future Prospects References Part II: Construction and Selection of Yeast Surface Display Libraries and Analysis of Isolated Variants Chapter 2: Yeast Surface Display for Protein Engineering: Library Generation, Screening, and Affinity Maturation 1 Introduction 2 Materials 2.1 Yeast Library Growth and Induction 2.2 Library Enrichment by Magnetic Bead Selections 2.3 FACS Selection (Equilibrium) 2.4 Identification and Characterization of Single Clones 2.5 Library Generation 3 Methods 3.1 Yeast Library Growth and Induction 3.2 Library Enrichment by Magnetic Bead Selections 3.2.1 Antigen Biotinylation 3.2.2 Negative Selection Against Magnetic Beads 3.2.3 Preparation of Antigen-Coated Beads 3.2.4 Positive Selection Against Target Antigen 3.3 FACS Selection (Equilibrium) 3.3.1 FACS Selection for Enrichment of Full-Length Binders 3.3.2 FACS Selection for Enrichment of Higher-Affinity Binders to Target Antigen 3.4 Identification and Characterization of Single Clones 3.4.1 Identification of Single Clones via DNA Extraction, Transformation, and Sequencing 3.4.2 Transformation and Characterization of Single Clones Displayed on Yeast 3.4.3 Further Characterization of Single Clones 3.5 Library Generation 3.5.1 Library Construction via Error-Prone PCR 3.5.2 Preparation and Transformation of Electrocompetent Yeast 3.6 FACS Enrichment of Binders for Affinity Maturation 3.6.1 Perform Equilibrium Sort(s) to Enrich for Full-Length Clones that Bind to Antigen of Interest 3.6.2 Measure koff of the Library to Determine the Proper Time Scale for Kinetic Sorting 3.6.3 Perform Kinetic Sorts to Enrich for Higher-Affinity Binders Based on koff 4 Notes References Chapter 3: Site-wise Diversification of Combinatorial Libraries Using Insights from Structure-guided Stability Calculations 1 Introduction 2 Materials 3 Methods 3.1 Choosing Residues of Interest 3.2 FoldX Stability Prediction 3.3 Rosetta ddg_monomer Stability Predicition 3.3.1 Structural File Preparation for High- and Low-Resolution ddg_monomer 3.3.2 Low-Resolution ddg_monomer: (See Note 14) 3.3.3 High-Resolution ddG_monomer 3.4 Library Design for Overlap Extension PCR and Electroporation into Yeast 4 Notes References Chapter 4: Ancestral Sequence Reconstruction and Alternate Amino Acid States Guide Protein Library Design for Directed Evoluti... 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 5: Machine Learning-driven Protein Library Design: A Path Toward Smarter Libraries 1 Introduction 1.1 Providing a Better Starting Point for Directed Evolution 1.2 Investigating Unexplored Parts of the Fitness Landscape 1.3 Estimating Degenerate Codon Performance via Fitness Distribution Analysis 2 Materials 3 Methods 3.1 Data Processing as an Initial Yet Pivotal Step in Any DL Algorithm 3.1.1 Input Data Refinement 3.1.2 Input Data Representation 3.1.3 Output Data Representation 3.2 Deep Learning Algorithm Selection Requires an Understanding of Each Algorithm Structure 3.2.1 Overview 3.2.2 Guidance for Building a Deep Learning Structure 3.3 Visualization Guidance 3.4 Decision Making and Evaluating Parameters 3.4.1 Hyperparameter Optimization 3.4.2 K-Fold Cross-Validation 3.5 Protein Library Construction 4 Notes References Chapter 6: Kinetic Competition Screening of Yeast-Displayed Libraries for Isolating High Affinity Binders 1 Introduction 2 Materials 2.1 Yeast Library 2.2 Media and Plates 2.3 Reagents and Buffers 2.4 Other Consumables and Equipment 3 Methods 3.1 Growth and Induction of Yeast 3.2 Measurement of Dissociation Rate Constants on the Yeast Surface 3.3 Kinetic Competition Sorting by Fluorescence-Activated Cell Sorting (FACS) 4 Notes References Chapter 7: Engineering Proteins by Combining Deep Mutational Scanning and Yeast Display 1 Introduction 2 Materials 2.1 Yeast Display Strain and Plasmid 2.2 Bacterial Strain for Propagating Plasmid DNA 2.3 DNA Purification 2.4 Restriction Enzymes 2.5 Polymerase Chain Reaction 2.6 Culture Media 2.7 Reagents for Transforming Yeast with Plasmid DNA by Heat Shock 2.8 Reagents for Preparation of Electrocompetent Yeast for Making Libraries 2.9 Electroporation 2.10 Flow Cytometry and Sorting 2.11 Illumina Deep Sequencing 2.12 Software for Deep Sequencing Data Analysis 3 Methods 3.1 Designing the Construct for Yeast Surface Display of a Protein of Interest 3.2 Transformation of EBY100 with Yeast Display Plasmid Containing the Gene of Interest 3.3 Analysis of Yeast Cells Transformed with the Gene of Interest 3.3.1 Sequencing Analysis 3.3.2 Flow Cytometric Analysis 3.4 Construction of Single Codon Libraries 3.4.1 Primer Design 3.4.2 Pre-SOE PCR 3.4.3 SOE PCR 3.4.4 Preparation of Cut Vector 3.4.5 Precipitate Vector and Insert 3.4.6 Preparation of Electrocompetent Yeast and Transformation of Library DNA 3.5 Sorting Libraries 3.6 Deep Sequencing 3.6.1 Preparation of DNA for Deep Sequencing 3.6.2 Design Primers to PCR Amplify Mutated Regions of the Gene for Paired End Illumina Sequencing 3.6.3 PCR to Add Flanking Sequences to DNA for Illumina Deep Sequencing 3.7 Deep Sequencing Data Analysis with Enrich 3.8 Targeted Mutagenesis Based on Deep Mutagenesis Data 4 Notes References Chapter 8: Engineering Binders with Exceptional Selectivity 1 Introduction 2 Materials 2.1 Yeast Labeling and Sorting 2.2 Screening of Single Yeast Clones 2.3 Sorting and Screening Instruments 3 Methods 3.1 Sorting Naïve or Enriched Yeast Library (First Round) 3.2 Fluorescence-Activated Cell Sorting Round 2 3.3 Analysis of Sorted Pool from Round 2 3.4 Negative Sorting Using Fluorescence-Activated Cell Sorting (Round 3) 3.5 Post-Negative Sorting Analysis and Optional Round 4 of Fluorescence-Activated Cell Sorting 3.6 Screening of Individual Clones 4 Notes References Chapter 9: Affinity and Stability Analysis of Yeast Displayed Proteins 1 Introduction 1.1 Ligand Depletion 1.2 Time to Equilibrium 1.3 Stability Analysis on the Surface of Yeast 2 Materials 2.1 Yeast Cells and Plasmids 2.2 Transformation of Yeast Cells 2.3 Yeast Media and Solutions 2.4 Proteins and Antibodies 3 Methods 3.1 Transformation of Yeast Cells 3.2 Culturing Yeast Cells Prior to Analysis 3.3 Determining the Affinity on the Surface of Yeast 3.4 Determining the Thermal Stability on the Surface of Yeast 4 Notes References Part III: Selection of Yeast Surface Display Libraries for Binding to Mammalian Cells or Extracellular Matrix Chapter 10: Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting 1 Introduction 2 Materials 2.1 Mammalian Cells and Media 2.2 FACS-Assisted Sorting and Panning 2.3 Reformatting, Production and Characterization 2.3.1 Reformatting via Golden Gate Assembly 2.3.2 Production and Purification of mAbs 2.3.3 Characterization of Isolated Antibodies 3 Methods 3.1 Yeast Libraries 3.2 Pre-screening: Cell Staining and Library Sorting by FACS 3.3 Yeast Biopanning 3.3.1 Cultivation of Mammalian Cells 3.3.2 Staining of A431 Cells 3.3.3 FACS-Assisted Biopanning of Mammalian and Yeast Cells 3.3.4 Target Binding Analysis of Isolated Single Clones 3.4 Reformatting, Antibody Production, and Characterization 3.4.1 Reformatting via Golden Gate Assembly 3.4.2 Production and Purification of mAbs 3.4.3 Size Exclusion Chromatography (SEC) 3.4.4 Thermal Stability 3.4.5 Unspecific Binding ELISA 3.4.6 On-Cell EC50 Determination 4 Notes References Chapter 11: A Hybrid Adherent/Suspension Cell-Based Selection Strategy for Discovery of Antibodies Targeting Membrane Proteins 1 Introduction 2 Materials 2.1 Plasticware, Glassware, and Consumables 2.2 Buffers and Yeast Media 2.3 Cell Staining Reagents 2.4 Other Reagents 2.5 Instruments 3 Methods 3.1 Round 1 Biopanning Selection (Debulking the Library) 3.2 Round 2+ MACS Selections (Enriching the Library) 3.3 Later Round FACS Selections (Fine-Tuning the Library) 3.4 Screening Individual Yeast Clones Via Biofloating 4 Notes References Chapter 12: Ligand Selection by Combination of Recombinant and Cell Panning Selection Techniques 1 Introduction 2 Materials 2.1 Yeast Surface Display Selection with Recombinant Antigen 2.1.1 Media, Buffers, and Reagents 2.1.2 Equipment and Consumables 2.1.3 Cell Lines 2.2 Yeast Surface Display Selection with Adhered Mammalian Cells 2.2.1 Media, Buffers, and Reagents 2.2.2 Equipment and Consumables 2.2.3 Cell Lines 2.3 Library Selections Combining Recombinant and Cell-Based Selection Techniques 2.3.1 Media, Buffers, and Reagents 2.3.2 Equipment and Consumables 2.3.3 Cell Lines 2.4 Clonal Specificity Characterization Using Microscopy and Cell Panning 2.4.1 Media, Buffers, and Reagents 2.4.2 Equipment and Consumables 2.4.3 Cell Lines 3 Methods 3.1 Yeast Surface Display Selection with Recombinant Antigen 3.1.1 Preparation of Biotinylated Recombinant Target Protein 3.1.2 Yeast Cell Preparation 3.1.3 Preparation of Target-Coated Magnetic Beads 3.1.4 Depletion of Non-Specific Binding Ligands from Naïve Library by Magnetic Bead Depletion 3.1.5 Enrichment of Target Binding Ligands by Magnetic Bead Selection 3.2 Yeast Surface Display Selection with Adhered Mammalian Cells 3.2.1 Target-Expressing Mammalian Cell Preparation 3.2.2 Yeast Library Preparation 3.2.3 Cell Panning Selection 3.3 Library Selections Combining Recombinant and Cell-Based Selection Techniques 3.3.1 Selection of a Target-Specific Binding Population from a Naïve Yeast Surface Display Library Using Recombinant Target 3.3.2 Selection of a Target-Specific Binding Population from an Enriched Yeast Surface Display Library Using Cell-Expressed Ta... 3.4 Clonal Specificity Characterization Using Microscopy and Cell Panning 3.4.1 Mammalian Cell Preparation 3.4.2 Yeast Clone Preparation 3.4.3 Clonal Specificity Characterization with Cell Panning and Microscopy 4 Notes References Chapter 13: Identification of Brain ECM Binding Variable Lymphocyte Receptors Using Yeast Surface Display 1 Introduction 2 Materials 2.1 Generating Mammalian ECM 2.1.1 Mammalian Cell Culture 2.1.2 Decellularizing Mammalian Cultures to Expose ECM 2.2 ECM Biopannning with a VLR YSD Library 2.2.1 Yeast Culture 2.2.2 bEnd.3 ECM Biopanning 2.3 ELISA-Based Screening for Identifying VLRs That Demonstrate Preferential Binding to bEnd.3 ECM 2.4 Verifying VLR Binding to Brain ECM Using Murine Brain Sections 3 Methods 3.1 Generating Mammalian ECM 3.1.1 Mammalian Cell Culture 3.1.2 Decellularizing Mammalian Culture Substrates to Expose ECM 3.2 ECM Biopannning with a VLR YSD Library 3.2.1 Yeast Culture 3.2.2 bEnd.3 ECM Biopanning 3.3 ELISA-Based Screen for Identifying Clonal VLRs That Demonstrate Preferential Accumulation in bEnd.3 ECM 3.4 Verification of VLR Brain ECM Binding Using Murine Brain Sections 4 Notes References Part IV: Specialized Yeast Surface Display Applications Chapter 14: Guidelines, Strategies, and Principles for the Directed Evolution of Cross-Reactive Antibodies Using Yeast Surface... 1 Introduction 2 Isolation of Cross-Reactive Antibodies from a Naïve Yeast Display Library 2.1 Choosing the Targets, the Labeling Strategy, and Antibody Library to Be Used 2.2 Isolation of Cross-Reactive Antibodies Using Highly Avid Magnetic Beads 2.3 Selection Cross-Reactive Antibodies Using Fluorescence-Activated Cell Sorting 2.4 Single Clone Analysis and Characterization Using Yeast Surface Titrations 3 Molecular Co-Evolution of Antibody Affinity and Cross-Reactivity 3.1 Generation of Genetic Diversity of Cross-Reactive Antibody Clones 3.2 Flow Cytometry Sorting to Enrich for Cross-Reactive Antibody Clones with Higher Affinity to Multiple Targets 3.3 Single-Clone Analysis and Combinatorial Site-Directed Mutagenesis 4 Conclusions References Chapter 15: Yeast Display for the Identification of Peptide-MHC Ligands of Immune Receptors 1 Introduction 2 Materials 2.1 Cloning and DNA Preparation 2.2 Yeast Construct Validation and Library Creation 2.3 Magnetic Selections 2.4 Next-Generation Sequencing Preparation 3 Methods 3.1 Designing and Validating a Peptide-MHC Construct for Yeast Display 3.1.1 Formatting MHCs for Yeast Expression 3.1.2 Validating MHC Expression and Folding on Yeast: Tetramer Staining 3.1.3 Validating MHC Expression and Folding on Yeast: Tag Enrichment 3.1.4 Optimizing MHCs for Expression on Yeast 3.2 Designing and Constructing Libraries 3.2.1 Insert Preparation: Error-Prone Mutagenesis 3.2.2 Insert Preparation: Peptide Library 3.2.3 Insert Preparation: Scale Up PCR 3.2.4 Vector Preparation 3.2.5 Library Creation 3.3 Conducting Selections 3.3.1 Inducing the Library 3.3.2 Assessing Induction 3.3.3 Starting Selections: Removing Non-specific Binders 3.3.4 TCR-Specific Selection: Round 1 3.3.5 TCR-Specific Selection: Rounds 2-3 3.3.6 TCR-Specific Selection: Rounds 4+ 3.3.7 Growing and Sequencing Selected Yeasts: Individual Yeast Colonies 3.3.8 Growing and Sequencing Selected Yeasts: Bacteria Colonies 3.4 Preparing, Processing, and Analyzing NGS Data 3.4.1 Preparing Sample for NGS 3.4.2 Processing NGS Data 3.4.3 Analyzing NGS Data 4 Notes References Chapter 16: Yeast Display Guided Selection of pH-Dependent Binders 1 Introduction 2 Materials 2.1 Strains and Libraries 2.2 Media and Buffers 2.3 Reagents 2.4 Equipment 3 Methods 3.1 Yeast Library Preparation and Display 3.2 Selection of Binder Pool Through Magnetic-Activated Cell Sorting 3.2.1 Preparation of Beads 3.2.2 Preparation of Cells 3.2.3 Magnetic Selection 3.3 FACS: Selection of Higher Affinity Binders to the Antigen of Interest 3.3.1 Cell Preparation 3.3.2 Antigen and Primary Antibody Labeling 3.3.3 Secondary Antibody Labeling (See Note 11) 3.3.4 Sorting 3.4 FACS: Isolation of Mutants That Lose Binding at Low pH 3.4.1 Cell Preparation 3.4.2 Antigen and Primary Antibody Labeling 3.4.3 Secondary Antibody Labeling 3.4.4 Sorting 3.5 FACS: Isolation of pH-Dependent Binders Through Endpoint Sorting 3.5.1 Cell Preparation 3.5.2 Antigen and Primary Antibody Labeling 3.5.3 Secondary Antibody Labeling 3.5.4 Sorting 3.6 Characterization 3.6.1 Sequencing 3.6.2 Single Clone Analysis 3.6.3 Biophysical Characterization of Selected Clones 4 Notes References Chapter 17: Yeast Mating as a Tool for Highly Effective Discovery and Engineering of Antibodies via Display Methodologies 1 Introduction 2 Materials 2.1 Yeast Strains 2.2 Media 2.3 Reagents and Equipment 2.4 Primers 3 Methods 3.1 Amplification of Insert DNA for HC and LC Libraries 3.2 Enzymatic Digestion for Vector Preparation 3.3 Electroporation for Haploid Library Construction 3.4 Purification of KRasG12D 3.5 GppNHp and GDP Incubation with Purified KRasG12D 3.6 MACS Screening of HC Library Against KRasG12D-GppNHp 3.7 FACS Screening of HC Library Against KRasG12D-GppNHp 3.8 Mating of the Enriched HC Library 3.8.1 Mating of the Enriched HC Library with a Fixed LC with Cytosol-Penetrating Ability 3.8.2 Mating of the Enriched HC Library with the Initial LC Library 3.9 Sequencing of VH and VL 4 Notes References Chapter 18: Humanization of Chicken-Derived Antibodies by Yeast Surface Display 1 Introduction 2 Materials 2.1 Cloning of Humanized VH and VL Domains 2.2 Construction of Yeast Surface Display Library 2.2.1 Humanized scFv Library-Specific Materials 2.2.2 Humanized Fab Library Specific Materials 2.3 Library Screening of Humanized scFvs/Fabs 2.4 Next-Generation Sequencing 2.5 Reformatting, Expression, Purification, and Characterization 3 Methods 3.1 In Silico Humanization of Chicken-Derived Antibodies 3.2 Cloning of Humanized VH and VL Domains 3.3 Construction of Yeast Surface Display Library 3.3.1 Preparation for the Generation of Humanized scFv Libraries 3.3.2 Preparation for the Generation of Humanized Fab Libraries 3.3.3 Yeast Transformation 3.4 Library Staining and Sorting 3.4.1 Cell Staining 3.4.2 Screening by FACS 3.5 Next-Generation Sequencing 3.5.1 Sample Preparation 3.5.2 NGS Data Interpretation 3.6 Reformatting, Expression, Purification, and Characterization 3.6.1 Reformatting of Humanized scFv- and Fab-Based Antibodies 3.6.2 Production and Purification of Humanized Antibodies 3.6.3 Characterization of Humanized Antibodies 4 Notes References Chapter 19: Engineering Tissue Inhibitors of Metalloproteinases Using Yeast Surface Display 1 Introduction 2 Materials 2.1 Expression of MMP-3 Catalytic Domain 2.2 Inclusion Body Extraction and Solubilization of MMP-3 Catalytic Domain 2.3 Purification of MMP-3 Catalytic Domain 2.4 Refolding of MMP-3 Catalytic Domain 2.5 Re-concentration of MMP-3 Catalytic Domain 2.6 APMA Activation of MMP-3 Catalytic Domain 2.7 Desalting of MMP-3 Catalytic Domain 2.8 Biotinylation of MMP-3 Catalytic Domain 2.9 Generation of TIMP-1 Library and Cell Growth 2.10 Yeast Surface Display and Cell Growth 2.11 Cell Preparation for Flow Cytometry 2.12 Flow Cytometry 2.13 DNA Preparation and Evaluation of Individual Clones 3 Methods 3.1 Expression, Purification, Solubilization, and Biotinylation of MMP-3 Catalytic Domain (MMP-3cd) 3.1.1 Expression of MMP-3cd Protein 3.1.2 Extraction of Insoluble MMP-cd from Inclusion Bodies 3.1.3 Purify and Refold MMP-3cd 3.1.4 Re-concentration, Activation, and Desalting of MMP-3cd 3.1.5 Biotinylation of MMP-3cd 3.2 Design and Generation of TIMP-1 Variant Library 3.2.1 Inserting the TIMP-1 Gene in the pCHA Yeast Display Vector 3.2.2 Design Targeted Library of TIMP-1 Random Mutants 3.2.3 Construct a Library of Human TIMP-1 Gene Variants 3.2.4 Preparation of EBY100 Culture for Electrotransformation 3.2.5 Electrotransformation of the TIMP-1 Variant Library into the Yeast Strain EBY100 Cells 3.3 Preparation of TIMP-1 Variant Yeast Surface Display Library 3.3.1 Passage of the TIMP-1 Variant Library 3.3.2 Preparation of Frozen Glycerol Stocks 3.3.3 Induction of the TIMP-1 Variant Library 3.4 FACS Screen of TIMP-1 Variant Library Toward MMP-3cd Binding 3.4.1 Preparation of Induced TIMP-1 Variant Library for Immunolabeling 3.4.2 Bind Biotinylated MMP-3cd to the TIMP-1 Variant Displayed Protein, and Strep-AF647 Labeling 3.4.3 Run Flow Cytometry and Screen for TIMP-1 Variant Populations of Interest 3.4.4 Set the Sorting Gate 3.4.5 Screen the TIMP-1 Variant Library Population 3.4.6 Recover the Sorted TIMP-1 Variant Library 3.4.7 Test the Sorted TIMP-1 Variant Library for Improved MMP-3 Binding 3.5 DNA Preparation and Evaluation of Individual TIMP-1 Variant Clones 3.5.1 Extraction of DNA Plasmids from the Sorted TIMP-1 Variant Clones and Transformation into E. coli Cells 3.5.2 Purification of Extracted TIMP Variant DNA from Yeast Library and Sanger Sequencing 3.6 Evaluating Isolated TIMP Variants for Improved MMP Binding 3.6.1 Growth and Induction of TIMP-1 Variant Clones 3.6.2 Labeling the Yeast Displayed TIMP-1 Variant Clones and Testing for Improved MMP-3cd Binding via Flow Cytometry 3.6.3 Drawing the Binding Curve 3.6.4 Comparison of TIMP-1 Variants and WT-TIMP-1 for Binding to MMP-3cd Binding 4 Notes References Chapter 20: Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries 1 Introduction 2 Materials 2.1 Yeast Strains and Plasmids 2.2 Yeast Media and Plates 2.3 Molecular Cloning to Achieve Peptide Yeast Surface Display 2.4 Yeast Transformation and Freezing 2.5 Cyclization of Yeast-Displayed Linear Peptide Precursors 2.6 Preparation of DNA for Constructing a Yeast Display Combinatorial Library of Linear Peptide Precursors 2.7 Construction of a Yeast Display Combinatorial Library of Linear Peptide Precursors 2.8 Magnetic Selection of Yeast Combinatorial Library 2.9 Fluorescence-Activated Cell Sorting of Yeast Combinatorial Library 2.10 Identification of Individual Clones Isolated from Combinatorial Screen 2.11 Binding Affinity Estimation of Individual Cyclic Peptide Mutants via Yeast Surface Titration 3 Methods 3.1 Cloning a Linear Peptide Sequence into pCTCON-Nterm-Peptide for Yeast Display 3.2 Yeast Transformation and Preparation of Frozen Yeast Stocks 3.3 Cyclization of Linear Peptide Precursors Displayed as Yeast Surface Fusions Using DSG 3.4 Preparation of DNA When Constructing a Yeast Display Combinatorial Library of Linear Peptide Precursors 3.5 Construction of a Yeast Display Combinatorial Library of Linear Peptide Precursors 3.6 Magnetic Selection of a Chemically Crosslinked Yeast Peptide Library 3.7 Fluorescence-Activated Cell Sorting of a Chemically Crosslinked Yeast Peptide Library 3.8 Identification of Individual Clones Isolated from Combinatorial Screening 3.9 Binding Affinity Estimation of Individual Cyclic Peptide Mutants via Yeast Surface Titration 4 Notes References Chapter 21: Generation of Thermally Stable Affinity Pairs for Sensitive, Specific Immunoassays 1 Introduction 2 Materials 2.1 Buffer/Medium Solutions 2.2 Plasmids and Cells 3 Methods 3.1 Antigen Sourcing, Yeast Library Culture, and Magnetic Bead Sorting 3.1.1 Library Revival, Passaging, and Induction (4 Days) 3.1.2 Positive Magnetic Bead Sorting (2 Days) 3.1.3 Library Density/Cell Viability Determination 3.1.4 Remove Dynabeads and Passage Cells (1 Day) 3.1.5 Determine Library Diversity, Passage, and Induce Library (2 Days) 3.1.6 Negative and Positive Bead Sort (1 Day) 3.1.7 Continued Magnetic Sorting (Variable) 3.2 Fluorescence-Activated Cell Sorting (FACS) 3.2.1 Culture and Induce Yeast Display Library (Variable) 3.2.2 FACS Sample Preparation (1 Day) 3.2.3 FACS Sorting 3.2.4 Additional FACS Sorts 3.3 Variant Identification 3.3.1 Variant Isolation and Sequence Determination 3.3.2 Sequence Individual Clones 3.3.3 Generation of Competent Yeast Cells 3.3.4 Transformation into Yeast Competent Cells 3.4 Flow Cytometry Titration Assay to Determine Apparent Dissociation Constant 3.4.1 Yeast Culture and Induction 3.4.2 Yeast Display Flow Cytometry Titration Analysis 3.5 Cloning Selected Variants into E. coli Expression Vector 3.5.1 Engineering the Capture Binder Plasmid Backbone 3.5.2 Engineering Reporter Binder Plasmid Backbone 3.5.3 Clone Different Variants of rcSso7d into Engineered Plasmid Backbones 3.6 Binder Protein Overexpression and Purification 3.6.1 Binder Growth and Overexpression 3.6.2 Binder Purification 3.7 Selection of Affinity Pairs 3.7.1 Synthesis and Purification of Biotinylated E1 Binder 3.7.2 Negative and Positive Magnetic Bead Sorts (E2 Binder) 3.7.3 FACS for E2 Binders 3.7.4 Identification and Characterization of E2 Binders and Cloning 3.8 Assay Development 3.8.1 Preparation of Commercially Available Cellulose Paper to Produce Vertical Flow Assay Strips 3.8.2 Assay Methodology and Standard Curve Development 4 Notes References Chapter 22: Isolating Anti-amyloid Antibodies from Yeast-Displayed Libraries 1 Introduction 2 Materials 2.1 Initial Library Preparation and Sorting 2.2 Antibody Identification and Characterization 2.2.1 Cloning Antibody Genes into Mammalian Expression Plasmid 2.2.2 Antibody Expression, Purification, and Characterization 2.2.3 Antibody Binding 2.2.4 Affinity Maturation 3 Methods 3.1 Library Preparation and Sorting 3.1.1 Antigen Bead Preparation 3.1.2 Library Preparation and Screening 3.1.3 Initial Library Sorting 3.2 Antibody Identification and Characterization 3.2.1 Antibody Library Sub-Cloning 3.2.2 Antibody Expression and Purification 3.2.3 Antibody Binding Analysis 3.3 Affinity Maturation 3.3.1 Design of Sub-Libraries 3.3.2 Preparing Libraries 3.3.3 Affinity Maturation Library Sorting 3.3.4 Clone Evaluation of Affinity and Conformational Specificity 4 Notes References Chapter 23: Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface Abbreviations 1 Introduction 2 Materials 2.1 Site-Specific Incorporation of NcAAs into Proteins of Interest in Yeast 2.2 Design and Construction of Protein Libraries Containing NcAAs 2.3 Bioorthogonal Reactions and Techniques for Evaluating Efficiency of Click Chemistry Reactions 2.4 Library Screening on the Yeast Surface 2.5 Secreting Soluble Proteins Containing NcAAs 3 Methods 3.1 Site-Specific Incorporation of NcAAs into Proteins of Interest in Yeast 3.2 Design and Construction of Protein Libraries Containing NcAAs 3.2.1 Libraries for Isolating Protein Binders Containing ncAAs 3.2.2 Yeast Display Library Construction Via Homologous Recombination 3.2.3 Libraries for Evolving AaRSs with Desired Specificity Profiles 3.3 Bioorthogonal Reactions and Techniques for Evaluating Efficiency of Click Chemistry Reactions 3.4 Library Screening on the Yeast Surface 3.4.1 Bead-Based Techniques for Identifying Binders from ncAA-Containing Yeast Display Libraries (Fig. 4) 3.4.2 Isolation of Mutant aaRSs with Desired Specificity Profiles Via FACS (Fig. 5) 3.5 Secreting Soluble Proteins Containing NcAAs 4 Notes References Chapter 24: Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of C... 1 Introduction 1.1 Biogenesis and Function of Extracellular Vesicles 1.2 EV Tetraspanins and Their Structural Elements 1.3 CD81 LEL as a Stand-Alone Antigen Recognition Unit 1.3.1 Structural Features of CD81 LEL 1.3.2 Peptide Grafting for Functionalization of CD81 LEL Towards Antigen Recognition 1.3.3 Stabilization of CD81 LEL 1.3.4 Display of Combinatorial Libraries and Affinity Maturation of CD81 LEL-Based Binders 1.3.5 Conversion of Yeast-Display Identified CD81 LEL Binders to EV Surface Molecules 1.4 Future Prospects in the Development of CD81 LEL as an Antigen-Recognition Unit 2 Materials 2.1 Reagents 2.2 Solutions and Buffers 2.3 Media 2.4 Kits 2.5 Equipment 2.6 Plasmids, Bacterial Strains, Yeast Strains, and Cell Lines 3 Methods 3.1 CD81 LEL Yeast Display Library Construction 3.1.1 Design of Recipient Vector 3.1.2 Library-Encoding PCR Fragment Preparation 3.1.3 Yeast Transformation 3.2 Quality Control and Sorting of CD81 LEL Yeast Display Libraries 3.2.1 Sequencing of Library Clones 3.2.2 Staining of Displayed CD81 Mutants 3.2.3 Selection of CD81 LEL Libraries 3.3 Determining Thermal Stability of CD81 LEL Mutants Displayed on Yeast Surface 3.4 Expression of Selected Library Clones in Mammalian Expression System 4 Notes References Chapter 25: Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display 1 Introduction 2 Materials 2.1 General 2.2 Molecular Biology 2.3 Library Expression, Screening, and Selection in Yeast 2.4 Expression and Purification of FAST Variants 3 Methods 3.1 Construction of the Library 3.1.1 Saturation Mutagenesis 3.1.2 Error-Prone PCR 3.1.3 DNA Shuffling 3.1.4 Vector and Insert Digestion 3.1.5 Construction of a Test Library 3.1.6 Analysis of the Mutagenesis Quality of the Test Library 3.1.7 Construction of the Large-Scale Library in Bacteria 3.1.8 Construction of the Large-Scale Library in Yeast 3.2 Library Expression in Yeast 3.2.1 Amplification and Induction 3.2.2 Immunolabeling 3.2.3 Sorting and Iterative Rounds of Flow Cytometry 3.3 Screening of Clones After FACS Rounds 3.3.1 Fluorescence Analysis of Selected Clones 3.3.2 DNA Analysis of the Selected Clones 3.4 Characterization of the Selected Clones 3.4.1 Cloning into pET28a 3.4.2 Protein Production and Purification 3.4.3 Determination of the Dissociation Constant of the Tag:Fluorogens Assembly 3.4.4 Determination of the Fluorescence Quantum Yield of the Tag:Fluorogens Assembly 4 Notes References Chapter 26: Simultaneous Display of Multiple Kinds of Enzymes on the Yeast Cell Surface for Multistep Reactions 1 Introduction 2 Materials 2.1 Media 2.2 Strains and Plasmids 2.3 Plasmid Construction 2.4 Sequencing Primers 2.5 Yeast Transformation with the Constructed Plasmid for Cell Surface Display 2.6 Immunofluorescence Labeling of Cells 2.7 Measurement of Display Efficiency 2.8 Measurement of Cellulase Activity 3 Methods 3.1 Construction of the Plasmids for Yeast Surface Display of Cellulases 3.1.1 Construction on a Multi-copy Plasmid 3.1.2 Transfer the Fusion Genes to a Genome Integration Plasmid 3.2 Yeast Transformation with the Constructed Plasmid for Cell Surface Display 3.3 Immunofluorescence Labeling of Cells 3.4 Measurement of Display Efficiency 3.5 Measurement of Activity of Displayed Cellulases on the Yeast Cell Surface 3.5.1 Measurement of β-Glucosidase Activity 3.5.2 Measurement of Endoglucanase and Cellobiohydrolase Activity 4 Notes References Correction to: Yeast Surface Display for Protein Engineering: Library Generation, Screening, and Affinity Maturation Index