Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols (Methods in Molecular Biology, 2676)

✍ Scribed by Yu-Hsuan Tsai (editor), Simon J. Elsässer (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 287
• Category: Library

✦ Synopsis

This detailed volume explores non-canonical amino acids (ncAAs) through their site-specific incorporation by genetic code expansion (GCE). The collection provides a broad resource of methods for implementing GCE in E. coli, mammalian cells, and animals, highlighting specific applications ranging from fluorescence labeling to photocontrol and the study of protein post-translational modification. 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 and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols serves as an ideal source of methodologies that can be adapted and extended, migrated to different model systems, and combined in new ways to help explore a wide range of biological questions and to augment industrial and pharmaceutical protein engineering.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Genetic Code Expansion in E. coli and Its Applications
Chapter 1: Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Var...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Chemical Competent E. coli Cells and Transformation
2.3 Gene Randomization
2.4 Screening by Intact Cell Fluorescence Readout
3 Methods
3.1 Production of Chemically Competent Cells
3.2 Focused MbPylRS Gene Randomization
3.3 Digestion and Ligation of Randomized PylRS Gene Plasmid
3.4 Transformation of Chemically Competent Cells
3.5 Screening by Intact Cell Fluorescence Readout
3.6 Comparison of Unique Constructs by Intact Cell Fluorescence Readout
4 Notes
References
Chapter 2: Engineering Homogeneous Photoactive Antibody Fragments
1 Introduction
2 Materials
2.1 Cell Lines and Strains
2.2 Equipment
2.3 Software
2.4 Reagents
2.5 Solutions and Buffers
3 Methods
3.1 Identification of Candidate Tyrosine Residues in the Antigen Binding Site of an Antibody Fragment for Replacement with Pho...
3.2 Expression of Photocaged Tyrosine-Containing Antibody Fragments in E. coli
3.2.1 Cloning of Antibody Fragment and Its Amber Mutants into pSANG10 Plasmid
3.2.2 Cloning of Photocaged Tyrosine-Specific Methanocaldococcus jannaschii Tyrosyl-tRNA Synthetase into pULTRA Suppressor Pla...
3.2.3 Test Expression of Photocaged Tyrosine Containing Antibody Fragments
3.2.4 Large Scale Expression of Photocaged Tyrosine Containing Antibody Fragments
3.3 Light-Mediated Decaging of Photoactive Antibody Fragments
3.4 Measuring the Binding of (Photocaged) Antibody Fragments Against Antigens Expressed on the Surface of Live Cells
4 Notes
References
Chapter 3: Repurposing Photosensitizer Proteins Through Genetic Code Expansion to Facilitate Photo-Biocatalysis
1 Introduction
2 Materials
2.1 Reagents, Cell Strains, and Plasmids
2.2 Instruments
2.3 Culture Medium, Stock Solution, and Buffers
3 Methods
3.1 Protein Expression Through Genetic Code Expansion
3.1.1 Plasmid Construction
3.1.2 Cell Culture and Protein Expression
3.2 Protein Purification
3.3 Characterization of the Proteins
3.3.1 UV-Vis Spectra
3.3.2 Mass Spectrometry Analysis
3.4 Artificial Photoenzyme for CO2/CO Conversion
3.4.1 Terpyridine Modification of Single-Cysteine PSP2 Mutant
3.4.2 Photo-Activated CO2/CO Conversion with PSP2-Ni(Terpyridine)
3.5 Artificial Photoenzyme for CO2/HCOOH Conversion
3.5.1 Reconstitution of the [4Fe-4S] Clusters in mPCE
3.5.2 Photo-Activated CO2/HCOOH Conversion with mPCE
3.6 Photo-Activated Dehalogenation with PSP2-Ni(Bipyridine)
4 Notes
References
Chapter 4: Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activi...
1 Introduction
2 Materials
2.1 ncAA and Plasmids
2.2 Site-Specific Incorporation of AFCouK into Proteins in E. coli
2.3 Purification of the UbAFCouK-sfGFP Probe
2.4 Analysis of Deubiquitinase Activities and Screening of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe
3 Methods
3.1 Site-Specific Incorporation of AFCouK into Proteins in E. coli
3.2 Purification of the UbAFCouK-sfGFP Probe
3.3 Fluorescence Detection of Deubiquitinase Activities Using the UbAFCouK-sfGFP Probe
3.4 Screening of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe
3.5 IC50 Analysis of Deubiquitinase Inhibitors Using the UbAFCouK-sfGFP Probe
4 Notes
References
Chapter 5: Creating Selenocysteine-Specific Reporters Using Inteins
1 Introduction
2 Creating Sec-Specific Reporters
2.1 Equipment
2.2 Reagents and Materials
3 Methods
3.1 Choosing a Reporter System
3.2 Cloning the Reporter Plasmid and Intein-Containing Variants
3.2.1 Transfer the T7-sfGFP Cassette from pET-15b to pBAD Vector
3.2.2 Identify Sites of Insertion for the M86 DnaB Mini-Intein
3.2.3 Create Cys-to-Ser Substitution
3.3 Developing the Intein Assay with Cys and Ser Variants
3.4 Test the Sec-Intein Assay for Its Specificity
4 Notes
References
Chapter 6: Protein Expression with Biosynthesized Noncanonical Amino Acids
1 Introduction
2 Materials
2.1 Aromatic Thiol Precursors
2.2 Protein Expression in E. coli
2.3 Protein Purification
2.4 Protein Site-Specific Modification
2.5 Selection
3 Methods
3.1 Synthesis of Thiol Precursors Containing Keto (Fig. 3a) or Azide Group (Fig. 3b)
3.1.1 Synthesis of 1-(4-Mercaptophenyl)Ethan-1-One
3.1.2 Synthesis of 4-Azidobenzenethiol
3.2 Expression
3.3 Purification
3.4 Reaction
3.4.1 Conjugation Reaction of Azido Group Containing Proteins and DBCO-mPEG20K
3.4.2 Conjugation Reaction of Azido Group Containing Proteins and DBCO-CY5
3.4.3 Conjugation Reaction of Ketone Group Containing Poteins and Hydroxyamine-mPEG20K
3.5 Selection
4 Notes
References
Chapter 7: Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into ...
1 Introduction
2 Materials
2.1 Co-transformation
2.2 Protein Expression
2.3 Protein Purification
2.4 Protein Labeling
3 Methods
3.1 Co-transformation
3.2 Protein Expression
3.3 Protein Purification
3.4 Protein Labeling by Copper-Catalyzed Azide-Alkyne Cycloaddition
3.5 Protein Labeling by Oxime Ligation
4 Notes
References
Chapter 8: Encoding Noncanonical Amino Acids into Phage Displayed Proteins
1 Introduction
2 Materials
2.1 Cloning of the Target ScFv into the Phagemid
2.2 Phage Expression with ncAAs
2.3 ELISA for Functional Analysis of Displayed scFv
2.4 Western Blot Analysis and Quantification
2.5 ncAA Conjugation Reactions
3 Methods
3.1 Cloning of the Target scFv into the Phagemid
3.2 Phage Expression with ncAAs
3.3 Titration (See Note 4)
3.4 ELISA for Functional Analysis of Displayed scFv
3.5 Western Blot Analysis and Quantification
3.6 ncAA Conjugation Reactions with Fluorophores or Biotin (See Notes 7 and 9)
3.7 In-Gel Fluorescence
4 Notes
References
Chapter 9: Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation
1 Introduction
2 Materials
2.1 Cell Lines, Plasmids, and ncAAs
2.2 Expression and Purification
2.3 19F NMR
2.4 Debenzoylation
2.5 Transfection and Imaging
3 Methods
3.1 Plasmid Preparation
3.2 Protein Expression
3.2.1 Expression and Purification of Kbz-Containing Ubiquitin Under Native Conditions
3.2.2 Expression and Purification of Kbz-Containing Histone H3 Under Denaturing Conditions
3.2.3 Expression and Purification of Wild-Type SIRT2 Protein
3.3 Debenzoylation of Modified Histone H3 by SIRT2
3.4 19F NMR Experiments for Proteins with Fluorinated Benzoyllysine
3.5 Transfection and Imaging of H2B-mKate2 Protein Containing a Kbz
4 Notes
References
Chapter 10: Semisynthesis of Glutamine-Methylated Proteins Enabled by Genetic Code Expansion
1 Introduction
2 Materials
2.1 Recombinant Expression of BnE-Containing Proteins
2.2 Protein Purification from Expression Host
2.3 Reactions Performed on H3-Q56BnE
3 Methods
3.1 Preparation of Electrocompetent Cells
3.2 Plasmid Transformation
3.3 Protein Expression
3.4 Protein Purification
3.5 Conversion of BnE on a Protein to Methylated Gln
4 Notes
References
Part II: Applications in Mammalian Cells
Chapter 11: Genetic Code Expansion in Mammalian Cells
1 Introduction
2 Materials
2.1 Materials for Molecular Biology
2.2 Materials for Cell Culture
2.3 Materials for Transfection
3 Methods
3.1 Preliminary Test for Incorporation of a ncAA into the POI in Mammalian Cells
3.1.1 Demonstrate the Solubility and Cytotoxicity of ncAAs
3.1.2 Demonstrate the Incorporation of BocK on a Model Protein (See Note 3)
3.1.3 For SDS Page and Immunoblotting Analysis
3.2 Genetic Code Expansion in Mammalian Cells by the Two-Plasmid System
3.2.1 Incorporation of a ncAA in Mammalian Cells
3.3 Genetic Code Expansion in Mammalian Cells via Stable Cell Line
3.3.1 Preparation of the Lentivirus
3.3.2 Preparation of the Stable Cell Line
4 Notes
References
Chapter 12: Generation of Amber Suppression Cell Lines Using CRISPR-Cas9
1 Introduction
2 Materials
3 Methods
3.1 Transfection of Adherent Human Cell Lines
3.2 Selection of Polyclonal Pools
3.3 Selection of Individual Clones
3.4 Validation of AAVS1 Knock-In Cell Line
3.5 Implementing AAVS1 Knock-In Cell Lines in Amber Suppression Experiments
3.6 Generating Full Stable Amber Suppression Cell Lines from PylRS Knock-In Cell Lines
4 Notes
References
Chapter 13: Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding
1 Introduction
2 Materials
2.1 Noncanonical Amino Acid
2.2 Plasmids
2.3 Cell Culturing
2.4 Confocal Imaging and Flow Cytometry Analysis
3 Methods
3.1 Cell Culture
3.2 Transfection
3.3 Sample Preparation for Confocal/Fluorescence Imaging
3.4 Flow Cytometry Analysis Sample Preparation
4 Notes
References
Chapter 14: Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensa...
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture and Transfection
2.3 In-Gel Fluorescence Analysis and Immunoblotting
2.4 Immunofluorescence Reagents
2.5 Equipment
3 Methods
3.1 Transfection
3.2 In-Gel Fluorescence and Western Blot
3.3 Immunofluorescence
4 Notes
References
Chapter 15: Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids
1 Introduction
2 Materials
2.1 Cloning of Amber Codon-Bearing Receptor (FZD6) Constructs
2.2 Cell Culture
2.3 Counting and Plating
2.4 Amber Codon Suppression
2.5 Transfection
2.6 Immunoblotting
2.7 Surface Expression
2.8 Fluorescence Labeling
2.9 BRET Measurement
2.10 Data Analysis
3 Methods
3.1 Cloning of FZD Constructs
3.2 Cell Culture, Counting, Seeding, and Transfection
3.3 Immunoblotting to Assess for Amber Codon Suppression
3.4 Whole-Cell ELISA to Confirm Receptor Surface Expression
3.5 Assessment of Fluorescence Labeling Efficiency
3.6 Detecting Ligand-Induced Conformational Changes in Nluc-FZD with BRET
3.7 Data Analysis
4 Notes
References
Chapter 16: Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering
1 Introduction
2 Materials
2.1 Small-Molecule Conjugate Tet-PKA
2.2 Plasmids
2.3 Reagents and Consumables
2.3.1 Cell Culture and Transfection
2.3.2 Cell Lysis and SDS-PAGE
2.3.3 Immunoblotting
2.4 Equipment
3 Methods
3.1 Design of Bioorthogonal Ligand Tethering
3.2 Synthesis of the Small-Molecule Conjugate Tet-PKA
3.2.1 Intermediate A1
3.2.2 Intermediate A2
3.2.3 Intermediate A3
3.2.4 Intermediate A4
3.2.5 Intermediate A5
3.2.6 Small-Molecule Conjugate Tet-PKA
3.3 Cell Culture
3.4 Transfection
3.5 Detection of Kinase Inhibition by Immunoblotting
3.6 High-Content Live Cell Imaging
4 Notes
References
Chapter 17: Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells
1 Introduction
2 Materials
2.1 Sulfotyrosine
2.2 Cell Line
2.3 Plasmids
2.4 Media and Reagents
2.5 Equipment
3 Methods
3.1 Revive and Maintain Cell Culture
3.2 Transfection
3.3 Confocal Imaging
3.4 Flow Cytometry Analysis
4 Notes
References
Part III: Genetic Code Expansion in Other Models
Chapter 18: Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code
1 Introduction
2 Materials
2.1 In Vitro Transcription
2.2 Injection Preparation
2.3 Zebrafish Embryo Microinjection
2.4 Zebrafish Embryo Luciferase Assays
2.5 Conditional Activation of a Protease with Phosphine Treatment
3 Methods
3.1 In Vitro Transcription of mRNA
3.2 In Vitro Transcription of the PylT
3.3 Zebrafish Microinjections
3.4 Activating Luciferase with 2DPBM
3.5 Conditional Control of Protease Activity
4 Notes
References
Chapter 19: Noncanonical Amino Acid Incorporation in Mice
1 Introduction
2 Materials
2.1 Molecular Cloning and Protein Immunoblotting
2.2 Cell Culture
2.3 Animal
2.4 Quantification
3 Methods
3.1 Plasmid Construction and Linearization
3.2 Vasectomized Male Mice
3.3 Preparing Female Mice of Pregnancy and Pseudopregnancy
3.4 Taking Fertilized Egg
3.5 Microinjection of Linearized Targeting Vectors and Donor Template
3.6 Embryo Transfer
3.7 Genotype Identification of Transgenic Mice by PCR Analysis
3.8 Noncanonical Amino Acid-Dependent Fluorescent Protein Expression in Transgenic Mice
3.9 Generation and Characterization of trans/mdx Mice
3.10 Restoration of Dystrophin in trans/mdx Mice
4 Notes
References
Index