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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.
List of contents
Focused Engineering of Pyrrolysyl-tRNA Synthetase-Based Orthogonal Translation Systems for the Incorporation of Various Non-Canonical Amino Acids.- Engineering Homogeneous Photoactive Antibodies Fragments.- Repurposing Photosensitizer Proteins through Genetic Code Expansion to Facilitate Photo-Biocatalysis.- Genetic Encoding of a Fluorescent Non-Canonical Amino Acid as a FRET Donor for Analysis of Deubiquitinase Activities.- Creating Selenocysteine-Specific Reporters Using Inteins.- Protein Expression with Biosynthesized Non-Canonical Amino Acids.- Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Non-Canonical Amino Acids into Proteins in E. coli.- Encoding Non-Canonical Amino Acids into Phage Displayed Proteins.- Genetically Encoded Non-Canonical Amino Acids in Proteins to Investigate Lysine Benzoylation.- Semi-Synthesis of Glutamine-Methylated Proteins Enabled by Genetic Code Expansion.- Genetic Code Expansion in Mammalian Cells.- Generation of Amber Suppression Cell Lines Using CRISPR-Cas9.- Genetic Code Expansion in Mammalian Cells through Quadruplet Codon Decoding.- Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation.- Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Non-Canonical Amino Acids.- Selective Inhibition of Kinase Activity in Mammalian Cells by Bioorthogonal Ligand Tethering.- Site-Specific Incorporation of Sulfotyrosine into Proteins in Mammalian Cells.- Small Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code.- Non-Canonical Amino Acid Incorporation in Mice.
