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This book provides a comprehensive overview of Expressed Protein Ligation (EPL), detailing methods and protocols to generate site-specifically modified proteins. Chapters include an overview of the protein semi-synthesis field, as well as related areas that have contributed to the development of EPL such as protein splicing and peptide synthesis. Following the introductory chapters, the rest of the book guides readers through protocols to perform EPL reactions, methods to synthesize peptide thioesters and to perform peptide and protein ligations, label proteins inside living cells, protocols for the semi-synthesis of phorphorylated, glycosylated and ubiquitylated proteins, synthesis and assembly of assymetrically modified nucleosomes, use of ligation auxiliaries and synthesis of cyclic proteins, as well as novel desulfurization strategies and use of selective Cys side chain protection to obtain precisely modified proteins.Written in the highly successful Methods in MolecularBiology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Expressed Protein Ligation: Methods and Protocols will ensure successful implementation of protein semi-synthesis methods to further study the structure and function of proteins.
List of contents
Methods and Applications of Expressed Protein Ligation.- Protein Splicing: From the Foundations to the Development of Biotechnological Applications.- Identification, Characterization, and Optimization of Split Inteins.- Methods to Study the Structure and Catalytic Activity of cis-splicing Inteins.- Expressed Protein Ligation: General Experimental Protocols.- Peptide Hydrazides as Thioester Equivalents for the Chemical Synthesis of Proteins.- Native Chemical Ligation Via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid Phase Peptide Synthesis.-Fused Split Inteins: Tools for Introducing Multiple Protein Modifications.- Streamlined Expressed Protein Ligation: Site-specific Antibody-drug Conjugate.- In vivo Histone Labeling Using Ultrafast trans-Splicing Inteins.- Site-Directed Modification of Yeast-Produced Proteins Using Expressed Protein Ligation.- Site-specific Phosphorylation of PDZ Domains.- Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications.- Facile Semisynthesis of Ubiquitylated Peptides with the Ligation Auxiliary 2-Aminooxyethanethiol.- Investigating the Effects of O-GlcNAc Modifications in Parkinson's Disease using Semi-synthetic -synuclein.- Recombinant Expression of Cyclotides Using Expressed Protein Ligation.- Highly Precise Protein Semisynthesis through Ligation-Desulfurization Chemistry in Combination with Phenacyl Protection of Native Cysteines.
