Expressed Protein Ligation, 1st ed. 2020
Methods and Protocols
Methods in Molecular Biology Series, Vol. 2133

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 Molecular Biology 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.

1. Methods and Applications of Expressed Protein Ligation

Zhipeng A. Wang and Philip A. Cole

2. Protein Splicing: From the Foundations to the Development of Biotechnological Applications

Neel H. Shah and Adam J. Stevens

Jing Zhao, Zhenming Du, Chunyu Wang, and Kenneth V. Mills

6. Peptide Hydrazides as Thioester Equivalents for the Chemical Synthesis of Proteins

Yu Wang and Yi-Ming Li

7. Native Chemical Ligation Via N-Acylurea Thioester Surrogates Obtained by Fmoc Solid Phase Peptide Synthesis

Judith Palà-Pujadas  and Juan B. Blanco-Canosa

8. Fused Split Inteins: Tools for Introducing Multiple Protein Modifications

Byung Joon Lim, Raymond F. Berkeley, and Galia T. Debelouchina

Silvia Frutos

10. In vivo Histone Labeling Using Ultrafast trans-Splicing Inteins

11. Site-Directed Modification of Yeast-Produced Proteins Using Expressed Protein Ligation

Benjamin J. Umlauf and Eric V. Shusta

12. Site-specific Phosphorylation of PDZ Domains

13. Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications

Nora Guidotti and Beat Fierz

14. Facile Semisynthesis of Ubiquitylated Peptides with the Ligation Auxiliary 2-Aminooxyethanethiol

Caroline E. Weller and Champak Chatterjee

15. Investigating the Effects of O-GlcNAc Modifications in Parkinson’s Disease using Semi-synthetic α-synuclein

16. Recombinant Expression of Cyclotides Using Expressed Protein Ligation

Maria Jose Campbell, Jingtan Su, and Julio A. Camarero

Somnath Mukherjee, Maria Matveenko, and Christian F.W. Becker

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts