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This volume focuses on applications of split inteins, and the progress that has been made in the past 5 years on discovery and engineering of fast and more efficient split inteins. The first few chapters in Split Inteins: Methods and Protocols explore new techniques on how to use split inteins for affinity purification of overproduced proteins, and split-intein based technologies to prepare cyclic peptides and proteins. The next few chapters discuss semisynthetic protein trans-splicing using one synthetic intein piece, synthetic intein-extein pieces used to deliver other cargos for chemical modification both of purified proteins and of proteins in living cells, as well as isotopic labeling of proteins for NMR studies, and a discussion on how protein block copolymers can be generated by protein trans-splicing to form protein hydrogels. The last few chapters deal with intein applications in transgenic plants and conditional inteins that can be regulated in artificialways by small molecules or light, a cassette-based approach to quickly test many intein insertion positions, and a computational approach to predict new intein split sites (the approach also works for other proteins). Written in the highly successful Methods in Molecular Biology series format, chapters include introduction 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.
Cutting-edge and thorough, Split Inteins: Methods and Protocols is a valuable resource that will provide guidance toward possibilities of split intein applications, explore proven and detailed protocols adaptable to various research projects, and inspire new method developments.
List of contents
Affinity Purification of Proteins in Tag-Free Form: Split-Intein-Mediated Ultra-Rapid Purification (SIRP).- Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.- Intracellular Production of Cyclic Libraries with SICLOPPS.- Recombinant Expression of Cyclotides Using Split Inteins.- Ribosomal Synthesis of Thioether-Bridges Bicyclic Peptides.- Preparation of Semisynthetic Peptides Macrocycles Using Split Inteins.- Semisynthesis of Membrane-Attached Proteins Using Split Inteins.- Protein Chemical Modification Inside Living Cells Using Split Inteins.- Segmental Isotopic Labeling of Proteins for NMR Study Using Intein Technology.- Segmental Isotope Labeling of Insoluble Proteins for Solid-State NMR by Protein Trans-Splicing.- Split-Intein Triggered Protein Hydrogels.- A Recessive Pollination Control System for Wheat-Based on Intein-Mediated Protein Splicing.- Conditional Toxin Splicing Using a Split Intein System.- Photocontrol of the Src Kinase in mammalian Cells with a Photocaged Intein.- LOV2-Controlled Photoactivation of Protein Trans-Splicing.- A Cassette Approach for the Identification of Intein Insertion Sites.- Computational Prediction of New Intein Split Sites.
Summary
This volume focuses on applications of split inteins, and the progress that has been made in the past 5 years on discovery and engineering of fast and more efficient split inteins. The first few chapters in Split Inteins: Methods and Protocols explore new techniques on how to use split inteins for affinity purification of overproduced proteins, and split-intein based technologies to prepare cyclic peptides and proteins. The next few chapters discuss semisynthetic protein trans-splicing using one synthetic intein piece, synthetic intein-extein pieces used to deliver other cargos for chemical modification both of purified proteins and of proteins in living cells, as well as isotopic labeling of proteins for NMR studies, and a discussion on how protein block copolymers can be generated by protein trans-splicing to form protein hydrogels. The last few chapters deal with intein applications in transgenic plants and conditional inteins that can be regulated in artificialways by small molecules or light, a cassette-based approach to quickly test many intein insertion positions, and a computational approach to predict new intein split sites (the approach also works for other proteins). Written in the highly successful Methods in Molecular Biology series format, chapters include introduction 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.
Cutting-edge and thorough, Split Inteins: Methods and Protocols is a valuable resource that will provide guidance toward possibilities of split intein applications, explore proven and detailed protocols adaptable to various research projects, and inspire new method developments.
