Split Inteins: Methods and Protocols (Methods in Molecular Biology, 1495)

✍ Scribed by Henning D. Mootz (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 275
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Preface
References
Contents
Contributors
Chapter 1: Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP)
1 Introduction
2 Materials
2.1 Protein Expression
2.2 SIRP
3 Methods
3.1 Protein Expression
3.2 Preparation of NC1A-CBD-­Chitin Affinity Column
3.3 Purification of Protein-of-­Interest in Sample via SIRP
3.4 Regeneration of NC1A-CBD-­Chitin Affinity Column
4 Notes
References
Chapter 2: Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System
1 Introduction
2 Materials
2.1 Construction of Recombinant pET/EI0C-TP Plasmid
2.2 Protein Expression
2.3 Protein Purification
3 Method
3.1 Construction of the Recombinant pET/EI0C-TP Expression Plasmid
3.2 Expression of ELP-Intein Segment Fusion Proteins
3.3 Purification and Cleavage of the Target Protein
3.3.1 Standard Two-Step Purification Method
3.3.2 Alternate One-Step Purification Method
4 Notes
References
Chapter 3: Intracellular Production of Cyclic Peptide Libraries with SICLOPPS
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 SICLOPPS Library Construction
3.2 Splicing Assay
4 Notes
References
Chapter 4: Recombinant Expression of Cyclotides Using Split Inteins
1 Introduction
2 Materials
2.1 Instruments
2.2 Cloning of MCoTI-Intein Contruct 1
2.3 Cyclotide Expression, Purification, and Characterization
3 Methods
3.1 Construction of Intein-MCoTI-I Construct 1
3.1.1 Annealing of the DNA Fragments Encoding Split Intein Npu DnaE and Cyclotide MCoTI-I
3.1.2 Ligation and Amplification of the DNA Fragment Encoding Construct 1
3.1.3 Preparation of Expression Plasmid pET28-MCoTI-TS
3.2 Expression and Purification of Cyclotides
3.2.1 Expression of Precursor Protein Encoding the MCoTI-Intein Construct 1
3.2.2 Protein Extraction
3.2.3 Preparation of Trypsin-­Immobilized Agarose Beads for Affinity Chromatography
3.2.4 Affinity-Purification of Cyclotide MCoTI-I from Bacterial Soluble Cell Lysate
4 Notes
References
Chapter 5: Ribosomal Synthesis of Thioether-Bridged Bicyclic Peptides
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvents
3 Methods
3.1 Synthesis of O-(2-­Bromoethyl)Tyrosine (O2beY)
3.2 Cloning of the Expression Vectors for the Precursor Protein
3.3 Expression of the O2beY-
3.4 Isolation and Characterization of the DnaE Trans Splicing Products
3.5 Isolation and Characterization of the Macrocyclic Peptide Products
4 Notes
References
Chapter 6: Preparation of Semisynthetic Peptide Macrocycles Using Split Inteins
1 Introduction
2 Material
2.1 Plasmid Construction
2.2 Protein Expression and Purification
2.3 Solid-Phase Peptide Synthesis
2.4 Protein Trans-­Splicing and Peptide Cyclization
2.5 Analysis and Purification of Cyclic Peptide
3 Method
3.1 Plasmid Construction
3.2 Protein Expression and Purification
3.3 Solid-Phase Peptide Synthesis
3.4 Protein Trans-­Splicing and Peptide Cyclization
3.5 Analysis of Trans-Splicing Reaction and Purification of Cyclic Peptide
4 Notes
References
Chapter 7: Semisynthesis of Membrane-Attached Proteins Using Split Inteins
1 Introduction
2 Materials
2.1 Peptide Synthesis
2.2 Expression of Intein Fusion Constructs
2.3 Trans-Splicing
3 Methods
3.1 Peptide Synthesis and Ligation
3.1.1 Synthesis of Membrane Anchor Peptide H-CKGENLYFQSKPalm AAKPalmK-(PPO)3-A-OH
3.1.2 Synthesis of C-Terminal Intein Segment
3.1.3 Native Chemical Ligation of C-Terminal Intein and Membrane Anchor Peptide
3.1.4 HPLC Analysis
3.2 Protein Expression and Purification
3.2.1 Cloning
3.2.2 Expression
3.2.3 Folding
3.3 Protein Trans-Splicing
3.3.1 Liposomes and Lipid-Coated Particles (See Note 21)
3.3.2 Trans-Splicing Reactions
3.3.3 Folding of PrP-MA Constructs
4 Notes
References
Chapter 8: Protein Chemical Modification Inside Living Cells Using Split Inteins
1 Introduction
2 Materials
2.1 Instruments
2.2 Cloning of YY1-IN Construct
2.3 Bacterial Expression YY1-IN Construct
2.4 In Vitro Trans-Splicing
2.5 In-Cell Trans-
3 Methods
3.1 Cloning of DNA Encoding Npu DnaE IN into Expression Vector pET28a(+)
3.2 Cloning of DNA Encoding YY1-IN Construct into Expression Vector pET28a
3.3 Bacterial Expression of the YY1-IN Construct
3.4 Purification of YY1-IN
3.5 In Vitro Labeling of YY1 Using Protein Trans-Splicing
3.6 Cloning of DNA Encoding YY1-IN Construct into Mammalian Expression Vector pcDNA4/TO/myc-His
3.7 Expression of YY1-IN Fusion Protein in U2OS and HeLa Cells
3.8 In-Cell Labeling Using Protein Trans-Splicing
4 Notes
References
Chapter 9: Segmental Isotopic Labeling of Proteins for NMR Study Using Intein Technology
1 Introduction
2 Materials
2.1 Equipment
3 Methods
3.1 Cloning Segment1 into Expression Vector
3.2 Cloning Segment2 into Expression Vector
3.3 Expression and Purification of the Protein Segments
3.4 Protein Ligation and Purification
3.5 NMR Spectroscopy
4 Notes
References
Chapter 10: Segmental Isotope Labeling of Insoluble Proteins for Solid-State NMR by Protein Trans-Splicing
1 Introduction
2 Materials
2.1 Cloning of Intein Fusion Protein Expression Vectors
2.2 Expression of Intein Fusion Proteins
2.3 Fusion Protein Purification and Refolding
2.4 Protein trans-­Splicing and Purification of the Splicing Product
2.5 Refolding and NMR Sample Preparation
3 Methods
3.1 Cloning of Plasmids for the Expression of POI-Intein Fusion Constructs
3.2 Intein Fusion Protein Expression and Purification
3.3 In Vitro Protein Trans-Splicing
3.4 Purification of Spliced Protein
3.5 Protein Refolding
3.6 NMR Rotor Packing
4 Notes
References
Chapter 11: Split-Intein Triggered Protein Hydrogels
1 Introduction
2 Materials
2.1 Protein Expression Components
2.2 Protein Purification
2.3 Hydrogel Formation
2.4 Hydrogel Characterization
3 Methods
3.1 Protein Expression
3.2 Protein Purification
3.3 Hydrogel Synthesis
3.4 Hydrogel Rheology Characterization
4 Notes
References
Chapter 12: A Recessive Pollination Control System for Wheat Based on Intein-Mediated Protein Splicing
1 Introduction
2 Materials
2.1 DNA Vectors and Cloning Procedures
2.2 Plant Material
2.3 Transient Assays Using Agroinfiltration
2.4 Analysis of GUS-­Expression Level
2.5 Analysis of Barnase Expression via Quantitative Reverse Transcription PCR (qRT-PCR)
2.6 Transformation of Arabidopsis by Floral Dipping
2.7 Transformation of Wheat Plants
2.8 Molecular Analysis of Transgenic Plants
2.8.1 DNA Isolation
2.8.2 Detection and Analysis of Transgenic Events by PCR
2.8.3 DNA Gel Blot Analysis
2.8.4 Hybridization with Radiolabeled Probe
3 Methods
3.1 General Design of Split-Gene Expression Vectors
3.2 Analysis of GUS-­Expression from Split-Genes Using a Rapid Infiltration Assay
3.2.1 Agroinfiltration
3.2.2 Tissue Extraction
3.2.3 GUS Assay
3.3 Transient Plant Assays for the Analysis of Split-Barnase Vector Functionality
3.3.1 Assessment of Split-Transgene Expression via Quantitative Reverse Transcription
3.4 Analysis of the Functionality of Intein-Mediated Trans-Splicing in Transgenic Arabidopsis thaliana
3.4.1 Transformation of Arabidopsis thaliana
3.5 Analysis of the Effects of Intron Insertion on Barnase Expression in Transgenic Wheat Plants: Transformation of Wheat Plants
3.5.1 Isolation of Wheat Embryos and Callus Culture Maintenance
3.5.2 Ballistic Transformation of Wheat Plants
3.6 Molecular Analysis of Transgenic Plants for Gene Identification and Copy-­Number Estimation via PCR and Southern-­Blot Analysis
3.6.1 DNA Isolation
3.6.2 PCR Analysis (See Fig. 6)
3.6.3 DNA Gel Blot Analysis (See Fig. 6)
3.6.4 Hybridization of Membrane with Radiolabeled Probe
3.7 Crossing of Winter Wheat Plants to Create a Male-Sterile Female Line and Hybrid F1 Progeny
3.7.1 Cultivation of Plants
3.7.2 Demasculinization
3.7.3 Cross-Pollination
4 Notes
References
Chapter 13: Conditional Toxin Splicing Using a Split Intein System
1 Introduction
2 Materials
2.1 Reagents and Supplies
2.1.1 Genes Encoding CTS Constructs, GFP Splicing Constructs, and Diagnostic Constructs (NMBP and CHis)
2.1.2 Rapamycin Solution (See Note 3)
2.1.3 Transfection Reagent
2.1.4 Cell Culture Media and Reagents
2.1.5 Molecular Biology Reagents
2.1.6 Western Blot Reagents
2.1.7 Apoptosis Assay
2.1.8 Equipment
2.1.9 Flow Cytometry
2.2 Cell Culture and Media Preparation
3 Methods
3.1 Gene Assembly of Splicing Constructs
3.2 Biochemical Confirmation of In Vitro Splicing Conditions (See Notes 11 and 12)
3.3 Determination of CTox Splicing
3.4 Determination of NTox Splicing
3.5 Rapamycin-­Mediated Activation of Toxin Splicing In Vitro
3.6 Determination of Optimal Rapamycin Concentration for In Vitro Splicing
3.7 Determination of Time Required for Rapamycin-­Induced Toxin Splicing
3.8 Determination of Optimal Permissive Temperature for Toxin Splicing
3.9 Detection of Apoptosis Using Flow Cytometry
4 Notes
References
Chapter 14: Photocontrol of the Src Kinase in Mammalian Cells with a Photocaged Intein
1 Introduction
2 Materials
2.1 Plasmids
2.2 Chemicals for Synthesis of S-(1-(4,5-
2.3 Other Materials
2.4 Equipment and Software
3 Methods
3.1 Synthesis of S-(1-(4,5-
3.2 Cell Culture and Transfection
3.3 Photoactivation and the Subsequent Imaging on an Epi-fluorescence Microscope
3.4 Image Processing
3.5 Photoactivation of Bulk Samples for Subsequent Characterizations
4 Notes
References
Chapter 15: LOV2-Controlled Photoactivation of Protein Trans-Splicing
1 Introduction
2 Materials
2.1 Visualizing and Predicting Protein Structure
2.2 Target Amplification
2.3 Subcloning
2.4 Cell Culture and Transfection
2.5 Imaging and Illumination
3 Methods
3.1 Designing LOV2-Activated Target Proteins (e.g., Inteins)
3.2 Choosing Where to Split the Extein
3.3 Using LOVInC-Extein
4 Notes
References
Chapter 16: A Cassette Approach for the Identification of Intein Insertion Sites
1 Introduction
1.1 Split Inteins as Tools for Protein Biochemists: PTS and CPS
1.2 Split Intein Insertion: Limitations
1.3 The PTS and CPS Intein Cassette Design
2 Materials
2.1 Plasmid Construction
2.2 Expression and CPS in S. cerevisiae
2.3 Expression and PTS in E. coli
2.4 CPS and PTS Evaluation
3 Methods
3.1 Primer Design and Initial Intein Cassette Amplification
3.2 CPS Cassette Integration via Homologous Recombination (HR)
3.3 Restriction-Free (RF) Cloning of the PTS Cassette
3.4 CPS of the TEV Protease in S. cerevisiae
3.5 PTS of CREB in E. coli
4 Notes
References
Chapter 17: Computational Prediction of New Intein Split Sites
1 Introduction
2 Description of the Computational Method
2.1 The Concept of CPred Program
2.2 Characterization of Residues at CP Sites
3 Method
4 Case Study
References
Index