Inclusion Bodies: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Inclusion Bodies: Status Quo and Perspectives
1 Introduction
2 State-of-the-Art Biopharmaceutical IB Process: An Overview
3 State-of-the-Art Biopharmaceutical IB Process: Challenges
4 State-of-the-Art IB Process: Recent Developments and Perspectives
4.1 Cell Lysis
4.2 IB Harvest and Wash
4.3 Solubilization
4.4 Refolding
4.5 Succinctly, the Current Pressing Challenges for State-of-the-Art IB Processes
5 Alternatives to Current State-of-the-Art IB Processes
References
Part I: Upstream Processing
Chapter 2: Thermoinducible E. coli for Recombinant Protein Production in Inclusion Bodies
1 Introduction
1.1 Bioprocesses Using λpL/pR-cI857 Thermoinducible System
1.2 Molecular Responses and IBs Formation
2 Materials
3 Methods
3.1 Bioreactor Conditions and Thermoinduction
3.2 Cell Concentration Estimation
3.3 Recovery and Purification of IBs
4 Notes
References
Chapter 3: High-Throughput Expression of Inclusion Bodies on an Automated Platform
1 Introduction
2 Materials
3 Methods
3.1 Preparation
3.2 Preculture
3.3 Set-Up and Start of the Batch Phase
3.4 Cultivation Conditions
3.5 Batch Phase
3.6 Fed-Batch Phase
3.7 Measurements
3.8 Mathematical Methods
4 Notes
References
Chapter 4: Design, Production, and Characterization of Catalytically Active Inclusion Bodies
1 Introduction
2 Materials
2.1 Cloning
2.2 Bacterial Cultivation and Expression
2.3 Microscopic Analysis of CatIB Formation
2.4 Preparation of CatIBs and Cell Fractions
2.5 Determination of CatIB Formation Efficiency for Fluorescent CatIBs
2.6 Determination of CatIB Formation Efficiency for RADH CatIBs
2.7 Preparation of Lyophilized CatIBs
2.8 Determination of the Protein Content of Lyophilized CatIBs
2.9 Determination of Residual Activity Using Lyophilized RADH CatIBs
3 Methods
3.1 Selection of CatIB-Inducing Tags
3.2 Cloning
3.3 Cultivation and Expression
3.4 Microscopic Analysis of CatIB Formation
3.5 Preparation of CatIBs and Cell Fractions
3.6 Determination of CatIB Formation Efficiency for Fluorescent CatIBs
3.7 Determination of CatIB Formation Efficiency for RADH CatIBs
3.8 Preparation of Lyophilized CatIBs
3.9 Determination of the Protein Content of CatIBs
3.10 Determination of Residual Activity Using Lyophilized RADH CatIBs
4 Notes
References
Chapter 5: Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli
1 Introduction
1.1 Design of the BIDI Promoter Library and the Acceptor Plasmid
1.2 Assembly Strategy for the Expression Constructs
1.3 Proof-of-Concept
2 Materials
2.1 Gene Preparation
2.2 One-Pot Assembly and Transformation
2.3 Co-expression and Downstream Processing
2.4 Enzyme Activity Assay
3 Methods
3.1 Gene Preparation
3.2 One-Pot Assembly and Transformation
3.3 CYP505x Activity Assay
3.3.1 Cultivation and Downstream Processing
3.3.2 Biotransformation
4 Notes
References
Chapter 6: Inclusion Body Production in Fed-Batch and Continuous Cultivation
1 Introduction
2 Materials
2.1 Host Organism
2.2 Cultivation Media
2.3 Devices and Equipment for Cultivation
2.4 Process Analysis
2.4.1 Biomass Determination
2.4.2 Metabolite Determination
2.4.3 IB Concentration Determination
3 Methods
3.1 Reactor Set-Up and Cultivation Scheme
3.1.1 Pre-culture and Batch
3.1.2 Fed-Batch vs Chemostat After Batch Phase
3.2 Sampling and Analysis
3.2.1 Biomass Determination
3.2.2 Metabolite Determination
3.2.3 Product Determination
3.3 Calculation of Process Parameters
3.3.1 Calculation of Feed Rate for Fed-Batch Cultivations
3.3.2 Calculation of Dilution Rate for Chemostat Cultivations
3.3.3 Calculation of Substrate Uptake Rates
4 Notes
References
Chapter 7: Avoiding the All-or-None Response in Gene Expression During E. coli Continuous Cultivation Based on the On-Line Mon...
1 Introduction
2 Materials
2.1 Microbial Strain
2.2 Cultivation Conditions
2.3 Equipment (Segregostat)
2.4 Software and Data Repository
3 Methods
3.1 Controlling the Induction of the Arabinose Operon of E. coli Based on Segregostat
3.2 Other Applications Involving the Use of the Segregostat for the Control of Cell Population Dynamics
4 Notes
References
Chapter 8: Optimization of Inclusion Body Formation and Purification in Multi-well Plates
1 Introduction
2 Materials
3 Methods
3.1 Optimization of Protein Expression Using 24-Well Plates
3.1.1 Analysis of Protein Expression in OD583 = 10 Samples
3.2 Purification of CatIBs
3.3 Quantification of Purified CatIBs
3.4 SDS Polyacrylamide Gel Electrophoresis (PAGE)
4 Notes
References
Part II: Downstream Processing
Chapter 9: High Pressure Homogenization for Inclusion Body Isolation
1 Introduction
2 Materials
2.1 Biomass Containing the Target Product as IBs
2.2 High Pressure Homogenization
2.3 Analytical Tools
2.4 Experimental Design and Multivariate Data Assessment
3 Methods
3.1 Experimental Planning and DoE
3.2 Resuspending the Biomass
3.3 HPH Experiments for the DoE
3.4 Centrifugation Step
3.5 Washing
3.6 Aliquotation and Storage of Washed IBs
3.7 Analytics
3.7.1 RP-HPLC
3.7.2 SDS-PAGE
3.7.3 DNA Concentration
3.8 Constant DSP
3.9 Analysis of the DoE
4 Notes
References
Chapter 10: Development of Solubilization and Refolding Buffers
1 Introduction
2 Materials
2.1 IB Isolation
2.2 Development of Solubilization Buffer (See Note 1)
2.3 Development of Refolding Buffer
3 Methods
3.1 IBs Isolation
3.2 Development of Solubilization Buffer
3.2.1 Studying the Effect of Type and Concentration of Denaturant Agent on Solubilizing IBs
3.2.2 Studying the Effect of pH on Solubilizing IBs
3.2.3 Studying the Effect of Chemical Additives on Solubilizing IBs
3.3 Development of Refolding Buffer
4 Notes
References
Chapter 11: Unit Operation-Spanning Investigation of the Redox System
1 Introduction
2 Materials
2.1 Prerequisites
2.2 Unit Operations
2.3 Analytics
2.4 DoE
3 Methods
3.1 Experimental Planning and Design of Experiments
3.2 Performing the DoE
3.3 Analytics
3.4 Analysis of the Performed DoE
3.5 Validation (Reactor Setup)
4 Notes
References
Chapter 12: Using High Pressure and Alkaline pH for Refolding
1 Introduction
2 Materials
2.1 Cultivation
2.2 Bacterial Lysis and IB Washing
2.3 Solubilization of IB and Refolding
3 Methods
3.1 Bacterial Culture
3.2 Bacterial Lysis and IB Washing
3.3 Determination of pH Adequate for IB Solubilization at High Pressure
3.4 Refolding at High Pressure
4 Notes
References
Chapter 13: Intensification of Inclusion Body Processing via Temperature-Based Refolding
1 Introduction
1.1 Inclusion Bodies (IB)
1.2 Recovery of Refolded Protein from IB
1.3 Temperature-Based Refolding of Core Streptavidin (cSAV) from IB
2 Materials
2.1 Expression of cSAV
2.2 Preparation of Inclusion Bodies (IB)
2.3 Temperature-Based Refolding
2.4 Buffer Exchange into PBS
2.5 Characterization of Refolded cSAV
2.5.1 Biotin-4-Fluorescein (B-4-F) Fluorescence Binding Assay
2.5.2 Reversed-Phase-High-Performance Liquid Chromatography (RP-HPLC)
2.5.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3 Methods
3.1 Expression of cSAV
3.2 Preparation of cSAV Inclusion Bodies (cSAV IB)
3.3 Densitometric Analysis for cSAV IB Quantification
3.4 Temperature-Based Refolding of cSAV
3.5 Buffer Exchange into PBS
3.6 Removal of Aggregates and Host-Cell Protein Impurities
3.7 Characterization of Refolded cSAV
3.7.1 Determination of Biotin-Binding Capability with B-4-F Fluorescence Binding Assay (See Note 12)
3.7.2 Analysis for Purity by RP-HPLC (See Note 16)
3.7.3 Determination of Structure, Molecular Weight, Solubility, and Quantitation by SDS-PAGE
4 Notes
References
Chapter 14: Refolding of Proteins Expressed as Inclusion Bodies in E. coli
1 Introduction
2 Materials
2.1 Solubilization
2.2 Refolding and Reversed-Phase (RP) HPLC
3 Methods
3.1 Solubilization
3.2 Refolding
3.3 RP-HPLC Analysis
4 Notes
References
Chapter 15: Infrared Spectroscopy for Structure Analysis of Protein Inclusion Bodies
1 Introduction
1.1 Secondary Structure Analysis by Infrared Spectroscopy
1.2 Acquisition of an Infrared Spectrum
1.3 Modes of Infrared Spectra Acquisition
1.4 Structure Analysis of Inclusion Bodies
2 Materials
2.1 Basic Lab Equipment and Chemicals
2.2 IR Instrumentation and Accessories
3 Methods
3.1 IR Spectra Acquisition
3.1.1 Common Operator-Set Parameters for Recording FTIR Spectra
3.1.2 (Micro)Spectroscopy of Dried IBs in Transmission
3.1.3 ATR-IR Spectroscopy of Dried IBs
3.1.4 Transmission Spectroscopy of IBs in Aqueous Solution
3.1.5 ATR-IR Spectroscopy of IBs in Aqueous Solution
3.1.6 Transmission Spectroscopy of IBs in D2O Solution
3.2 IR Spectra Processing
3.2.1 Correction for Atmospheric Water Vapor
3.2.2 Difference Spectra
3.2.3 Second Derivative Spectra
3.3 Secondary Structure Analysis by Curve Fitting
4 Notes
References
Part III: Success Stories
Chapter 16: The Purification of Heme Peroxidases from Escherichia coli Inclusion Bodies: A Success Story Shown by the Example ...
1 Introduction
2 Materials
2.1 Equipment
2.2 Isolation and Solubilization
2.3 Refolding
2.4 Chromatography
2.5 Enzyme Activity Measurements
2.6 RP-HPLC
2.7 SE-HPLC
3 Methods
3.1 Isolation and Solubilization
3.2 Refolding
3.3 Chromatography
3.4 Enzyme Activity Measurements
3.5 Reinheitszahl
3.6 RP-HPLC
3.7 SE-HPLC
4 Notes
References
Chapter 17: Production of α-Synuclein Fibrillar-Specific scFv from Inclusion Bodies
1 Introduction
2 Materials
3 Methods
3.1 Cloning E. coli Expression Construct
3.2 Expression of scFv-pF
3.3 Comparison of Inclusion Body Solubilization Using Different Methods
3.4 On-Column Refolding of Solubilized scFv-pF Followed by Affinity Purification
3.5 Characterization of Purified scFv-pF Using Western Blotting and DOT-Blot
4 Notes
References
Chapter 18: Method for Inclusion Bodies Production via E. coli Host System: rGCSF as Model Biotherapeutic Protein
1 Introduction
2 Materials
2.1 Fermentation
2.2 Cell Disruption
3 Methods
3.1 Fermentation
3.2 Cell Disruption
4 Notes
References
Chapter 19: Antimicrobial Applications of Inclusion Bodies
1 Introduction
2 Materials
2.1 Production of IBs using Escherichia coli
2.2 IB Purification
2.3 Western Blot and Coomassie
2.4 Determination of Antibacterial Activity of IBs
2.5 Determination of Antibiofilm Activity of IBs
2.6 Equipment
3 Methods
3.1 IB Production using E. coli
3.2 IB Purification
3.3 Determination of IB Yields by Western Blot and Coomassie
3.3.1 Gel Preparation
3.3.2 Sample Preparation and Electrophoresis
3.3.3 Coomassie Staining
3.3.4 Gel Transfer to a PVDF Membrane
3.3.5 Western Blot
3.4 Determination of Antibacterial Activity of IBs
3.5 Determination of Antibiofilm Activity of IBs
4 Notes
References
Index